Journal Pre-proof

Overcoming barriers by local drug delivery with liposomes

S.G. Antimisiaris, A. Marazioti, M. Kannavou, E. Natsaridis, F.

Gkartziou, G. Kogkos, S. Mourtas

PII: S0169-409X(21)00031-4
DOI: https://doi.org/10.1016/j.addr.2021.01.019
Reference: ADR 13717

To appear in: Advanced Drug Delivery Reviews

Received date: 10 November 2020

Revised date: 17 January 2021
Accepted date: 23 January 2021

Please cite this article as: S.G. Antimisiaris, A. Marazioti, M. Kannavou, et al.,
Overcoming barriers by local drug delivery with liposomes, Advanced Drug Delivery
Reviews (2021), https://doi.org/10.1016/j.addr.2021.01.019

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© 2021 Published by Elsevier.

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Overcoming barriers by local Drug Delivery with liposomes

S.G. Antimisiaris1,2,* santimis@upatras.gr, A. Marazioti1, M. Kannavou1,2, E. Natsaridis1,2, F. Gkartziou2, G. Kogkos1,2, S. Mourtas1

1
Laboratory of Pharmaceutical Technology, Dept. of Pharmacy, School of Health Sciences, University of Patras, 26504 Rio Patras,
Greece
2

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Institute of Chemical Engineering Sciences, Foundation for Research and Technology Hellas (FORTH/IEC-HT), 26504 Rio Patras,

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Greece
*
Corresponding author.

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Abstract
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Localized or topical administration of drugs may be considered as a potential approach for overcoming the problems caused by the various

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biological barriers encountered in drug delivery. The combination of using localized administration routes and delivering drugs in nanoparticulate
formulations, such as liposomes, may have additional advantages; Such advantages include prolonged retention of high drug loads at the site of

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action and controlled release of the drug, ensuring prolonged therapeutic effect; decreased potential for side-effects and toxicity (due to the high

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topical concentrations of drugs); and increased protection of drugs from possible harsh environments at the site. The use of targeted liposomal

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formulations may further potentiate any acquired therapeutic advantages. In this review we present the most advanced cases of localized delivery
of liposomal formulations of drugs, which have been investigated pre-clinically and clinically in the last ten years, together with the reported
therapeutic advantages, in each case.

Keywords: Liposomes, Drug, Topical routes, Local Delivery, Administration, Ocular, Pulmonary, Nasal, Oral, Intratumoral, Vaginal, Vaccine

1. Introduction

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Positive patient outcomes across a wide range of pathologies highly rely on the ability to direct drugs to their specific site of action, at
concentrations that will be sufficient to elucidate a therapeutic effect. Thereby, a very important current challenge in the drug delivery field is the
enhancement of drug transport across the various biological barriers (BBs) they encounter on route from their administration site to the site where
they express their therapeutic action. It is logical then to conclude, that such BBs are mostly encountered when drugs are administered by the so
called “non-invasive” of „less-invasive” routes that do not directly place the full fraction of the administered dose in the systematic circulation;

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examples of such BBs are the mucosal epithelium of the GI-track (encountered after oral administration), the nasal or lung epithelia (encountered
following nasal or pulmonary administration, respectively), the skin (after dermal/transdermal administration) as well as the various mucosal

o
epithelia in the mouth (encountered after sublingual, buccal, dental and oromucosal administration). In general, the intensity of the drug delivery

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challenges increase with the non-invasive nature of the drug administration method. From a pharmaceutical point of view the characterized as

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„less-invasive‟ routes come with important advantages such as, highly reduced manufacturing cost, longer product shelf-life, highly improved

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patient convenience (which leads to increased compliance), and others. However, depending on the pathology treated, persistent BBs may be
encountered even when drugs are injected directly in the bloodstream. Indeed, the most challenging BB today, is the Blood-Brain-Barrier (BBB)

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[1, 2], which is a particular problem for drugs that act in the CNS; thereby it involves a great number of therapeutics for neurodegenerative
pathologies, brain cancers etc. Other important barriers that are also encountered following intravenous injection are located in the eye, such as
the blood-retinal-barrier (BRB) [3-5].

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BBs are designed by nature as a security wall, to keep foreign substances (that may be toxic) out of the body and only allow entrance for specific

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types of -required by our body- molecules, such as nutrients, minerals, etc. which have specific structural characteristics that aid their transport
across the BBs, e.g. by receptor-mediated transcytosis or other mechanisms. Nevertheless, as the pharmaceutical industry moves towards
development of macromolecular biological substances as the next generation of therapeuticss [6], the drug delivery challenges as well as the
bottlenecks of overcoming biological barriers, are highly intensified.

Although many methodologies to overcome BBs are currently being considered [6-16], one simple approach for drugs with a direct mode of action
is to use localized administration. The latter is of course possible, only if the site of action can be reached without the need to overcome a BB.
Localized drug administration has several advantages compared to systemic delivery (Figure 1), such as increased bioavailability, reduction of
side effects/toxicity and off-target effects (due to reduced systemic exposure), , and reduced cost (in some cases) [17]. Nevertheless, in many

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situations, when local drug delivery in discrete body cavities is considered or when the local route is possible only by overcoming specific bodily
barriers and tissues, usually the administration is carried out by special injection methods which are difficult to perform and require specialized
personnel, or even special apparatus or dosing devices (even for non-injectable formulations). Additionally, the administration may involve
particular pain, and/or increased potential for side effects or infections due to wrong manipulations or other reasons (Figure 1, case 2). Thereby, in
such cases it is preferable to administer high drug doses that will stay in the area for prolonged time periods, reducing the need for frequent dosing.

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However, such high drug doses, sufficient to provide therapeutic drug concentrations locally for a few days, weeks or even months, are highly
likely to cause local toxicity or inflammation; it might additionally be a huge challenge to retain the drug at the site for the required time periods,

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depending on the drug clearance mechanisms available at the particular sites. As depicted in Figure 2, drug delivery systems (DDSs), with

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capability to retain high amounts of drugs at localized sites, and to release the drug in a controlled manner (to maintain therapeutic drug

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concentrations for prolonged time periods), come in handy for the particular purpose. Furthermore, such DDSs may additionally protect the

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surrounding tissues from potential toxicities and/or inflammation that may be else wise induced by the high concentrations of drugs. Additionally,
at the same time they can protect the drug from potential harsh environments at the local sites of administration [18]. Thereby, local

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administration of controlled DDSs may be considered as a potential strategy to overcome biological barriers.

Nanoparticulate DDSs or nanomedicines offer additional advantages compared with other DDS types, due to their nano-size (because of which

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they have higher capability to diffuse through different types of biological media), and the easier control of their surface properties which

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facilitates the construction of DDSs with potential to target specific cell types, if required. Various types of nanomedicines were studies as

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localized administered DDSs, and in many cases they have demonstrated improved therapeutic efficacy [19-22]. Between the various types of
nanomedicines, liposomes (LIP) (Figure 3) are considered to be highly safe, non-toxic and biocompatible, explaining their vast applicability as
drug carriers [23]. In the current review article we focus on localized delivery of liposomes, as an approach to overcome BBs.

2. Liposomes in Drug Delivery

The research on liposome DDSs has grown substantially in the past few decades [23-29]. Liposomes (LIPs) are vesicular nanosystems with
numerous potential advantages as drug cerriers over other nanomedicine types; mainly because they consist of natural substances as boilding

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blocks (Figure 3). For the latter reason LIPs are highly biocompatible and safe ( non-toxic and non-immunogenic) and also fully biodegradable,
[23], being thus ideal for local delivery of therapeutics.

Indeed, as seen in Figure 3, LIPs are composed of phospholipids and cholesterol (Chol), the main building blocks of biological membranes,
explaining their safety and biocompatibility. Another advantage of LIPs, compared to other types of drug carriers, is that they can incorporate
broad types of active substances and due to their structural versatility, modify their pharmacokinetics and consequently improve their therapeutic
profile [23-29].

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LIPs consist one or more (co-centric) membrane bilayer(s) encapsulating an aqueous volume in their core and also between the co-centric bilayers,

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(if they consist of more than one bilayer). Due to their structure LIPs are capable to encapsulate or incorporate high loads of almost any drug

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molecule, regardless of its physicochemical properties or molecular size; lipid soluble drugs may be accommodated in the membrane bilayer(s) of
LIP, and aqueous soluble drugs in their aqueous compartment(s). The numerous current methodologies for liposome preparation, makes it possible

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to select the most appropriate, depending on the properties and the stability of the drug to be loaded. Indeed, lipophilic, water soluble as well as
amphiphilic drug molecules may be efficiently loaded in LIPs; particularly in the latter case, the possibility to attain practically 100% loadings by

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using remote or active loading methodologies is an additional advantage [23, 28, 29]. In addition to solutions, LIPs can also encapsulate

bacteria for vaccine development [31].

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particulates, such as iron oxide NPs [30], gold NPs or quantum dots for therapeutic or diagnostic applications [23,28,29], or even attenuated

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In addition to their biocompatibility and to the fact that both, lipid soluble and aqueous soluble drugs can be accommodated by them, LIPs have
many more advantages as drug carriers, compared with other types of nanoparticles (NPs), as presented in Figure 3. One of them is that LIPs can
protect labile drugs from distortion by the harsh conditions that apply at the site of administration, or the site of action or in the blood, and can
additionally facilitate the transport oh hydrophilici drugs through biological membranes [19,23,28, 32]. In fact the use of liposomes for the
protection of actives that are sensitive in blood and cannot be transported through membranes (due to their poor permeability) is particularly
popular today since two of the first COVID-19 vaccines (that received approval for human use) that are mRNA vaccines (the BNT162b by
Biontech/Pfizer, and mRNA-1273 by Moderna), are delivered in liposomal form (more details about them are mentioned in part 4).

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As mentioned above, the biological advantages of LIPs are related to their biodegradability, biocompatibility and very low toxicity, since they are
composed of phospholipids and cholesterol [23, 28, 29]. Additionally, by shielding their surface (that is recognized by mononuclear phagocyte
system) with hydrophilic polymers, such as PEG, the circulation time of liposomal drugs after systemic administration, or in other words their
bioavailability, can be substantially increased (compared to the bioavailabilty of free drugs) [33].

When the topical delivery of LIP is considered, as in the case of formulations of polymer-composed NPs, the viscosity and rheological

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characteristics and perhaps also the mucoadhesive properties of the formulation may have to be appropriately adjusted; the latter can be easily
achieved by addition of gelling agents in LIP dispersions [29]. Other types of excipients/additives such as, co-solvents, penetration enhancers,

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surfactants, etc. may be perhaps additionally required for different purposes, depending on the drug that will be used as well as on the specific

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therapeutic application and route of delivery [23, 24]. More details about the various types of LIPs are given in the next section.

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Compared to other categories of nanomedicines and especially with polymeric nanoparticles (NPs), the most important disadvantage of LIPs is
their limited potential to retain drugs for prolonged time periods (Figure 3). Indeed, high MW polymers can prolong drug retention for long time

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periods (up to 6 months or even a year), compared to LIPs. Nevertheless, the latter limitation of liposomal drugs can be overcome by development

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of hybrid LIP formulations, consisted of polymer-coated LIPs, or LIPs that are embedded in polymeric hydrogels [23-29]; several examples of

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such “hybrid” LIP formulations will be presented in the following parts where local delivery of LIPs is discussed.

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2.1 Liposome Types and Biological Fate

Since this reviews focus is on liposomal drugs, it is useful to provide some more information and details about the various types of LIPs, and their
particular properties and biological fate.

Several types of LIPs exist, depending on their physicochemical properties (size, number of lamella, surface charge, integrity), and their biological
fate (following in vivo administration). As presented schematically in Figure 4, depending on the number of lamella that the vesicles contain, LIP
may be multi-lamellar (MLV), oligo-lamellar or uni-lamellar, while unilamellar LIPs. In regards to their size, LIPs can be characterized as small
(SUV) (<200nm), or large (LUV) (>200nm); Giant LIPs which may be multilamellar or not, or multicompartmental [31], may also be formulated.
Due to the increased number of lamella, multilamellar LIPs demonstrate higher integrity and retain entrapped drugs for longer time periods,

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compared to unilamellar LIPs, however their larger size minimizes their applicability as drug carriers, since the small size of liposomes is
particularly important for their circulation in blood, and also when they are intended to pass through biological barriers and/or to enhance the
cellular uptake of LIP-associated drugs [23]. LIPs that are smaller than 200 nm, are particularly efficient for the delivery of anti-cancer drugs, due
to the Enhanced Permeation and Retention (EPR) effect which is realized in some cancer types [22-28]. According to the EPR effect, liposomes
that have diameters below 200nm have the capability for extravasation through the tumour vasculature which is significantly more “leaky”
compared to the vasculature of normal tissues.

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LIPs may also be characterized by their surface charge or zeta-potential, as zwitteroionic or neutral (practically no charge), cationic (positive

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charge) or anionic (negative charge). Cationic LIPs are important for gene delivery, as mentioned in more detail below.

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Depending on their biological fate following in vivo administration, which is highly dependent on their lipidic composition and especially their

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surface properties, LIP can be characterized as Conventional or long circulating, depending on their integrity in the bloodstream and their
corresponding biological half-life. Conventional LIPs have very low integrity in blood (compared to Long-circulating LIPs) and are rapidly taken

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up by the macrophages of the reticuloendothelial system (RES) following in vivo administration, accumulating in the liver and spleen.

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Oppositely, Long-circulating or Stealth or sterically-stabilized LIPs, demonstrate prolonged circulation periods in the blood (following i.v.

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injection), as a result of their decreased interaction with blood components; the latter is attributed to the composition (and corresponding

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properties) of their surface which is (usually) coated with hydrophilic polymers, usually PEG (explaining the term “PEGylated” LIPs).

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Historically, other methods were tested in order to increase the integrity of liposomes in blood, such as size reduction, addition of high amounts of
cholesterol in their membrane, and the use of saturated phospholipids in order to increase LIP membrane rigidity; however LIP PEGylation results
in substantially higher blood circulation periods (compared to the other methods), and is currently the method of choice for development of long-
circulating LIPs.

Long circulating or Stealth or PEGylated LIPs have numerous application in cancer therapeutics, since due to their long blood circulation times
and the Enhanced Permeation and Retention (EPR) effect (mentioned already above), which is realized in some cancer types, higher amounts of
such LIP accumulate in cancer tissues (compared to normal tissues), resulting in higher local bioavailability of anticancer drugs (compared to
when the anticancer drug is administered as free drug or associated with other types of LIPs).

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Long circulating or Stealth or PEGylated LIPs are the LIP types (Fig. 4), that possess the most important therapeutic advantages (Fig. 3), since
they may modulate the biodistribution as well as the pharmacokinetic parameters of drugs (compared to those of the free drug) resulting thus in
reduced toxicity and side effects of drugs [33, 34]. A well-known example of reduced toxicity and side effects is the dramatically reduced
cardiotoxicity of liposomal doxorubicin, compared to the free drug [35]. Reduced frequency of administration, in cases when the LIP-associated
drug is released slowly from LIPs (that remain intact for prolonged time periods post-administration), is another potential advantage [34].

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However, the therapeutic advantages methioned above are mainly demonstrated in cases of highly toxic drugs and particularly when the
pharmacokinetics of the liposomal formulations are such, that the drug concentration in tissues where they express high toxicity is adequately

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reduced. Unfortunately this is not the case for all drugs and pathologies. In the latter cases, specific targeting ligands or homing molecules are

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required to realize high concentrations of LIP drug to the particular target cells or tissues, where they manifest their therapeutic action [35, 36].

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Thereby, according on the specific therapeutic application and drug to be delivered, long-circulating LIPs may be engineered to have one or more

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(multitargeted) targeting ligand on their surface, to direct them to their therapeutic target at higher amounts (compared to the amounts distributed
in other tissues). Ligand-targeted LIPs, are being considered today in many cases, and many examples are mentioned in the following parts of this
review.

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Another sub- type of Long-circulating LIPs, are the Cationic LIPs or LIPs for gene delivery. These LIPs include special types of lipids in their
membrane (cationic or ionizable lipids), in order to be able to accommodate different types of oligonucleotides (which bear high negative charge)

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for gene-delivery applications. The first types of lipidic carriers developed as systems for the delivery of DNAs or RNAs were cationic LIPs (also
known as lipoplexes), and solid lipid NPs (SLNs). With the synthesis of new types of charged or ionizable lipids, novel types of vesicular
structures evolved [29]. Four major types of LIPs or lipidic nanoparticles (Fig. 4) with different structure are currently known [29] : (i) Lipoplexes,
also reffered to as cationic-LIPs, (ii) SNALPs, which is acronym for Stable nucleic acid–lipid particles, (iii) Lipopolyplexes, that can be
considered as more complex types of lipoplexes; also mentioned as Multifunctional-Envelope-type-Nano Devices (MENDs)from), and last (iv)
MCNPs, which is the acronym for Membrane-Core NPs [29]. All cationic-LIP types have more or less some type of vesicular structure, i.e. the
oligonucleotides (ONs) are in some way “encapsulated” in the vesicles, and thus protected from the environment they encounter after their in vivo
administration. When required, extra ON protection may be provided by coating the ON-encapualting vesicles with PEG (as an example) which
may also serve this role, in addition to providing long circulation in blood. Finally, various types of ionizable lipids provide the additional

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advantage of early endosomal escape after cell uptake of liposomes by endocytocis, preventing the lysosomal degradation of the LIP-entrapped
ONs.

Other special types of liposomes also exist, such as transformable or elastic LIP and triggered –release LIPs. The so called “Elastic” LIPs are
mainly intended for topical skin administration; these LIP types have special components in their membrane (edge activators) that make them
more elastic or “deformable”, or else able to squeeze through narrow passing‟s. More details about the former LIP types are discussed in the part

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of Skin Delivery of liposomal drugs (Part 3.4). Conserning the triggered-release LIPs, these are various types of LIPs (such as conventional, or
stealth or targeted, etc.), which are sensitive to specific envirormental triggers, such as pH (pH-sensitive LIPs) or temperature (temperature-

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sensitive LIPs), or light (photo-sensitive-LIPs), etc. Such sensitive to environment LIP types are able to release higher amounts of encapsulated

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drugs at specific sites in the body, either due to physiologically existing triggers at the specific areas (different pH or ionic strength in different

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body cavities or tissues, or cellular compartments) or by externally activated triggers (temperature, magnetic fields, light, etc). Some examples will
be discussed in different subsections of Part 3.

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Concerning the potential methods of interaction between LIPs and cells, depending on the LIP composition, surface properties and type, as well as

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on the type of cells considered, LIPs may: (i) Be endocytosed by the cells (particularly in the case of ligand-targeted LIP, where the ligand

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identifies and binds to receptors that are over-expressed of the cell membrane), through clathrin or caveolin mediated routes; in such cases early
endosomal escape is of great importance if the drug is destroyed by the lysosomal contents; (ii) Be adsorbed on the cell surface, or exchange some

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of its components with those of the cell membrane; the drug is released in close proximity with the cell and may be eventually taken up; (iii) Be
fused with the cell membrane, particularly when the liposomes have specific “fusogenic” components. The mechanism of interaction and cellular
uptake of LIP drugs may be clarified by quantitative and morphological methods.

According to the intended therapeutic application, the optimal LIP type can be selected, from all the different types mentioned above (Fig. 4). At
this point, it should be mentioned that other means of LIP categorization or naming are also possible and used in the relevant literature. Indeed,
LIPs can be named depending on the specific method used for their production (such as REV (reverse evaporation vesicles), DRV (dehydration
rehydration vesicles), freeze-thawed, etc.), for the reduction of their size (sonicated, extruded, etc), or by the method used to load them drugs
(active loaded, remote loaded, etc.).

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3. Local Routes for Liposome administration. A way to overcome challenging Biological barriers

Several liposomal drug formulations are currently being used as therapeutics in clinical practice, and others are under clinical testing, as reviewed
elsewhere [25-27]. Additionally, several preclinical studies report significant improvement of the therapeutic activity of specific drugs when they
are targeted by the use of ligand-targeted LIPs. However, despite the intensive on-going research, and numerous encouraging reports, no ligand-(or
actively)-targeted LIPs (or any nanomedicine type) has been approved for clinical use up-to-date [35, 36], although several are under clinical

f
testing [27]. Thereby, in addition to overcoming BBs, localized administration of liposomal drugs may also be considered as a method of

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overcoming the current bottleneck in the field of ligand-targeted LIP development.

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An analysis on liposomal drug product submissions for clinical study was recently performed [37] and reported that the majority of the liposomal

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products (under clinical testing) are administered via the intravenous route (84%). In fact, it is suggested in the previous report that perhaps the

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exploration of local administration routes for liposomal drugs has not been adequately considered up-to-date. We hope to highlight this area with

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the current review, and encourage future research efforts on this topic.

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In this article we reviewed the recent literature, focusing on the last 10 years (2010-to-date), concerning the applicability of liposomal drugs for
controlled localized drug delivery. We focus on approaches to overcome barriers for specific types of diseases, by using special types of localized

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routes/methods of administration. As depicted in Figure 5, the methodology included initial searches in Pub Med database

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(https://pubmed.ncbi.nlm.nih.gov/ ) which were performed using the key words:“ROUTE” + “LIPOSOME” + (“DELIVERY” OR

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“ADMINISTRATION”) + (“TOPICAL” OR “LOCAL”); + “IN VIVO”. At a second step the specific hits (numbers of hits appear in Table 1)
were reviewed and evaluated for their relativity with the aim of this article. For some routes when the term (“TOPICAL” OR “LOCAL”) was
removed, the number of hits increased highly (Table 1); in these cases all references were screened. For all cases of routes of administration, many
or several of the hits screened, were not further evaluated since they were not relevant with the aim of the current review, mostly because non-
localized administration was applied, or the reports were about other types of nanomedicines and not LIPs. The last step was the selection of the
hits that were finally evaluated for each route of administration.

As seen in Table 1, the local administration routes considered included: Intra-articular (IA) delivery, Intranasal (IN) delivery, Intra-tumoral (IT)
administration, Skin delivery (Dermal and Transdermal delivery), Ocular or Intraocular delivery (to anterior and posterior eye segments), Oral

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delivery (including oral and oromucosal, sublingual vaccine delivery and Dental delivery), Pulmonary or Lung administration (including injection
to the pleural cavity), Vaginal and Uterine administration and other routes. All routes are analyzed in the following part.

3.1 Intra-articular delivery

Intra-articular (IA) delivery is intended for locally acting drugs. As discrete cavities, di-arthrodial joints are ideal sites for local drug administration

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by direct intra-articular injections, thereby IA delivery is particularly important for the therapy of diarthrodial joint disorders [17]. In fact, in most

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of these pathologies (with the exception of rheumatoid arthritis) there are no clinically significant extra-articular symptoms which would require
systemic availability of the drugs; making IA delivery the route of choice [17]. Poly-articular inflammatory pathologies, such as rheumatoid

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arthritis (RA), gout, and osteoarthritis (OA), affect individual joints, and also have high incidence and long-term therapeutic needs. The currently

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available treatments for these pathologies are not sufficient, and cost-effective methods to achieve long-term retention of the drugs at the particular

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anatomical sites (for enhanced efficacy), are urgently needed.

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In general, the concept of intra-articular injection of drugs, as free drug molecules or in various types of particulate formulations, is schematically
depicted in Figure 6 [20]. As seen, the main advantage of injection of liposomal formulations is the prolonged retention of the drug at the site.

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This is particularly important for low-molecular weight drugs, which are cleared rapidly from the site via blood circulation, nevertheless even

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large proteins can be retained for substantially higher time periods when injected as LIP formulations, compared to free molecules. Another
advantage of liposomes is their safety profile and their ability to retain high drug concentrations in them, decreasing potential drug-related tissue

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toxicity.

Several recent reports confirm the therapeutic advantages of intra-articular delivery of LIP formulations (Table S1). The majority of the latter
reports involve therapeutics intended for the treatment of arthritic diseases, while reports of advanced studies for pain management and for bone
tuberculosis were also found. Since IA injection is particularly painful, it is of particular importance to retain high amounts of drugs at the site for
as long as possible. Thereby, the prolongation of LIP retention by development of hybrid LIP formulations is particularly useful for this route of
administration. A brief description of the various recent cases of IA delivery of LIP drugs (Table S1) follows.

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VX-745 is a p38 MAPK inhibitor with high selectivity that has similar activity with dexamethasone. When VX-745-LIPs were
delivered intra-articularly in an antigen-induced arthritic (AIA) mouse model, therapeutic drug concentrations were rapidly achieved,
and extended pain management for up to 2-3 months was observed [38].

Human lactoferin (hLf), a glycoprotein possessing anti-inflammatory and antimicrobial activities towards RA was injected IA in a
collagen-induced arthritis mice model, entrapped in LIPs (Lf-LIPs); negatively charged, positively charged as well as pH-sensitive

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LIP were tested [39]. Positively charged LIPs were found to be more efficient in prolonging hLf residence in inflamed joints

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(compared to the other formulation). In another study (with the same formulations) dendritic cells (DCs) in draining lymph nodes were

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identified as the targets of the LIPs. The Lf-LIPs significantly decreased the arthritic score, continuously for 2 weeks (compared to 3-4

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d for free Lf). Furthermore the LIPs resulted in decreased expression of some co-stimulatory molecules on the surface of dendritic

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cells and

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in the reduction of pro-inflammatory (TNF) cytokine concentrations. LIPs also increased the production of anti-
inflammatory cytokines (IL-10) [40], verifying the increased efficacy and decreased toxicity of the liposomal formulation, compared
to the free drug

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In another report, optimized paclitaxel-LIPs were tested for their efficacy in an antigen-induced rat model of arthritis, and were

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compared with direct injections of the free drug [41]. Histopathological studies demonstrated that IA delivery of LIPs led to

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significant improvement (compared to free drug) of gait scores and synovial inflammation in rats, 28d post-injection.

As mentioned before, the retention of liposomal drugs can be prolonged by the development of hybrid LIP formulations consisted of LIP, in gels.
In this context, IA injection of celecoxib (Clx) LIP embedded in hyaluronic acid (HA) gel, was evaluated against cartilage degeneration in an OA
model in rabbit knees. Rabbits were IA injected with free drug, HA and Clx-LIPs separately, and also HA and Clx-LIPs combination (hybrid-
LIPs). It was verified that the combination of Clx-LIPs and HA, or in other words the hybrid LIP system, was significantly more effective for pain
control (compared to the controls), Furthermore, it is of particular importance that the LIP formulation was found to additionally provide
increased cartilage protection [42].

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In another case, targeted liposomes were tested for IA delivery of a drug. Novel types of LIPs encapulatimg Ni(2+)-(N-5-amino-1-carboxypentyl)-
iminodiacetic acid, and additionally also targeted with APO2L/TRAIL (Apoptosis ligand 2 tumor necrosis factor (TNF)-related apoptosis-inducing
ligand), were IA injected into knees of rabbits with adjuvant-induced arthritis (AIA). The targeted LIPs (APO2L/TRAIL-LIPs) demonstrated
increased bioactivity compared with non-targeted LIPs, and substantially reduced synovial hyperplasia and inflammation. Results indicated that
the increased bioactivity was due to the higher concentration of the recombinant protein at the site. Additionally, it is particularly important to
highlight that the treatment with targeted LIPs, lacked systemic toxicity and was not hepatotoxic [43].

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The anti-hyperalgesic effect of LIP formulations of two novel NSAID-CAI (nonsteroidal anti-inflammatory drugs and carbonic inhibitors) hybrid

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molecules (Sulindac and Diclofenac) was investigated in a rat model of RA. LIP/Diclofenac after only one intra-articular injection, had higher

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efficacy compared to repeated oral treatments, demonstrating significant localized activity [44].

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In another case, RvD1 (therapeutic) and macrophage depletion with Clodronate-loaded LIPs (CL) were tested for therapy of post-traumatic OA
(PTOA) in obese mice; results showed that CL treatment reduced synovitis and cartilage destruction, while IA treatment with RvD1 diminished

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the progression of OA in the knee joint, by decreasing macrophage infltration in synovium [45].

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IA administration of bupivacaine (which is a local anaesthetic), encapsulated in LIPs was evaluated for the management of post-operative pain in

n
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exercised horses. The retention of the drug at the joints and the plasma clearance were evaluated together with measurements of markers of

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collagen degradation and synthesis of cartilage matrix in synovial fluid. Bupivacaine concentrations in the joint were still detectable after 96 h.

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The sustained concentrations of IA delivered bupivacaine-LIPs proved their potential as an IA analgesic; however some effects on the animal
chondrocytes need further study [46]. It should be mentioned at this point that particularly for pain management with intra-articular injection of
Bupivacaine-LIPs, many clinical studies have been (or are currently being) carried out, as discussed in Part 4 (below).

In another report, the treatment of localized bone tuberculosis (TB) was attempted by IA injection of LIPs encapsulating an isoniazid derivative
(DINH). For prolongation of their retention at the site, a hybrid LIP formulation was developed by embedment in a PLGA-PEG-PLGA co-
polymer hydrogel. In addition to its therapeutic advantages, the hybrid system demonstrated thermo-responsive and self-healing properties. The
latter properties are particularly useful for IA administration, since the system could retain high amounts of drugs at the site, even under increased

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pressure. In fact, sustained drug release for several days was demonstrated in rabbits, providing evidence that the system can provide important
therapeutic advantages for the therapy of bone TB [47].

Summarizing, many recent studies [38 – 47] prove the therapeutic advantages, the safety and lack of toxicity of IA injected LIP formulations. The
use of hybrid LIP systems (such as LIPs embedded in hydrogels, or in thre-block copolymer gels) as methods to extend the retention of LIP drugs
at the site of injection, has been successfully applied. Targeted LIPs were also evaluated and found to have therapeutic advantages (compared to
non-targeted ones).

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3.2 Intranasal (IN) Delivery and nose-to-brain delivery following nasal administration

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Nasal or intranasal (IN) delivery is an interesting route not only for locally acting drugs but also for systemically acting ones, since due to the

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permeability of the nasal mucosa it can exhibit high drug concentrations in blood, while avoiding the problems of first-pass effect, enzymatic
destruction, and patient compliance. IN delivery has recently attracted much attention due to its potential for direct nose-to-brain delivery (N-B),

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bypassing one of the most important BBs, the BBB [14,15,16,48] (Figure 7). Indeed, IN delivered drugs can be directly transferred to the brain,

l
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and demonstrate enhanced therapeutic efficacy and reduced side effects (as a consequence of the reduced systemic exposure). Furthermore, several

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types of DDSs, such as polymeric nanoparticles (NPs), LIPs, micelles, micro and nano-emulsions, have been investigated for the delivery of drugs
to the brain via the intranasal route, as reviewed elsewhere [14,15,16,48].

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As seen in Table 1, the search for hits of LIP and nasal or intranasal delivery (excluding the term “topical or local”) gave 42 hits. Following an
initial review to validate the relativity of the hits with the scope of this article, we have included the most relative and advanced cases of IN
delivered liposomal drugs in Table S2. As seen, most of the reports involve N-B delivery of therapeutics, and the majority of them are intended as
therapeutics for neurodegenerative diseases.

IN delivery of LIPs intended as therapeutics for Alzheimer disease (AD), has been evaluated in several cases. The effects of Donepezil (DNP)
LIPs [49], Rivastigmine (RV) LIPs and also targeted LIPs, using cell-penetrating-peptides (CPP) as targeting ligands [50], and H102 (a beta sheet
disrupting peptide) LIPs [51], were studied after IN delivery in rat models of AD. In vivo study results confirmed significantly higher plasma
levels of DNP [49], and higher brain levels of DNP and RV (especially in hippocampus and cortex which are most affected in AD) compared to

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free drug administration, proving the potential of LIPs for N-B delivery. Furthermore IN-delivered H102 LIPs ameliorated spatial memory and
inhibited plaque deposition, even in a lower dose compared with the free drug. Finally it is extremely important that all the LIP formulations were
not toxic for the nasal mucosa.

IN delivery has also been considered for liposomal therapeutics against Parkinson‟s disease (PK). One example is the IN delivery of cationic-LIPs
entrapping Glial cell-derived neurotrophic factor (GDNF) which is known to exert significant neuroprotective effects. Indeed, GDNF cationic-

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LIPs demonstrated significantly increased brain levels (throughout the brain and along the rostral-to-caudal axis), indicating that IN GDNF-LIPs
can reach target areas following a single dose [52]. The same LIPs also demonstrated significant neuroprotective efficacy in and in vivo model of
PD [53]

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N-B delivery of LIsP encapsulating antipsychotic drugs has also been reported. Novel phospholipid based colloidal nanocubic vesicles combined

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with Poloxamer 188 or 407 and encapsulating olanzapine, a highly effective antipsychotic drug, demonstrated increased drug levels in brain after
IN delivery [54]. In another study, Quetiapine fumarate (QTF, an anti-schizophrenic drug), was formulated in LIPs and demonstrated superior

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brain levels [55] and brain/plasma ratio [56] compared to QTF dispersions. Finally, Risperidone-LIPs administered IN in wistar albino rats,

l
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showed enhanced brain levels of drug, compared to systemic exposure; among formulations tested, PEGylated LIPs showed higher brain

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bioavailability after IN administration compared to IV, proving direct NB delivery [57].

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LIP therapeutics for other pathologies have also been investigated by IN N-B delivery. A single IN dose of deuterium-labeled cholesterol (Chol-

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D6)-LIP, in wild-type (WT) mice, demonstrated high delivery of Chol-D6 in brain (striatum, cortex, and cerebellum), indicating their potential as
Huntington disease (HD) therapeutics since the synthesis of cholesterol in the brain is modulated in HD [58]. IN delivery of NGF (nerve growth
factor)-LIPs to the hypothalamus of mice with age-related hypogonadism, resulted in improvement of the sperm quality of the mice and also
enhanced their sexual drive and fertility [59], suggesting that IN delivery of NGF-LIPs could be considered as a potential treatment of of age-
related hypogonadism. Ethylacetate fraction (EF)-LIPs administered by IN route in mice, produced significant neuroprotection at only 10% of the
oral dose [60]. In another study, Baicalin (BA)-LIPs after IN delivery prolonged BA plasma concentrations and improve its bioavailability,
compared to IV delivery, and additionally the neurological deficits, cerebral infarction and brain pathological status in rats with artery occlusion
(MCAO) surgery were significantly improved. No toxicity to the nasal mucosa was observed after prolonged treatment [61].

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The neurological consequences related to Iron deficiency are difficult to treat, due to inefficient iron delivery to brain. Ferric ammonium citrate
LIPs (FAC-LIP) were administered IN in Sprague–Dawley (SD) rats, and it was found that the concentrations of iron in the olfactory bulb,
striatum, cerebral cortex, cerebellum and hippocampus were significantly increased; the formulation was also found non-toxic [62]. In another
study, Nitric Oxide donor (NO)-LIPs were delivered IN in sheep model of chronic rhinosinusitis (CRS) and demonstrated localized and systemic
effects, and also decreased inflammation, ciliary preservation and lower biofilm biomass, although high NO concentrations were used [63].

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Several recent reports consider IN delivery of LIP for enhanced BBB crossing. The bioavailability of cationic-LIPs associated rhodamine-B in the
brain after IN delivery of the LIPs was higher than when the LIPs were administered intravenously. IN delivery of cationic-LIPs encapsulating 2-

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PAM to rats (challenged with paraoxon) was shown to reactivate central AChE, suggesting that the reactivation of AChE can be achieved non-

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invasively via the N-B pathway [64]. A peptide ligand with good affinity for low-density lipoprotein receptor-related protein-1 (LRP1), Angiopep-

-
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2 (ANG), was used as a brain targeting ligand on LIPs that were loaded with Cyclovirobuxine D (CVB-D), which were additionally coated with

compared to non-targeted LIPs [65].

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polysorbate 80 on their surface. The targeted LIPs demonstrated increased transport of CVB-D across a cellular model of the BBB (in vitro),

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Concerning formulation aspects, a linear correlation was found between the elasticity of LIPs and their capability to deliver LIP-asscoiated drugs

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to the brain after IN delivery in Wister albino rats, using transfersomes with sodium deoxycholate (SDC) and Span® 60 in their lipid membranes
[66] (this LIP type is discussed in more detail in paragraph 3.4, below). Furthermore, it has been demonstrated that IN delivery of LIPs can be

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further improved by embedding them in in situ forming thermosensitive gels [48]. In a recent report, a mixture composed of Poloxamer 407 and
188 (15/1%, w/w) showed good mucoadhesive properties, which were further increased after addition of LIPs in the gel [67].

In addition to N-B delivery, IN administration has also been recently explored for vaccination, and it has been demonstrated to be a promising
alternative to classic parenteral vaccination. Indeed, several LIP types delivered by the IN route, were reported to increase local and systemic
immunity, and are considered to be promising novel immunization strategies. Cationic LIPs were investigated as adjuvants of IN delivered
mucosal vaccines, for treatment of infectious diseases, and were found to induce localized cell death; extracellular leakage of host dsDNA was
also observed. Treatment with DNAse I markedly impaired the cationic LIPs exerted ovalbumin (OVA)-specific mucosal and systemic antibody
production, indicating the potential mechanism of action [68].

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Many other cases of LIPs for intranasal immunization have been reported, such as: Cationic triple adjuvant (TriAdj)- LIPs that enabled stronger
responses at lower doses of antigen compared to non-lipidic preparations [69]. Ovalbumin (OVA)- beta-galactosylated LIPs were demonstrated to
mediate the development of an effective immunity against tumors [70]. The innate immune responses demonstrated in cats were effectively
activated after mucosal administration of novel TLR-LIP-complexes, suggesting that such complexes may be considered as mucosally-delivered
immunotherapies to treat respiratory tract infections (viral and bacterial) [71]. Haemagglutinin (HA) complexed LIPs for IN delivery of hepatitis B

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surface antigen (HBsAg) [72]. Pegylated cationic LIPs containing D-alpha-Tocopheryl polyethylene glycol 1000 succinate (TPGS) [73]. Hepatitis
B surface antigen (HBsAg)–immuno-LIPs (targeted by IgG antibody immobilized on their surface), which generate both humoral (i.e., systemic

o
and mucosal) and cellular immune responses upon IN administration in mice or cats, inducing higher levels of pro-inflammatory cytokines [74].

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Summarizing this part, the recent studies prove the high potential of IN delivery of LIPs as therapeutics towards neurodegenerative and other
diseases, by the direct N-B roue [49 – 66]. Additionally, many recent reports prove that intanasal immunization with LIPs is a promising

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alternative (to classical parenteral vaccination) immunization strategy [68 – 74]. Concerning the LIP types used and the formulation aspects, it is

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concluded from the studies discussed above that for the IN route the use of hybrid LIP types embedded in polymeric gels and/or mixed or coated
with mucoadhesive polymers, etc., is of particular importance for enhanced retention of the LIP at the site of administration. Additionally, for

a
delivery to the brain, targeted-LIP had better results compared to non-targeted ones. Importantly, the LIP formulations did not show toxicity

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towards the nasal mucosa, in all the studies which reported toxicity results.

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3.3 Intra Tumoral Delivery

A major challenge for effective cancer therapy using liposomal drugs is the limited capability to distribute the therapeutic throughout the entire
tumor mass. Direct injection of LIP drugs in the tumor (intratumoral, IT), or convection-enhanced-delivery (CED), have been proposed as
strategies to by-pass the RES and the barriers of the tumor vasculature. Such options to treat the primary tumor site in order to control local cancer
invasion, could certainly be of benefit for cancer types that spread locally or regionally. For loco-regional administration, it is critical to be able to
access the tumor; thereby this type of administration is mostly applicable towards superficial tumors. IT injection of LIPs can allow the drug to
enter directly inside the solid tumor, overcoming the barriers of tumor mass and reducing adverse effects [75]. Another possibility is to place
interventional catheters for guided delivery of therapeutics by imaging-assisted technologies. Although IT administration is less prevalent for LIP

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chemotherapeutics, it has gained recent interest for cationic LIP-based gene therapeutics (Figure 4), as a methodology to increase the concentration
of LIPs in tumors [76].

Some recent reports utilizing IT administration of LIPs, are presented in Table S3, categorized according to the particular type of cancer
challenged. As seen, hepatocellular carcinoma, melanoma, and lung cancer are between the most studied tumor types. A significant number of
papers from the hits of the search (Table 1) were not included in Table S3, since IV injection of LIPs was used, and not IT injection.

f
In two reports, IT injected curcumin cationic LIPs modified with glycyrrhetinic acid (GAMCLCL) were compared with free curcumin, for the

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o
therapy of hepatocellular carcinoma [75,77]. GAMCLCL exhibited stronger antitumor activity (inhibited tumor growth), by reducing the tumor

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microvascular density, after downregulating VEGF-protein expression, and upregulating caspase-3 protein. The efficacy of GAMCLCL was

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slightly weaker after IV administration, compared to IT [77].

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Another strategy for treatment of hepatocellular carcinoma was the IT injection of SN-38 (an active metabolite of irinotecan) LIP. The use of plain

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LIPs was compared with that of a hybrid LIP formulation consisted of a thermo-sensitive hydrogel system with acidic SN-38 LIP (SN-38-Lip-Gel)

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[78]. The SN-38-Lip-Gel improved the drug efficacy by stabilizing its lactone form; it additionally provided thus high local drug concentrations
and reduced the systemic toxicity of the drug (due to the increased retention of the drug at the injection site).

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Skin cancer and melanoma treatment is seriously challenged by the skin barrier. In a recent study, IT injection was compared with topical

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iontophoretic application, for a curcumin-loaded cationic liposome-STAT3 siRNA complex. It was found that the co-delivery of curcumin and

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STAT3 siRNA, significantly inhibited the progression of the tumor, compared to curcumin-LIPs as well as naked siRNA; while IT administration
demonstrated similar efficacy with iontophoretic administration [79]. Another nanocarrier-based strategy for melanoma therapy considered IT-
injected DOPE/CHEMS lipoplexes loaded with CpG-motif-containing oligodeoxynucleotides (ODNs), which could not effectively stimulate the
required immune response as free molecules, due to their low stability. IT administration of the lipoplexes induced anti-tumor activity by
infiltration of T cells and natural killer cells, in B16 melanoma-bearing mice. In fact the efficacy demonstrated following IT administration of the
lipoplexes was considerably higher compared to that observed after administration of the same therapeutic by the other routes tested [80].

In addition to treatment of other cancer types, gene therapy has been widely considered for treatment of lung cancer. In two recent studies, IT
delivery of DNA-cationic LIP complexes (LPs) with tumor suppressor genes LKB1 [81] or LKB1 and FUS1 (co-encapsulated in LIP) [82], was

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examined. The LPs were found to mediate LKB1 gene therapy and also sensitize the lung cancer cells towards cisplatin; LPs also significantly
inhibited proliferation, invasion and metastasis and induced the apoptosis of the cancer cells. Similar results were found with genes that co-
expressed LKB1 and FUS1. Substantial inhibition of the growth of tumors was observed in vivo after IT administration of both LP types, or by
combined treatment with intraperitoneal injection of low-dose cisplatin. In another study, IT-injected pH-sensitive LIPs were used for the delivery
of an anticancer agent in a lung cancer mice model. Enhanced pGFP transfection efficiency of the pH-sensitive LIPs (compared to non-pH-
sensitive LIPs) was demonstrated [83].

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Another promising approach for lung cancer treatment is siRNA-assisted (downregulation of the human antigen R (HuR) protein. LIPs

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encapsulating HuR-siRNA and targeting the transferrin receptor (Tf) were compared to control siRNA (C)-Tf targeted LIPs, in in vivo models of

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lung cancer. It was seen that IT administration of HuR-Tf-LIP significantly inhibited the subcutaneous tumor growth from A549 cells (>55%

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inhibition) and HCC827 cells (>45% inhibition) compared with the control formulation (C-Tf-LIPs). The HuR-Tf-LIP formulation was also active

fibroblasts, demonstrating a tumor-specific efficacy [84].

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in a lung metastatic model (A549-luciferase), and it sufficiently silenced HuR and HuR-regulated proteins in cancer cells but not in normal lung

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IT injection of LIP formulations may also be combined with physical methods for enhanced antitumor effect. In one case, in order to enhance the

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anticancer effect local hyperthermia (temperatures up to 80oC) was combined with radiation or chemotherapy. For this, temperature sensitive LIPs
(LTSL) loaded with docetaxel and indocyanine green (LTSL/DI), were injected IT in SCC7 subcutaneous tumor bearing mice. Interestingly,

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when near-infrared (NIR) laser irradiation was applied after treatment, the anticancer activity was significantly increased and recurrence was
inhibited. This was due to the localized heat-induced burst release of docetaxel from the temperature sensitive LIPs [85].

In another case, a single direct IT injection of 99mTc-LIP to nude rats bearing human head and neck squamous cell carcinoma (xenograft),
demonstrated anticancer activity which was proportional to the injected radionuclide dose. For larger tumors, the use of multiple locations for
infusion of the LIPs was found to be much more efficient, in terms of the demonstrated tumoricidal effect [86]; the ideal locations for the IT
injections were identified by a modelling approach.

Magnetite NP-LIPs (MNPs) and magnetic fluid-LIP (MFLs) were tested as IT injected contrast agents for MRI imaging in a rat model of renal cell
carcinoma. Effective ranges of magnetite concentration in MNPs (for contrasting at both T2 and T1 relaxation) were determined, and no

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significant changes were observed in the morphology of internal organs of the rat upon IT injection of MFLs, suggesting that such LIP are
relatively safe as potential contrasting agents for MRI tumor imaging [87].

In another study, YSK05 (a novel pH-sensitive cationic lipid), improved the early endosomal escape of MEND - type LIPs compared to MENDs
that were composed by other cationic lipids. IT administration of YSK05-MENDs in renal cell carcinoma-bearing mice, demonstrated enhanced
silencing of genes compared with the other types of MENDs (that did not contain YSK05)[88].

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As in other local administration routes, IT injection of paclitaxel (PTX) LIPs embedded in a Poloxamer 407 and 188 thermosensitive hydrogel

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provided higher local drug concentration. Additionally, extended drug retention in tumor, increased antitumor efficacy and systemic safety were

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demonstrated by the Poloxamer-embedded LIPs (in a mice model of sarcoma), compared to the control formulations, [89].

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Finally, IT administration of siGFP (luciferase GL2 siRNA) using trilysinoyl oleylamide (TLO)-based LIPs (TLOL) demonstrated significant

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reduction of fluorescence in green fluorescent protein (GFP)-expressing tumor tissue, compared with control siGL2. When using intravenous

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injection, the same LIPs were found to reduce tumor growth (compared to control formulations) only when siRNA together with an anticancer

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drug (suberoylanilide hydroxamic acid) were co-encapsulated [90], proving the advantage of the IT route.

Summarizing this part, it is proven from numerous recent studies that when possible, direct IT injection of LIP therapeutics is an effective and safe

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method for treatment of cancer [75-90]. The use of ligand-targeted-LIPs or other types of trigger-sensitive LIPs (temperature or pH sensitive), as

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well as hybrid LIPs embedded in gels (for enhanced retention at the injection site) is particularly interesting for the delivery of oligonucleotide

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and/or conventional therapeutics by IT injection. Concluding, it should be emphasized that it is probably easier to translate such advanced LIP
technologies to products intended for IT injection, compared to parenterally administered ones.

3.4 Dermal and Transdermal delivery

For drug administration to the skin, two potential cases are possible, depending on the site of drug action. The first case is transdermal delivery
where drugs are applied topically in order to be finally transported across the skin barrier and diffuse into the systemic circulation for systemic
drug action. Transdermal drug delivery is of particular interest for drugs that cannot be administered orally, due to first pass effect, enzymatic
degradation and other potential problems [91].

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The second case is dermal administration, where drugs are applied topically for penetration in deeper skin levels, in order to act locally in the skin,
for treatment of skin localized pathologies such as topical infections and inflammations, psoriasis, alopecia, skin cancer, etc. [92].

Transdermal drug delivery (TDDS) has been widely explored in the last decades due to the numerous benefits of this route, such as avoidance of
pre-systemic metabolism (first-pass effect), potential for systemic drug action after local painless administration, increased patient compliance,
safety, etc. However, an important BB, the skin, prevents the absorption of drugs after topical administration, limiting the applicability of this
route, even in some cases when only local drug action is required (dermal delivery) [91].

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In more detail, the epidermis which is the outermost skin layer, constructs a physical barrier preventing the transport of molecules through the skin

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(Figure 8.A). The epidermis is composed by four distinct layers, which are: (i) Stratum corneum (SC) that is the main barrier; (ii) Stratum

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granulosum (SG); (iii) Stratum spinosum (SS), and (iv) Stratum basale (SB). SC has a thickness between 15 and 20 um, and is composed of 5-8
layers of corneocytes or keratinocytes (dead cells) that have proteins and lipids between them. The lipids are mostly ceramides, cholesterol, and

ones with molecular sizes > 500 kDa [91].

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other free fatty acids. Due to its composition the SC is a very hydrophobic layer that blocks the passive absorption of most drugs, especially the

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The problem of limited drug penetration via topical application to the skin makes the search for safe drug carriers that may enhance drug

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permeation through the skin and provide an extended therapeutic effect very important. Several nanomedicine-types, some of them having

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numerous advantages, such as good biocompatibility, low cytotoxicity, capability of incorporating high drug loads, capability of enhancing drug

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stability, and most important, ability to increase the penetration of drugs into the skin have been developed in the last years [91, 92]. Between the
nanomedicine types, LIPs are the most important, due to their numerous advantages as drug carriers, as mentioned already before (Figure 3).

The potential routes for the transport of nanomedicines through the SC (Figure 8.B) are three, the intercellular route, the transcellular route and the
follicular route [91]. For LIPs (and many other types of nanomedicines) the most important route is the transcellular route, thereby the small size
or LIP and especially their ability to squeeze through narrow passing‟s is of great importance, determining their potential to facilitate the transport
of drugs to deeper skin layers (dermis), from where they can be absorbed into the blood capillaries. The latter is especially important for
transdermal drug delivery, where systemic absorption of the drug is required. However even in some cases of dermal delivery, depending on the
skin pathology to be treated, it may be required for therapeutics to reach deeper skin layers (if the pathology is manifested or extends in these

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layers). In the latter cases, it is a particularly difficult challenge to prevent systemic absorption of the drugs and retain them in the deeper skin
layers, as discussed below.

The special types of LIPs applied for skin delivery, are LIP that consist of phospholipids (with or without Chol), and other ingredients that increase
their elasticity or ability to change their shape. The main types of such elastic LIPs are presented in Fig. 8C, and are transferosomes, ethosomes
and niosomes. Transfersomes are ultra-deformable vesicles that in addition to phospholipids and (in some cases) cholesterol, also contain other

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substances in their membranes, such as single-chain surfactants. These are reffered to as “edge activators” and enhance the deformability of the
bilayers, mostly by modulating their interfacial tension [91]. Some examples of edge activators used for preparation of transfersomes are Span 60,

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65 0r 80 and sodium cholate or sodium deoxycholate, etc. Ethosomes are similar to conventional liposomes with the only difference that they

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contain also ethanol (20-50%); their major advantage compared to transferosomes is that they can enhance the transport of drugs across skin under

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occlusive and also under non-occlusive conditions, while transferosome function only under non-occlusive conditions. Finally, niosomes differ

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from LIPs since they are composed of non-ionic surfactants instead of phospholipids; however they also contain cholesterol and may be uni- of
multilamellar. The surfactants act by modifying the SC structure, making it more permeable; however potential toxicity issues should be carefully
investigated.

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review.
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Other types of lipid NPs or nanoemulsions have also been used for enhancement of drug transport through the skin, but were not included in this

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After reviewing the hits of the bibliographic search for Skin,Dermal and Transdermal delivery (Table 1), we tabulated in Table 4S.A and Table
4S.B, the most relevant cases, respectively.

3.4.1 Dermal Delivery

As mentioned above, dermal delivery using LIPs or lipid-based vesicles is mostly intended for treatment of skin localized pathologies such as
acne, psoriasis, skin infections, inflammatory diseases, and cancer. In the following part we mention in more detail the reports presented in Table
S4.A. A general observation is that in most cases the liposomal drugs used for dermal delivery are embedded in various types of gels that provide

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extended retention on the skin and improved skin bioavailabity, which is a major challenge for dermal delivery with LIPs. Additionally, the same
ingredients in some cases may also preserve the stability of the LIP-associated drug and improve drug penetration in deeper skin layers.

In one report, Benzoyl peroxide (BPO) and Adapalene (AD) LIPs (BPO-AD-LIPs) embedded in carbopol gel, were evaluated for acne treatment
and showed higher dermal bioavailability and lower skin irritation, compared to the free drug and a marketed acne treatment product (Epiduo)
[93]. In another study, liposomal Kynurenic acid (KA), a drug known to prevent scarring by modulating extracellular matrix deposition, was

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topically applied on healthy skin of volunteers to assess skin sensitivity after single and chronic use. Results proved the safety and tolerability of
topically delivered KA-LIPs. Additionally, KA was not detectable in blood indicating its high retention in the skin [94].

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For treatment of alopecia, finasteride and baicalin LIPs, hyalurosomes, glycerosomes and glycerol-hyalurosomes (which are LIPs coated with

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hyaluronic acid or glycerols or both), were developed. All vesicular systems realized improved hair growth after dermal delivery in C57BL/6 mice

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[95]. In another case, Minoxidil (MXD) and Tretinoin (TRET) LIPs embedded in a hydrogel were evaluated as a treatment of androgenic alopecia.
Association of LIPs with the hydrogel enhanced MXD permeation through skin, but TRET was retained, while the LIP hydrogel was non-irritant
to skin [96].

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Several studies in Table 4SA involve the treatment of Psoriasis. Zedoary turmeric oil (ZTO) and tretinoin (TRE)-LIPs which were embedded in a

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Carbopol gel matrix, demonstrated prolonged penetration of drugs into the hair follicles of mice and higher drug retention in skin compared to

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conventional gel formulations. In vivo, the LIP gel was more effective than conventional gels for the treatement of psoriasis, in a dose-dependent

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manner [97].

In another report, curcumin loaded propylene glycol–based ethosomes linked with hyaluronic acid (HA-ES) realized higher skin permeability and
skin retention of curcumin compared to plain ethosomes, while the two formulations had similar anti-psoriatic activity. After topical application
the HA-ES succeeded to reduce inflammation [98].

PAMAM dendrimer and siRNA-loaded cationic LIPs were stable in serum and in the presence of RNAse, and resulted in improved in vivo
efficacy on a psoriatic plaque model, compared to treatment with the drug of choice for psoriasis, Imiquimod. These findings suggest that
dendrimers and cationic LIP can be potential carriers for topically applied gene therapeutics [99].

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Topical administration of sponge Haliclona sp. spicules (SHS) is known to have a penetration enhancement effect on skin [100, 101]. When SHS
was used in combination with cationic flexible LIPs (CFL) for the delivery of siRNA, improved skin penetration of siRNA was observed. In vivo,
a similar effect (knockdown rate of glyceraldehyde-3-phosphate dehydrogenase) was observed by topical SHS-CFL and subcutaneous injection of
siRNA-LIPs, proving the potential for topical administration of siRNA by the combined use of SHS and CFL [100]. In another study, the
combination of SHS and flexible LIPs (SFLS) was used as a formulation that could enhance the penetration of hyaluronic acid (HA) in deep skin

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layers. In porcine skin (in vitro) the SFLS system increased the penetration of FITC-labelled HA. Furthermore, it significantly increased the
deposition of HA into deep skin layers of BALB/c mice (in vivo). SFLS treatment caused minimal skin redness which recovered in short time.

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Concluding, such compinations of SHS and flexible-LIPs seem to be promising DDSs for safe and effective delivery of hydrophilic

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macromolecules in the skin [101].

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Anti-inflammatory agent encapsulating LIPs have also been delivered topically. Ammonium glycyrrhizinate, a derivative of glycyrrhizic acid

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(GA) with similar anti-inflammatory activity (with GA), was encapsulated in Soy phosphatidylcholine / sodium cholate ultradeformable LIPs. The
latter LIPs reduced skin inflammation on human volunteers, suggesting their potential as topical anti-inflammatory agents [102].

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Harmine (HAR) is an alkaloid with anti-inflammatory properties, but its low bioavailability and side effects severely limit its clinical use. HAR-

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ethosomes (HLE) exhibited good biocompatibility and higher skin deposition compared to HAR-LIP and solution (GS). When mixed with a gel,
HLE significantly inhibited inflammation (in paw edema rat model) compared with HS gel [103]. Thymoquinone (TQ)-LIPs incorporated in a

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chitosan gel showed superior anti-inflammatory activity (in a carrageenan-induced paw edema) in rats, compared with a TQ chitosan gel and
comparable effect with the marketed indomethacin gel. The gel formulation was clear and provided suitable skin permeation [104].

In a recent report, Vancomycin LIPs were evaluated for treatment of MRSA skin infections, and it was found to be more effective than free
Vancomycin. The LIPs were found to be non-cytotoxic on a human epidermoid cell line [105]. Also, Luliconazole LIPs based on elastic lipogel
and ethogel formulations were found to be more potent compared to marketed formulations against Candida albicans and dermatophytes. In vivo
antifungal activity studies on albino rats showed that the LIP based gel formulations were safe and more effective for topical treatment of fungal
infections, providing enhanced topical delivery of Luliconazole with no drug reaching the systemic circulation [106].

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Elsewhere, ultra-deformable LIP (UL) co-loaded with Miltefosine (MTF) and Amphotericin B (AmB) were optimized and tested as a potential
therapeutic system against of cutaneous Leishmaniasis (CL). The anti-leishmanial efficacy of the optimized UL-LIPs revealed a synergistic effect
of the two drugs against amastigotes of Lesihmania mexicana. Furthermore, the co-loaded UL-LIPs had a lower IC50 concentration compared to
the two free drugs, as solutions. A significant reduction of parasites has observed in vivo (in a model of CL), verifying the potential of proposed
UL-LIPs system as an effective topically delivered treatmens against CL [107].

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Topical chloro-aluminum-phthalocyanine-loaded LIPs showed similar efficacy to reduce the parasitic load in the lesion and spleen of infected
animals, with systemic pentavalent antimony, proving that photodynamic therapy with these LIP is this a promising strategy for treatment of CL

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[108]. Topical miltefosine (ML) loaded LIPs (with 4% ML) showed optimal ex-vivo penetration and in vivo anti-leishmanial activity against CL,
compared to ML cream and other LIP types [109].

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Nano-deformable LIPs encapsulating sodium stibogluconate (SSG) were embedded in a carbopol gel for optimal application to the skin. Such
SSG-LIP gels demonstrated increased permeation of the drug through the skin and prolonged its retention in deep skin layers (ex-vivo), without

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any need for penetration enhancers. In vivo studies confirmed the safety of the topically applied formulation. A decreased IC50 concentration of

l
a
the SSG-LIP gel system compared to that of free SSG was measured (in vivo) in an amastigote model of Leishmaniatropica [110].

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For the photothermal therapy (PTT) of melanoma, curcumin was entrapped in Au LIP. Au LIP + Laser realized an enhanced therapeutic coverage

u
compared to Au LIPs without laser. PTT studies in B16 mice, showed that the tumor volume was significantly reduced, as more curcumin was

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released from the LIP in the localalized site of the tumor [111].

Amino-functionalized silicon blue-emitting nanoparticles NPs (ASNP) were found to be nontoxic in vitro (in two tumor cell lines) and in vivo
(zebrafish larvae and embryos), at concentration < 100 μg/ml; they also demonstrated a photosensitizer effect for photodynamic therapy. It is
particularly interesting that when the abobe NPs were entrapped in ultradeformable (UF) LIPs they succeded to reach the viable epidermis layers
where neoplastic events occur [112].

Summarizing the above, it is evident that the use of various types of LIPs, combined in most cases with hydrogels for better application on the skin
and enhanced skin retention, offers many therapeutic advantages for local treatment of skin-localized pathologies. Furthermore, in all cases

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reported herein, the topical LIP formulations were found to be safe, non-toxic and non-irritant, and minimized the systemic absorption of the LIP-
associated therapeutics.

3.4.2 Transdermal Delivery (TD) of liposomal drugs

As mentioned before, transdermal drug delivery is an alternative administration route for drugs that cannot be delivered orally. TD has obvious

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advantages, especially for patients who do not tolerate oral dosage forms, including people who are unconscious or those with swallowing

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problems. Additionally TD is ideal for drugs that are rapidly metabolized (since first pass metabolism is avoided). It also allows for pain-free and
safe administration of drugs, in addition to reducing the frequency of dosing; increasing patient compliance. Special types of vesicles can increase

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drug permeability through the skin, as mentioned above and presented in Figure 8.C. More details about the categories, structure and mechanism

-
of transport of these and other vesicles used for TD can be found in relevant review articles [91, 92].

e
and discussed below.
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Several liposomal drugs (formulated as conventional LIPs or elastic/deformable LIPs), have been proposed for TD delivery, as seen in Table 4SB,

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The topical application of optimized Rivastigmine LIPs realized significantly higher Cmax and AUC values and prolonged plasma levels

[113].
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compared to control formulations, indicating the potential of the LIPs as topical skin applied formulations for treatment of Alzheimer disease

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In another case, the encapsulation of the Letrozole (LET) in LIPs, resulted in elevated blood levels of estrogen, in breast cancer patients. The LET-
LIP cream showed increased penetration ability, superior anti-proliferative action, enhanced apoptosis, and a four-fold increase in the LET plasma
concentration, compared to plain LET cream [114].

The topical application of Folate LIPs in rats showed an11-fold increase in the plasma folate levels within 2h, confirming the systemic delivery of
folate. Significantly higher plasma levels and significant skin depots were observed in folate-deficient rats treated topically with the LIPs,
compared to oral delivery. An in vivo (in healthy volunteers) patch test to evaluate skin irritation was carried out and proved that the proposed LIP
nutraceutic formulation is non-irritant [115].

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The use of targeted-LIPs has also been investigated for TD. Cationic Lipid-Polymer-RNA (LPR) lipopolyplexes targeting specific endocytic
receptors present on DCs with mannose containing glycolipids, after being functionalized with a tri-antennary mannopyranoside (triMN-LPR),
were demonstrated to be safe. Additionally, the transdermal delivery of the same LIPs encapsulating synthetic mRNAs encoding tumor antigens
induced tumor-specific immune and therapeutic responses in clinical trials. Furthermore, a dramatic decrease of tumor growth was observed after
more intensive TD vaccination [116].

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A similar strategy for targeted-TD of Vemurafenib (VE), a toxic chemotherapeutic recently approved by FDA to treat subcutaneous melanoma via
the skin, was explored with peptide-modified VE-LIPs (pVE-LIPs). Trandermal delivery of pVE-LIPs demonstrated higher inhibition of the

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growth of subcutaneous melanoma (in vivo), compared to i.v. injection and oral delivery. Additionally the TD treatment with pVE-LIPs was found

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to be safer (reduced damage to major organs), providing a new strategy for safe delivery of VE [117].

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A specific fusion gene, the MPRSS2/ERG (TE), is known to be present in 50% of prostate cancer patients. RGD- peptide-coated LIPs
encapsulating a siRNA that targets TE demonstrated silencing and decreased tumor growth in vivo (in xenograft cancer models) after systemic

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delivery. When Docetaxel was co-entrapped in the targeted LIPs its activity was significantly enhanced. Intersetingly, when the vasodilator NG

l
a
was simultaneously delivered by application to theskin the delivery of siRNA to target tissues was increased, as well as the knockdown activity
[118].

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In other cases, more elastic LIP types (compared to conventional LIPs) were used for enhanced TD of drugs. Dapsone (DPS) invasomes composed

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of PC, ethanol, and one or more terpenes enhanced the percutaneous permeation; the maximum in vivo skin deposition of DPS accomplished by
the invasomes was ~2.5-fold higher compared to the drug solution, suggesting that DPS invasomes can be used for effective treatment of acne
[119]. Thymosin β-4(Tβ-4) is a macromolecular protein with potential for wound repair. Optimized Tβ-4 ethosome gels demonstrated significantly
reduced wound healing times (half, compared to T-β4 gel) in vivo, due to increased percutaneous absorption of the drug. Ethosomal gel systems
are thus proposed for TD of macromolecular proteins or peptidic drugs [120]. An ethosome gel of Raloxifene (an estrogen modulator used as
therapeutic of breast cancer) was also found to realize high drug TD in vivo, realising a bioavailability equal to 157% compared to the oral
formulation [121]. Flurbiprofen is a NSAID that exhibits anti-inflammatory, analgesic and antipyretic action. Flurbiprofen ethosomes were found
to increase the transdermal flux of the drug (compared to controls), while an optimized ethosomal gel showed a marked analgesic activity and
inhibited any increase in paw edema (in vivo) [122]. Optimized Cytarabine (used for therapy of acute myeloid leukemia (AML)) “malleable LIP”

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or ethosomes demonstrated good blood levels (low lag time, and high plasma concentrations 3-12 h post administration) after TD. Skin irritation
was minimal, proving the usefulness of TD of CYT ethosomes, as an improved alternative to the oral delivery of CYT (that suffers from low
bioavailability) [123]. In another report, TD of Diflunisal (DIF) was attempted with ethosomes (with 30% ethanol) and transfersomes (with
sodium deoxycholate, as an edge activator) -both incorporated into hydrogels-. Compared to a similar LIP hydrogel, both elastic carriers were
superior in terms of DIF permeation and flux across skin and also exhibited high antinociceptive, anti-inflammatory effects and inhibition of paw

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edema. The latter elastic LIP types could thereby be considered for enhanced delivery of DIF [124]. A similar type of elastic LIPs of Iloperidone
(IL) (with transcutol in their lipid membrane) referred as PEVs (penetration enhancing vesicles) embedded in a gel, showed relative bioavailability

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of 62% and 166% compared to the oral formulation and the gel without vesicles, respectively. Thereby, the topical skin delivery of IL elastic LIPs

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p
can be considered as a potential startegy for its administration [125].

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In another study, Methotrexate (MTX) (the gold standard for Rheumatoid Arthritis) deformable LIP (with labrasol in their membranes), were

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formulated in a hydroxyethyl cellulose nanogel (MTX nanogel) and sustained systemic levels up to 48h were realized after TD; low drug
accumulation was found in the gut, the liver, and the kidneys. In a collagen-induced in vivo model of arthritis, the proposed system significantly

toxicity were observed [126].

a l
reduced the arthritic score, joint damage and paw swelling, and increased the concantrations TNF-α and IL-6 in serum.No signs of irritation or

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In another case, Felodipine (with the problem of first-pass metabolism), was formulated in labrasol (and sodium deoxycolate)-containing

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transferosomes which were embedded in HPMC gels that could be lyophilized for enhanced stability. The lyophilized gel demonstrated
bioavailability after TD, proving the potential of TD felodipine transferosomal gel as an alternative of oral delivery [127]. The antihypertensive
drug Olmesartan medoxomil (OLM) is another drug with the problem of extensive first-pass metabolism. OLM transethosomes (with ethanol,
TEs) demonstrated enhanced ex vivo skin levels compared to transferosomes (with Sodium Cholate, TFs) and OLM suspension. The formulation
maintained normal blood pressure in rats for 24 h and its safety was confirmed, proving its potential as a successful delivery vehicle for
transdermal administration of OLM [128].

Another anti-hypertensive drug, carvedilol (CA) was formulated in vesicular ethosomal gel (ethogel) systems for upgraded TD, and compared to
pure CA-gel. CA-ethosomes and CA-ethogel demonstrated prolonged drug retention in the skin (in vivo) and also high skin permeation (ex-vivo).
Furthermore, CA-ethogel realized a significant decline in rat arterial pressure for up to 6 h post-TD [129].

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The low solubility of lornoxicam, a NSAID, together with its gastric adverse effects, limits the potential for its oral delivery; thereby TD of
optimized proniosome type vesicles embedded in gels was explored. TD administration of the proniosomal gel significantly inhibited the hind-paw
edema (which was induced in rat paws by carrageenan), compared to oral drug administration, suggesting improved clinical efficacy of TD
delivery of lornoxicam-vesicular gel compared to oral therapy [130].

Rifampicin (RIF) in elastic LIPs (eLIP) (RIF-eLIPs) provides an alternative delivery method offering various benefits for the treatment of

(compared to oral delivery) [131].

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cutaneous and systemic tuberculosis. A lower Cmax after TD of RIF-eLIP proved sustained TD as an improved tuberculosis management

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Sinomenine (SIN) which is frequently used to treat rheumatoid arthritis, was formulated in PEGylated transfersomes which incorporated mixed

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monoterpenes in their membranes(MMPTs), as edge activators. In vitro and in vivo studies proved that optimized MMPTs enhance the

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percutaneous absorption of sinomenine [132]. Finally, factorial design was used for optimization of a special type of Methotrexate (MXT) and
ascorbyl-palmitate entrapping elastic LIPs (Aspasomes of MXT) for TD treatment of rheumatoid arthritis (RA). The optimized formulation could

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load high amounts of the drugs and excibited good permeation rates through skin. Compared with oral administration of the free drug, the

management [133].
n a
proposed system demonstrated better treatment of RA disease (in vivo), indicating that it is a more attractive therapeutic strategy for RA

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Summarizing this part, it is evident from the recent studies that transdermal delivery of various drugs can be markedly improved by special types

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of elastic liposomes. The transdermal administration of such LIP formulations could be considered as safe and effective alternatives in cases when
oral administration of drugs is problematic.

3.5 Ocular Delivery

Ocular administration is solely for local action of drugs in the two main segments (anterior and posterior) of the ocular cavity (Figure 9), for
treatment of locally manifested pathologies, such as local infections, glaucoma, macular degeneration, dry eye syndrome, etc. The use of systemic
administration (for treatment of ocular diseases) is practically impossible, since for achieving the required local drug concentration for therapeutic
efficacy, the corresponding drug levels in blood would be higher than the minimum toxic concentrations. Thereby, localized delivery is practically
a one way root in most cases of ocular diseases. Ocular delivered drugs may be formulated as solutions, ointments or hydrogels for topical

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administration. Numerous BBs are encountered in the eye and at different eye segments, as seen in Figure 9 [134]. More details about these BBs
can be found in recent review articles [3, 134].Consequently administration in the form of intra-ocular injections is unavoidable for treatment of
localized pathologies. Alternatively, LIP drugs may enhance the transport of drugs across such BBs in some caes. Specific examples from the
recent literature are discussed in the following parts.

In Table S5 the recent (2010-to-date) cases of ocular administration using liposomal drugs are presented in tabulated form. The table is divided in

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two parts one for delivery of liposomal drugs to the anterior eye segment, and one for the posterior eye segment.

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3.5.1 Anterior diseases

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Several types of LIPs have been investigated recently for ocular delivery of drugs intended for cataract treatment. Cyanidin-3-glycoside (C3G) (an
anti-oxidative drug)-LIPs coated with TMC demonstrated increased efficacy in a selenite-induced model of cataract in rats, due to the enhanced

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transepithelial transport of the coated LIPs (compared to the uncoated ones) in the cornea. Prolonged corneal residence time was additionally

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demonstrated for C3G when the coated LIPs were used [135]. In another report, chitosan-coated LIP co-entrapping Lanosterol and Hesperetin
were found to be substantially effective in delaying or preventing cataract. The progression of cataract was stopped in rats that received stromal

a
injections of coated-LIPs, and it was delayed in the groups treated with uncoated LIPs, possibly because of the slow release of the drugs from the

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LIPs; the antioxidant properties of chitosan may also be implicated [136].

Three recent reports (Table S5) involve ocular delivery of LIP drugs for treatment of Keratitis [137,138,139]. Amphotericin B (AmB) was

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administered in the form of a microneedle ocular patch (MOP) device containing free AmB or AmB-LIPs. AmB-LIP MOP significantly reduced
the Candida Albicans load of the cornea following intrastromal injection in a rabbit model of infection [137]. In another study, fluconazole-LIP
eye drops administered in the subconjunctiva suc were more efficient against C. albicans infection in a rabbit cornea model of experimental
keratomycosis, compared to the free drug, and provided longer contact time of the formulation allowing higher drug penetration [138].
Voriconazol (VOR)-LIPs were also demonstrated to be effective towards fungal keratitis (delivering ~ 48μg/cm2 of VOR in 30 min, to the cornea
(porcine model)), and were classified as „non-irritant‟ in cattle corneas after performing a HET-CAM‟s test [139].

For treatment of Dry eye (DE) syndrome, replenishment of the destabilized tear film was achieved by topical delivery (in rabbits) of a novel in situ
gel forming, eye drop formulation containing vitamin A and vitamin E LIPs. The formulation demonstrated good ability to spread over the entire

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surface of the eye, and it did not cause any discomfort, tear film instability or blurred vision [140]. Another ophthalmic liposomal formulation
composed of vitE-LIPs with 0.2% sodium hyaluronate (SH) (that resembles the mucin-aqueous phase of tear film, and prolongs cornea contact
time), was administered as eye drops in the cul-de-sac, and the treated animals showed no discomfort up to 24h post-administration. The cornea
remained transparent and the coloration of the conjunctiva remained normal [141]. Topical administration of vitamin A–LIPs containing, siRNA
against HSP47 (Heat shock protein 47) that may have broad therapeutic implications in various types of dry eye pathologies, were instilled as eye

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drops in Female BALB/c mice and were observed to deliver the siRNA to activated fibroblasts or myofibroblasts preferentially. The latter is
particularly important since these cell types are the cells in which vitamin-A is stored and/or degraded [142].

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Non-infectious anterior uveitis (AU) is an inflammatory condition that is potentially sight-threatening. Triamcinolone acetonide phosphate (TA)-

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LIsP or Prednisolone (PR)-LIPs were administered by subconjunctival injections to a rsabbit model of uveitis, and it was found that the LIP

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formulations resulted in significantly decreased mean inflammatory scores compared to untreated rabbits (controls),. The LIP-treated rabbits also

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had lower scores than rabbits that were treated with a topical marketed formulation (PredForte 1%), 4 times daily. After antigen re-challenge, the
groups that received subconjunctival steroid-LIPs continued to have greater suppression of inflammation. The localization of LIPs in the inflamed

l
ocular tissue persisted for at least one month post-administration [143].

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For the management of corneal pain, Tetrodotoxin (TTX) + Dexmedetomidine (TD)-LIPs were functionalized with succinyl-Concanavalin A
(sConA-LIP) (that binds to corneal glycans and should significantly prolong the LIPs retention in the cornea) and administered as eye drops in

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Sprague-Dawley rats. A single instillation of the targeted-LIPs achieved complete analgesia for 105 min and partial analgesia for 608 min (without
delaying cornea wound healing), significantly longer than analgesia with proparacaine, TTX+TD solution or unmodified TTX-TD-LIPs, proving
the therapeutic advantage of the targeted LIP formulation [144].

In another study, Usnic acid (UA)-LIPs showed healing activity in animal models of dermal burn lesions. After epithelial removal and treatment
with UA-LIP eye drops, a 49.4 % reduction in injury was found compared with 36.6 % by the control, while corneal expression of VEGF and
TGF-β1 increased by 70 % and 50 % respectively; no toxicity was reported after Draize test [145]. Recently, ozone-LIPs were evaluated (in dogs)
as a replacement of povidone iodine (5%), for pre-operative bacterial colonization reduction in the conjunctival sac. Significant decreases were
reported in the total microbial counts (in colony forming units (CFU)), but the coagulase-positive and coagulase-negative staphylococci

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countswere similar for both formulations. In other words, Ozone-LIPs were found to be as effective as povidone iodine for the reduction of
preoperative bacterial load on ocular surfaces [146].

Senicapoc-LIPs dispersed within a thermosensitive hydrogel (Pluronic F-127) demonstrated increase drug bioavailability and increased ocular
surface residence time. Hydrogels demonstrated greater drug retention capabilities compared with topical viscous solutions [147].

For investigation of the ocular distribution of hydrophilic and lipophilic drugs following after topical application, CF (5-Carboxyfluorescein) as a

f
model of hydrophilic drugs and Dil (1,10-dioctadecyl-3,30,30-tetramethylindocarbocyanine perchlorate) as a model of hydrophobic drugs, were

o
o
used, and. DiI-LIPs and CF-LIPs were instilled as eye drops in Sprague–Dawley rats. Dil-LIPs resulted in higher corneal concentration of Dil with

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less systemic absorption compared to Dil solution, while CF-LIPs gave lower corneal concentrations compared to the CF-solution [148]. In

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another case, silk fibroins (SFs) coated Ibuprofen (IBO)-LIP showed rapid and sustained adhesion and uptake in cornea cells without detectable
cytotoxicity, suggesting that SF-coated LIPs may be a promising ophthalmic drug delivery system [149].

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From all the discussed above recent reports [135-149], it is evident that LIP drugs have several advantages as topically applied ocular formulations

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(in most cases as eye drops for topical corneal or subconjunctival installation, or intrastromal injections) for the treatment of various pathologies of
the anterior eye segment. Depending on the drug properties, LIP formulations may enhance the corneal retention and/or permeation of drugs and

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thus increase (and perhaps also prolong) its therapeutic action. Targeted LIPs were found to further enhance drug activity in a few cases, while the

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addition of hydrogels of polymers that form hygrogels in-situ, have also been found to provide further therapeutic advantages. Most important, in

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all the studies there were no indications of toxicity, inflammation or irritation towards ocular tissues.

3.5.2 Posterior diseases

Concerning the treatment of posterior segment ocular pathologies, most of the relevant reports involve OD of LIP-drugs for treatment of
Glaucoma. The first line treatments for glaucoma are currently topically applied medications, but such medications can not achieve sustained
ocular drug delivery, and daily administration ie needed. Daily topical administration has many limitations, such as potential development of
ocular allergies from the repeated use of drugs as well as poor patient compliance. The latter limitation, leads to sub-optimal intra-ocular pressure
(IOP) control, disease progression, and eventual to blindness. Latanoprost-LIPs administered in the subconjunctival area as eye drops
demonstrated significant decreases of the IOP, but a single subconjunctival injection demonstrated a sustained IOP decrease for up to 50d, and the

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extent of IOP decrease was comparable to that achieved by daily administered eye drops [150]. In another study, Latanoprost-LIPs demonstrated
sustained low IOP values (compared with those achieved by daily topical administration of latanoprost solution) for even longer time periods
(90d), while no toxicity or localized inflammation were observed [151]. In another case, Citicoline (CIT)-LIP containing sodium hyaluronate were
administered as topical eye drops and resulted in an improvement of the retinal function in patients with open angle glaucoma (OAG) treated over
a 4-month period [152]. Acetazolamide (ACZ)-LIPs [153] and ACZ- PEHVs (penetration enhancing hybridized vesicles) [154] were also tested as

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topical eye drops, were found to decrease IOP (in rabbits); the ACZ-PEHVs provided higher decrease of IOP compared to plain ACZ-LIPs. No
modulations were observed in the cornea, the iris, the pupil region and the anterior chamber region of any animal over a period of 21d [153, 154].

o
The latter results propose the safe use of LIPs and PEHVs as topical therapeutics towards glaucoma. In another study, Timolol maleate (TM)- LIPs

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were found to significantly decrease IOP approximately for 1 week, in New Zealand albino male rabbits [155]. Melatonin analog (5- methoxy-

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carbonyl-amino-N-acetyltryptamine)-LIPs combined with a mucoadhesive (sodium hyaluronate (SH) or carboxymethylcellulose) or

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thermosensitive (poloxamer) polymer, were found to decrease IOP much more when they were dispersed in 0.2% SH, compared to the uncoated
LIPs or to the LIPs that were coated with the other polymers tested, while the effect lasted for more than 8h [156]. Another formulation of LIPs

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embedded in in-situ forming gel [5], was recently used for OD of TM. In more detail, TM LIPs were dispersed in an ion-sensititive, deacylated
gellan gum gel. The optimized formulation showed no eye irritation (in vivo), significant reduction of IOP with a minimum of 13.61 ± 0.95 mmHg

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at 2 h, and longer duration of effect compared with TM eye drops. The latter results are most probably attributed to the longer retention time and

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the 1.93 times greater cornea permeability of TM from the LIP-in-(in situ-forming)-gel formulation (compared to free drug) [157].

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Several reports involve potential applications of OD of LIP-drugs for treating macular edema or degeneration, or other retinal diseases. The
development of age-related macular degeneration (AMD) may be linked with the de-regulation of TGF-β1, the transforming growth factor-β1. In
this context, TGF-β1 delivery could be a possible therapeutic strategy. Annexin V and Ca2+ complemented TGF-β1-LIPs were found to be well
tolerated, while high vitreous levels of TGF-β1 up to 114.7 ± 12.40 pg/mL, were detected 1h post-topical application of the LIPs, proving their
potential usefulness in clinical practice for AMD [158]. In another case, a formulation of Triamcinolone acetonide (TA) chitosan-coated LIP (TA-
CH-LIP) was instilled as eye drops to the posterior eye segment in mice, and demonstrated good potency for drug delivery as observed by optical
coherence tomography (OCT). Cell uptake of CH-LIP was higher than non-coated LIP, indicating that TA-CH-LIP eye drops can be effective to
treat macular edema (ME) treatment [159]. In another report, patients with refractory pseudophakic cystoid macular edema (PCME) received one

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drop of TA-LIPs every 2h for 90d (or until best-corrected visual acuity (BCVA) was achieved). The patients had significant improvement in
BVCA and central foveal thickness (CFT) without and significant change of their intra-ocular pressure. TA-LIPs were additionally well tolerated
and no ocular abnormalities or adverse events were observed [160]. Recently, the protective effects of Edaravone (ED)-LIPs were evaluated in an
N-methyl-D-aspartate (NMDA)-induced retina disease model, after intravitreal injection. The ED-LIPs decreased NMDA-induced ganglion cell
layer (GCL) cell death, compared to free drug administration. The LIP protective effect was dependent on the lipid composition of the LIP, and

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increased when the membrane fluidity of the LIP was increased [161]. In another report, TA-LIPs administered topically, were able to efficiently
deliver TA to the retina, with peak levels 12h post-administration (that subsequently declined for up to 14d). Cell viability was unaffected by TA-
LIPs, and no increase in IOP or other ocular alterations were observed in rabbits [162].

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In another recent study it was found that Coenzyme Q10-LIP coated with TMC could be promising for protecting human lens epithelial cells

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(HLECs) against H2O2-induced oxidative damage. It was found (by microscopy studies) that the transepithelial transport was enhanced by the

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TMC coating of LIPs, depending on the concentration of TMC used for coating, and the contact time for TMC and LIPs (during coating). The
Coenzyme Q10-LIPs increased cell viability and reduced oxidative damage (decreased percent of apoptotic cells) in a concentration-dependent
way [163].

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The effect of LIP size for topically applied targeted LIPs (with transferrin to target the retinal pigment epithelium) on their penetration to the
posterior segment tissues was recently studied. LIPs with diameters <80nm (with high loading capacity for plasmid DNA), permeated all the way

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to the retina, whereas LIPs were only distributed in the choroidal endothelium if they had diameters>100 nm; while it was demonstrated that active
targeting was necessary for the LIPs to be retained in the target tissue [164].

A limitation of LIP-protamine-DNA complexes (LPD), when applied for gene therapy of retinal diseases, is their lack of cell specificity, since the
retina is composed of seven cell types. Cationic-LIPs demonstrated cell-specific expression of the reporter gene after subretinal injection via the
transscleral route, proving that cell-specific promoters could enable lipid-based nanoparticles to deliver genes to specific cells of the retina in vivo
[165].

In another study, recoverin (rec) (an important endogenous protein for visual phototransduction), or antibodies against rec were administered as
LIPs by intravitreal injection to reach the retina, and higher amounts of rec were found in the photoreceptor layer compared with other regions of

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the retina, proving the potential to deliver proteins to photoreceptors, and thus modulate phototransduction by using biocompatible LIP injected in
the vitreous [166].

In order to increase intravitreal retention and bioavailability of drugs following intravitreal injection, Bevacizumab-multivesicular LIP (Bev-LIP)
for choroidal neovascularization (CNV) treatment [167] and a novel Flurbiprofen (FLB)-in-LIP-in-hydrogel (Pluronic F- 127) system [168] were
developed. Both formulations demonstrated increased retention and bioavailability (compared to the corresponding free drugs). Bev-LIP

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effectively inhibited the thickness of CNV lesions (28d post-treatment) compared to free drug [167]. The drug-in-LIP-in-hydrogel system also
increased AUC and the mean residence time (MRT) of FLB in the vitreous cavity without any signs of inflammation [168]. In another study,

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calcein, FITC-dextran-4000 (FD4) and Flurbiprofen (FLB), were encapsulated in negatively charged LIP, and mixed with protamine to produce

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novel Liposome aggregates (LAP). In vivo studies demonstrated increased vitreous retention for all molecules when formulated in LAP, while no

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visible inflammation was demonstrated, suggesting that the LAP system has great potential as a platform for intravitreal sustained delivery of
drugs [169].

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Summarizing, the recent efforts for ocular delivery of LIP drugs applied topically in the form of eye drops [153-164] or as injections

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(subconjunctival, or subretinal or intraviteal) [150,151, 165-169] to treat pathologies of the posterior segment, are very promising. Results

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demonstrate the prolonged therapeutic effect of LIP drugs compared to the free drugs, in several cases, while when safety studies were additionally
performed no toxicity, irritation of inflammation by the formulations and/or the high drug amounts administered (which were well-tolerated), were

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noted. Bioadhesive polymers, used as LIP coatings, or hybrid LIP formulations embedded in gels were found to prolong the therapeutic results,
without causing toxicity. In addition to conventional small molecule drugs, oligonucleotides [164, 165] and proteins [158, 163, 166] were
sucesfully delivered in LIP form to the back of the eye, in some cases using targeted-LIP [164].

3.6 Oral Cavity Administration (Oral, Sublingual, Dental)

The oral cavity includes a number of different sub-routes for drug administration (Figure 10), that may also be considered for LIP delivery. In
particular, oral or oromucosal administration, sublingual administration and dental administration could be considered; the two first routes mainly
for delivery of systemically acting drugs (but also for topical action), and the last mainly for topical activity. In Table S6, the recent (2010-) cases
of preclinical studies of LIP drugs intended for oral administration are reported. The Table is divided in three parts, the first for oral administration

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(Table S6.A), the second for Sublingual administration (Table S6.B) and the third for Dental administration of oral anesthesia or treatments for
periodonditis (Table S6.C).

3.6.1 Οral delivery of LIP-drugs or vaccines

High oral bioavailability is of course desirable, however many drugs demonstrate poor and hughly variable oral bioavailability. Various factors

f
may be implicated for the former, as presented in Figure 10, such as low solubility and/or low permeability of the drugs, poor stability (enzymatic

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degradation of drugs), first-pass metabolism, existence of drug efflux transporters, etc. LIP formulations may enhance the potential for oral
delivery of drugs in cases in which the above mentioned problems may be resolved (such as first-pass metabolism which may be resolved if

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lymphatic pathways are involved in drug absorption, and low solubility if drug dissolution rate is increased by incorporation of the drug in LIP

-
formulations). In several cases oral bioavalability of drugs was highly improved by incorporating them into various LIP types; several examples

e
r
are tabulated in Table S6.A and mentioned in more detail below. Additionally, the GI-track mucosal is currently being highly exploited for the

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delivery of antigens towards oral vaccination.

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In one recent report bile-salt LIPs (LIPs that have bile salts in their membranes) were developed as a potemtial way to enhance the oral
bioavailablity of Paclitaxel (PTX). The PTX-BS-LIP were stable in the presence of GI enzymes and pH alternations, and realized a twofold

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increase in PTX lymphatic transport, and a 2.5 and 4-fold increase in PTX oral bioavailability, compared to conventional LIPs and free drug,

u
respectively. Additionally, the inhibition of tumor growth by oral LIPs was similar to that of the free drug administered intaveneously (as a

Jo
solution) [170].

In another study, a topotecan (TPT) novel lipid coreshell lipid nanoparticle (CLN) -containing formulation was found to increase the
bioavailability and maximum blood concentration of TPT compared with free TPT, proving that the oral absorption of the drug was improved. The
new formulation enhanced the intestinal lymphatic transport of the drug. Lower GI irritation of TPT-CLN and improved antitumor efficacy were
observed after oral delivery of TPT-CLN compared to free TPT [171].

The antihypertensive drug Carvedilol (CRV), has very low and highly variable oral bioavailability. CRV niosomal formulations (with or without
bile salts) were administered orally to rats and it was demonstrated that surface charge of the niosomes and also the concentration and the specific
type of bile salts used for their construction highly influenced their oral bioavailability. When the lymphatic absorption pathways were blocked the

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oral bioavailability of CRV LIPs was significantly reduced, proving that the overall two fold enhancement in bioavailability (compared to free
drug) is highly attributed to absorption of CRV by lymphatic pathways [172].

Another strategy for improving orally administered LIP-drug efficacy and safety is to coat them with polymers. In this context, the potential of
carboxymethyl chitosan (CMCS) and quaternary ammonium chitosan (TMC)-coated LIPs to improve the oral delivery of curcumin (CUR) was
explored by preparation of CMCS/TMC-LIPs by electrostatic adsorption in a layer-by-layer manner. The coated LIPs possessed favorable gastric

o f
acid tolerance due to its stable structure, and satisfactory biocompatibility. The oral bioavailability of CUR- CMCS/TMC-LIPs was about 38%,
which was ~6 folds and 3 folds higher than CUR-LIPs (uncoated) and CUR-TMC-LIPs, respectively. CUR retention time in systemic circulation

r
was also increased and high CUR concentrations were measured in liver, spleen and lung [173].
o
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In another case, targeted LIPs were tested for oral delivery of a drug. Improved pharmacokinetics of Asenapine maleate (ASPM) (which due to

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high hepatic metabolism exerts low oral bioavailability) were accomplished by RGD- LIPs that could target the intestinal Peyer's patches
achieving access into the lymphatic system of the intestins. The targeted LIP showed increased oral bioavailability and high drug concentration in

P
brain after oral administration in rats, compared to the plain drug. Microscopy and imaging studies veryfied that the RGD-LIPs could indeed target

l
a
the Peyer's patches (ex vivo and in vivo) [174].

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A second group of orally delivered LIP drugs involves oral vaccination (Table 7.A), especially for treatment of infectious diseases caused by

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mucosal tract infiltrations of pathogens. Mucosal tissue delivered actigens may provide protection by mimicking the natural infection mechanisms.

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Thus, mucosal delivery and especially oral delivery (as a more convenient route) is becoming the most preferred administration route for the
delivery of vaccines [175]. In addition, the fact that LIPs also demonstrate adjuvant activity, makes them ideal as antigen carriers for oral vaccines.
However, oral vaccines have to overcome all the barriers already mentioned above (Figure 10), and more particularly the very low stomach pH,
the presence of proteolytic enzymes and bile salts and their low permeability in the intestine. In addition, low uptake of antigens (particles) is also
due to their short exposure time to mucosal tissues. Stable oral vaccine formulations that are based on NP strategies are currently being
investigated. Oral immunization with antigen-loaded NPs induced mucosal and systemic antibody responses, resulting in complete immune
responses. Besides protecting antigens against the harsh environment of the gastro-intestinal tract, NPs are also efficiently taken up by M-cells,
which are important for the induction of mucosal immunity. Some types of LIP have shown good potential as oral vacines.

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Lectin-modified LIPs are promising as antigens intended for oral administration. In particular the lectin UEAI (Ulex europaeus agglutinin I), that
has high affinity for fucose residues which are overexpress on the M cells located on the apical surface of Peyer‟s patch (resulting in enhanced
macromolecule absorption across the intestinal epithelial barrier), was selected as a bio-adhesive and targeting-ligand for development of UEAI-
LIP-BSA (BSA was used as model antigen) in order to investigate their potential as oral vaccine carriers [176, 177].The bioadhesive properties of
UEAI-LIP were confirmed on mice GI mucosa (intestinal segments with Peyer‟s Patches (PPs)). Additionally after oral delivery in mice the in

o f
vivo immune-stimulating activity of the UEAI-LIP was proven by the increased IgG levels measured in serum, and the increased IgA levels
measured in intestinal secretions (compared to the corresponding levels measured in both media after administration of non-conjugated LIP). 42d

o
post-immunization, highest IgG and IgA antibody levels were produced by UEAI-LIP compared to BSA alone, proving the high potential of

r
p
UEAI-LIP as oral vaccine carriers [176, 177].

e -
In a similar approach, others synthesized a mannose- conjugated PEG-colesterol (MPC) as a LIP targeting ligand. MPC-LIP loaded with lipid A

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(as adjuvant) [MLLs] and BSA, were able to realize potent immune responses (high levels of IgG in the sera and IgA in the salivary, intestinal and
vaginal secretions of mice) without exerting any side effects after oral mucosal delivery. Oromucosal delivery of MLLs induced a mixed Th1/Th2

l
response against antigens in treated mice, and established humoral and cellular immunity. The MLLs could additionally be lyophilized reserving

a
n
their activity following rehydration [178].

r
Microneedle arrays (MAs) are micro-structures that can painlessly deliver vaccines in the epidermis or dermis by piercing the skin. In another

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report MLLs loaded with BSA were delivered orally by MAs (proMLL) [179]. The proMLL were able to pierce porcine skin, and after
rehydration (in the skin) they rapidly dissolved releasing the MLLs. ProMLL that were delivered orally to mice resulted in high systemic and
mucosal immunity, enhancing IgG2a and IFN-γ (mixed Th1/Th2 response). Cellular immunity was also confirmed, proving the potential of
proMLLs as effective oral vaccine adjuvant-delivery systems applicable in cases when it is difficult to maintain particularly low controlled
temperature chains [180]. MLLs for stable oral mucosal vaccine against hepatitis B virus (HBV) were developed by others with HBsAg
(recombinant subunit HBV antigen), and were also loaded on MA‟s and dried to form proHBsAg-MLLs (proHMAs). One single immunization of
mice (via oral mucosa) with proHMAs induced a robust systemic immune response, as evidenced by the high levels of HBsAg-specific IgG
(produced in sera) and IgA (produced in salivary, intestinal and vaginal secretions). Thus, proHMAs, that may be conveniently delivered by the

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oromucosal route and could produce multiple immune defenses against HBV invasion while being stable for wide temperature chain distribution
[181].

In another study, the effect of drug carrier surface roughness on their targeting potential was evaluated, in an application concerning the oral
delivery of a Hepatitis B surface antigen (HBsAg) targeting the intestinal antigen-presenting cells (APCs). Layer-by-layer coated LIP (LBL-Lipo)
were produced with oppositely charged VitaminB12 conjugated chitosan (VitB12-CH) and sodium alginate (SA) layerings on PC/Chol LIP.

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Surface roughness modifications were observed as pH changed from acidic (gastric) to neutral (intestinal) due to corresponding increases and
decreases in charge density of VitB12-CH. In vitro cell uptake by macrophages (J-744) showed increased uptake of rough LBL-LIP compared to

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smooth LBL-LIP. In vivo immunization revealed that rough LBL-LIP produced higher IgG and IgA levels (in serum and feces, respectively)
compared to smooth LBL-LIP [182].

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In regard to DNA vaccines, chitosan has been used for surface modification and as a mucoadhesive enhancer in biodegradable microspheres
carrying peptides and proteins for mucosal and oral delivery. The bioadhesive properties of chitosan (CH) increase the residence time and prolong

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the adsorption at the mucosal site. In this context, CH-coated polyplex-LIP (CH-PLIP) containing a polyplex (which is plasmid DNA condensed

l
a
using CH as the condensing agent), were prepared as an oral vaccine with potential to target Peyer‟s patcheses. For in vivo evaluation of the effect

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of the CH coating and the final charge of LIP, plasmid encoding green fluorescence protein (GFP) was used as a reporter gene, and 100 μg dose of
plasmid was intragastrically inoculated into BALB/c mice. GFP expression was detected after 24 h, and it was found that CH-PLIP were better for

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DNA delivery to the distal part of the intestine compared to control LIP, due to their decreased enzymatic degradation rate and their higher
surface charge (positive) [183].

a DNA-vaccine consisted of a pcDNA 3.1 plasmid was developed using the influenza A virus M1 gene.. Cationic LIP were developed as a
delivery vector and in vitro/ in vivo testing confirmed expression of M1 gene in cell lines and in intestines of orally vaccinated C57BL /6 mice.
Oral administration of the DNA vaccine could induce immune responses (humoral and cellular) and additionally augment the production of IFN-γ
production, proving the potential of such DNA-LIP oral vaccines against challenging respiratory infections [184].

Antioxidant enzyme manganese superoxide dismutase plasmid was entrapped in LIP (MnSOD-PL) as a radioprotective gene-therapy of squamous
cell head and neck cancer, and was additionally combined with three tumorocidal agents (ionizing irradiation (II), C225, and the hypoxic cell

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cytotoxin, TPZ). The latter system was evaluated in a CAL-33 human oral cavity squamous cell orthotopic carcinoma model, in nu/nu mice. While
in the untreated mice the tumor volume increased group, in mice receiving only II tumor reductions was observed. The mice that were treated with
all modalities (MnSOD-PL+ C225+TPZ + II), had the lowest tumor volume compared to irradiation only. The combined therapeutics optimized
the therapeutic ratio for control of the local hypoxic regions of the orthotopic tumors [185].

With the final aim being oral vaccination against chronic conditions such as various types of allergies, galactosylceramide (α-GalCer), a potent

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immunomodulating compound, was formulated in cationic LIP (cLIP), polymerized LIP (pLIP) or poly(lactic-co-glycolic acid) nanoparticles
(PLGA-NPs) and all systems evaluated for oral administration in a model of atopy , using ovalbumin (OVA)(as model antigen). cLIP

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encapsulating α-GalCer could induce immunostimulating effects systemically (increases in serum IgG antibody levels) following oral

p
administration, while local cytokine changes in the mesenteric lymph nodes were realized when α-GalCer was delivered in pLIP and PLGA-NPs.

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e
The responses varied between formulations, but they did not exhibit tolerogenic traits which could be useful for immuno-regulation [186].

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For stronger vaccines capable of inducing effective and long-lasting immunity, new adjuvants are being considered; between them, gypenoside
(GP) saponins have good adjuvant properties. The adjuvant activity of gypenosides LIP (GP-LIP) was investigated in vitro, and GP-LIP could

a l
significantly enhance T and B lymphocytes proliferation more than free GP and empty LIP. Chickens, vaccinated with Newcastle disease (ND)

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vaccine simultaneously received oral GP-LIP at three doses, free GP and free LIP; the chickens that received GP-LIP demonstrated increase
antibody titers and cytokine secretion, proving the better adjuvant activity of oral GR-LIP compared to free GP and LIP [187]

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For treatment of a mosquito-borne viral CNS disease, Japanese encephalitis (JE), AmB was delivered in a mouse model of JE, before a LIP
formulation of the NS1 protein of the JE-virus is (LIP vaccine) and the immune response of the LIP vaccine was enhanced (but not when AmB
was delivered together with the LIP) [188]. The levels of IL-1β, IL-6 and the expression TNFa produced by the cells from animals that were
treated with AmB and LIP vaccine were significantly higher compared to those produced by cells from non-treated mice. It was suggested that
AmB augments the adjuvant effect of the LIP vaccine, realizing enhanced immunity and [rptection against JE infection.

The alkylphosphocholine analogue of Miltefosine, Oleylphosphocholine (OlPC) has a potential incremental value (compared to miltefosine) for
the treatment of leishmaniasis. OlPC-LIP realized higher efficacy (in a hamster model of leismaniasis that was treated orally for 5 d) compared to

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Miltefosine-LIP. OlPC solution or OlPC LIP were able to reduced the parasites in liver by 96.2% and 99.3% respectively, and high parasite
reductions were also realized in spleen, and in bone marrow; No edverse effects or toxicity signs were orserved [189].

3.6.2 Sublingual delivery (SLD) of LIP-drugs or vaccines

Although placed in the oral cavity, administration of drugs via sublingual epithelia is systemic. Indeed, instead of passing through the GI tract,

f
drug can directly enter the blood through the sublingual membrane. Rapid therapeutic onset is possible if the drug is administered in a formulation

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type that confers rapid drug dissolution. SLD is also used for vaccination, although the sublingual (SL) mucosa is a barrier for vaccine penetration.
Nevertheless, the unique anatomical and physiological characteristic of the SL mucosa, make it a very good site for mucosal vaccination.

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Adjuvants and antigens can be delivered directly to the SL tissue located mediators of immune responses; thereby effective infectious disease

-
vaccines can be delivered by SL route. Examples of SL delivery of LIP are seen in Table S6.B, and they are discussed in more detail below.

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The co-delivery of LIP encapsulating CRX-601, a synthetic Toll-like receptor-4 agonist, together with influenza antigens by the SL route, realized
a high high immune response. LIP modification with PEG copolymers (Pluronics, PL), phospholipid-PEG conjugates (PEG) and chitosan (C) were

a l
tested for treir effect on the immune response exerted by the LIP, in a murine model of influenza. PEG-LIP were more stable than PL-LIP, and
also more effective. However, methylglycol chitosan coated LIP realized the highest flu-specific immune responses [190].

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a-GalCer-LIP were administered sublingualy for 7d, for evaluation of their adjuvant effect in an OVA (ovalbumin)-induced mouse model of

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Allergic Rhinitis (AR). The combination of OVA with a-GalCer-LIP realized a downregulation of cytokines (Th2 and Th17) and their gene

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expression in CD4+ T cells, while in CLN cells Th1 type cytokines and their gene expression were enhanced; suppression of all nasal symptoms
was also observed. Additionally, specific IgG2a‟s were increased while specific IgE and IgG1 were decreased in the serum. None of the previous
results were observed when free OVA of just LIP were delivered. All the previous results suggest that a-GC-LIP delivered SL together with an
allergen is a potential strategy for increased the vaccination efficacy [191].

LIP were also evaluated for SL administration of OVA, in a model of inflammation of airways by allergy (that was induced by OVA). Airway
eosinophilia and splenocyte proliferation were decreased with OVA-LIP, much more than the effect of plain OVA. Also, decreased levels of
interleukin-5 and interferon-γ were found in spleen cells of mice that received OVA-LIP, but not in the sham-treated group. Concluding, it is
suggested that SL delivered OVA-LIP offer increased protection towards allergic inflammation compared to the dame dose of free OVA [192].

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3.6.3 Dental delivery (SLD) of LIP-drugs (Topical Anesthesia, Periodontitis)

In Dentistry topical anesthesia is frequently used as a method to decrease the pain during injections of anesthetic drugs. In many cases, the
commercially available formulations are not effective and anesthesia is not realized. The current procedure is also feared by the majority of dental
patients, which skip dentist visits for this reason. Thereby, better solutions for local anesthesia are currently seeked. Drug delivery systems
(DDSs) may be used for enhancing the efficacy of topically applied agents [193]. The most common anesthetics used today for topical action are

o f
Lidocaine, Tetracaine hydrochloride and Benzocaine; combinations of Benzocaine, Tetracaine and Butamben, are also used. EMLA® cream
(AstraZeneca), which is applied for anesthesia on the skin, is also used as an anesthetic on the oral mucosa. EMLA is an eutectic mixture of

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Prilocaine and Lidocaine. Specific studies proved that topically applied anesthetics formulated as semi-solids, reduce the pain caused by needle

p
insertion in some areas of the buccal cavity, but not in the palate. Even EMLA, the positive control in studies evaluating efficacy of topical

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e
anesthetic, cannot efficiently manage the pain from injections in the palate. As seen in Table S6.C, several LIP formulations are being evaluated
as topical anesthetics, described in more detail here.

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Ropivacaine (ROP)-LIP embedded in Carbopol gel, improved the pain relief of needle insertion when applied to the oral mucosa prior to injection

a l
in healthy volunteers [194]. However, the same ROP-LIP gel could not reduce injection-induced pain in the palatal mucosa, as also some

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commercially available formulations, such as EMLA [195]. Nevertheless, in another report lidocaine (LID)-LIP (5%) and EMLA realized similar
efficacy, and non of them cou;d reduce the pain caused by needle insertion in the palatal area. Additionally, both formulations demonstrated higher

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in vitro permeability and in vivo efficacy (anesthesia) compared to Xylocaina and placebo. [196]. Lignocaine (LIG)-LIP (2%) were also compared
with a commercial gel (18% Benzocaine/2% Tetracaine) and showed better pain management during injection of anesthetics in the palatal area
[197]. Others hypothesized that perhaps some fraction of local anesthetic (LA)-LIP applied to the oral mucosa could penetrate the cortical bone,
and realize pulpal (tooth) anesthesia. To test this hypothesis, Benzocaine (BEN)-LIP [198] and ROP-LIP and LIP gels [194, 199] were compared
with commercial formulations, but were not found able to induce pulpal anesthesia, even after 30-min application [194].

Another LIP type evaluated for their analgesic properties, were actively loaded ROP-LIP, which were prepared using trans-membrane (ionic or
pH) gradients that are known to achieve high intra-liposome concentrations. The specific ROP-LIP formulations demonstrated increased drug
loadings and prolonged release, and consequently also prolonged therapeutic efficacy in vivo [200, 201].

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Furthermore, a 10% BEN-LIP formulation induced longer soft tissue anesthesia compared to a 20% BEN commercial gel, but neither formulation
induced pulpal anesthesia [198].

A Bupivacaine-LIP injectable suspension (EXPAREL, Pacira Pharmaceuticals) is a novel sustained-release formulation indicated for infiltration in
the surgical site for postsurgical analgesia. Approximately 97% of the drug in each vial is encapsulated in MLV LIP, in the structure of
DepoFoam delivery technology1 (Pacira, Biosciences Inc.). The analgesic efficacy and safety of bupivacaine-LIP (B-LIP) was evaluated on

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patients that were subjected to bilateral third molar extraction, and B-LIP significantly lowered the cumulative pain at all time points, compared to
placebo [202]. However, in another study, symptomatic patients diagnosed with pulpal necrosis that also experienced moderate to severe

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preoperative pain, received a buccal infiltration of B-LIP but there was no improvement of postoperative pain management, compared with an

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infiltration of bupivacaine [203]. Another B-LIP formulation did not decrease the pain in patients with irreversible pulpitis (symptomatic and

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untreated), although it had some effect on soft tissue anesthesia [204]. In another case, an opioid-sparing postsurgical pain management protocol

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showed that B-LIP caused statistically significant reduction of postsurgical pain and could reduce opioid consumption [205].

From all the above we can conclude that despite the conflicting results reported in the literature, in some cases LA-LIP demonstrate increased

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topical anesthesia efficacy, suggesting that this may be an important strategy that could be improved in order to achieve pain free dental
anesthesia.

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Periodontitis is a dental disease in which localized administration of LIP drugs is valuable. It is a complex disease and bacterial infection is the

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most common factor involved. Current strategies for the local delivery of antibiotics do not allow a complete clearance of bacteria fillings in
dentinal tubules and this limits their therapeutic efficacy. Therefore, there is a strong need for the development of new delivery strategies aimed at
improving the efficacy of antibiotic therapy for periodontitis with special reference to their ability to penetrate into the tubules. Some recent
strategies with LIP, are presented in Table S6.C.

In a recent study novel LIP types of Ketoprofen (KET-LIP) that contain surfactants, characterized as pro-niosomed were developed as gels by
embedment in carbopol gel. After optimization, its effectiveness was validated in an in vivo experimental periodontal disease (EPD) model.

1
The DepoFoam system consists of “microsized, spherical, lipid-based particles composed of a honeycomb of numerous, internal aqueous chambers containing
the encapsulated drug, which are separated by lipid membranes. Following injection, the DepoFoam system releases the drug over an extended time period due to
erosion and/or reorganization of the lipid membranes”.

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Results showed that disease condition were improved with preserved bone resorption process; the formulation was safe and more effective than
the currently available gels, suggesting that such KET-LIP can be effectively used for improved ketoprofen delivery in periodontal pockets [206].

The combined use of LIP localized delivery and Ultrasound (UL) activation, was attempted for delivery of naked plasmid DNA encoding
luciferase or enhanced green fluorescent protein (EGFP) into the lower labial gingiva of Wistar rats, by using Bubble LIP. The efficacy and
duration of gene expression in the gingiva were studied, and it was found that the luciferase activity was significantly higher in the gingiva that

delivery for periodontitis treatment [207].

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were treated with bubble LIP and ultrasound, than in the gingiva of the control groups, indicating the potential of this approach for gene or drug

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More recently Doxicycline LIP coated by electrostatic interactions with N,N,N-trimethyl chitosan (TMC) were evaluated for peridonditis

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treatment in vitro and in vivo (in a rat disease model). The results showed that the novel LIP formulation “achieved superb inhibition of free mixed

potential for use in treatment of periodontal diseases [208].

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bacteria and biofilm formation” and “strongly inhibited biofilm formation and prevented alveolar bone absorption in vivo” suggesting its high

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Finally, in a recent study a LIP-WNT3A protein therapeutic, was shown to accelerate healing of tooth extraction socket by 2-fold after single

a
application during extraction. The study was based on the fact that Wnt-responsive populations of stem cells were found in the periodontal

n
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ligament (PDL); these upregulate osteogenic protein expression and differentiate into osteoblasts. This new stem cell group in the intact

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periodontium is directly responsible for alveolar bone healing after tooth removal, opening new directions for periodontitis treatment, and bone

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regeneration [207].

Pain control during periodontal therapy is another potential indication of localized anesthesia; however, recently a placebo, a LID/PRI commercial
formulation (Oraqix®), and a LID/PRI-LIP formulation were compared, and results, did not demonstrate differences between intervention groups
in relation to pain frequency or intensity, providing limited indications for the use of non-invasive periodontal anesthesia [209].

Summarizing the recent efforts for oral delivery of LIP drugs, we may conclude that oromucosal, sublingual and dental administration of LIP
drugs may offer several therapeutic advantages, particularly for topically acting drugs, such as oral anesthesia or drug to treat periodonditis.
Nevertheless, in cases of systemically acting therapeutics when drug absorption through the GI track is limited (mostly due due to first pass-
effect, or low permeability), special types of LIP-drugs absorbed through lymphatic pathways, or targeting the Peyers patches, have been reported

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to increase the potential for their oral delivery. In the latter cases, LIP that are coated with different types of polymers or targeted-liposomes have
been mostly considered. However, the most promising use of LIP delivered by oral route is most probably as carriers of oral vaccines. Indeed,
many more cases of successful immunization by orally (oromucosal and sublingual) delivered LIP-vaccines (16) have been reported in the last
decade, compared to only 5 cases for delivery of systemically acting drugs.

3.7 Pulmonary (Lung and Pleural cavity)

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The pulmonary route has emerged as a route of choice for noninvasive delivery of drugs due to a number of advantages (Figure 11), however

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currently pulmonary delivery is used only for treatment of localized pathologies. LIP drugs have been considered for local pulmonary

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administration in the lungs (by inhalation through the mouth and the further deposition of the inhaled drug in lower airways), and also for direct

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administration into the pleural cavity (by intrapleural injection), as seen in Figure 11.

3.7.1 Local administration of LIP drugs in the lungs

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a l
Pulmonary drug delivery has many advantages over other routes of drug administration and drug absorption (Figure 11), including the large
alveolar surface area, extensive vascularization, low thickness of the epithelial barrier and limited enzymatic activity. However, the previous

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advantages may posse challenges, for treatment of localized pathologies. Respiratory diseases are prevalent around the world, and pulmonary

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delivery-based therapies provide an attractive, noninvasive method of directly delivering therapeutic agents to their site of action to improve the

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treatment efficacy and limit the adverse systemic side effects. In general, when delivered by the pulmonary route, drugs may be delivered locally
for treatment of lung-specific diseases such as lung cancer, chronic pulmonary diseases and bacterial infections (e.g. tuberculosis). Therapeutic
strategies for targeting lung cancer are of great importance not only because of possible remission, but also for reducing the potential of lung
cancer metastases, and thereby increasing the chance of success with surgical intervention or radiotherapy. However, the pulmonary delivery of
anticancer drugs has serious limitations, such as the poor water solubility of many chemotherapeutic agents, as well as the thinness of the
pulmonary epithelium that results in short residence of the delivered drug in the lung and high potential for systemic adverse effects (since the
drug may be systemically absorbed). The use of LIP as delivery systems could localize the drug action in the lung for prolonged periods [210].
When pulmonary infections are concerned, such as tuberculosis, the current treatment options present a serious impediment, since bacteria can

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survive intracellulary in the host alveolar macrophages for long time periods, while most of the anti-mycobacterial drugs presently in use fail to
penetrate macrophages. For this reason, many researchers are considering the use of delivery systems as well as their engineered versions, in order
to make them therapeutically effective [211].

Table S7, is divided in two parts, one for pulmonary delivery and one for the special category of local drug administration in the pleural cavity. In
Table S7A, we present in tabulated format the most advanced recent (2010-to-date) cases for preclinical studied liposomal drugs intended for

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pulmonary administration, after reviewing the corresponding hits of Table 1. Several references (from Table 1 included hits) were not included
since they were about irrelevant administration routes (in most cases systematic intravenous delivery) or they were about other types of drug
carriers, and not LIP.

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As seen, from a total of 27 studies tabulated in Table S7A, the majority (eleven) involve therapeutics intended for treatment of lung cancer, three

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reports for pulmonary infections and seven for other diseases. Moreover, six studies focused on improving the retention of liposomal drugs in the
lungs, a critical aspect of high therapeutic efficacy. The studies conserning the potential use of LIP as effective drug nanocarriers in the
pulmonary tract are discussed in more detail below.

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The development of more effective and local therapy is required for improved treatment of advanced lung cancer. Among new active agents,

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Camptothecin (CPT) and its derivative 9-nitrocamptohtecin (9-NC) acting as Topoisomerase I inhibitors are an attractive class of anticancer drugs.

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Zhang et al compared the effectiveness of 9-NC loaded LIP for lung cancer treatment after intravenous and pulmonary delivery in rats [212].

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Pulmonary delivery of 9-NC LIP could directly deliver the drug to the lung and make the drug accumulate in the lung with sustained-release
characteristics. LIP also modulated the in vivo drug disposition and reduced the severity of damage to the lungs following intratracheal instillation.
Additionally they decreased the toxicity and side effects on other tissues.

Quercetin (QR), 3,5,7,3',4'-pentahydroxylflavone, is a natural flavonoid known to possess anticancer activity. QR has been suggested as a
chemoprevention drug in lung cancer therapy because its involvement in cell-cycle arrest, apoptosis, replication and angiogenesis. The potential of
peptide-functionalized LIP as nanocarriers to overcome QR limitations in lung cancer therapy was studied by Riaz et al 2019 [213]. Specifically,
they loaded QR in DSPE-PEG LIP which were conjugated with T7 peptide (that has specific binding affinity for transferrin receptors), and studied
the in vivo biodistribution and the therapeutic potential of the targeted QR LIP towards lung cancer (in vitro and in vivo). In vitro studies

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significantly augmented cytotoxicity, higher apoptosis induction, cell-cycle arrest and tumor spheroid growth inhibition in A549 cancer cells. The
in vivo biodistribution study via pulmonary delivery of T7 –QR- LIP demonstrated accumulation in the lungs and a sustained release behavior up
to 96 h. Furthermore, T7-QR-LIP significantly enhanced the anticancer activity of QR and the lifespan of mice in an orthotopic lung tumor-
bearing mice model, without any systemic toxicity.

Another approach of increasing drug lung targeting efficiency is the use of anti-carbonic anhydrase IX (CA IX) antibody decorated LIP. Lin et al

o f
incorporated in such surface modified LIP the anticancer drug Triptolide (TPL) and studied its therapeutic effects in lung cancer therapy after
pulmonary administration [214]. They found that the CA IX-TPL-LIP significantly improved the cellular uptake in A549cells and A549 tumor

r o
spheroids, resulting in more efficient cell killing compared with free TPL and non-targeted TPL-LIP. In vivo, CA IX-LIP showed specificity and

p
sustained release of TPL in the lungs up to 96 h, resulting in improved efficiency to slow down the tumor growth and prolong the survival of mice

-
e
with lung tumors.

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Interestingly, functional liposome administration via the pulmonary route seems to be effective in the treatment of lung cancer. When Poloxamer
188, a block copolymer used as a medical additive, was incorporated into liposome membranes, it greatly facilitated the sustained and targeted

a l
release of the anticancer drug doxorubicin in the lung epithelium and enhanced its cytotoxic effects in A549 cells. These results suggest that DOX-

n
loaded DPPC/P188 LIP administered via the pulmonary route may be useful for treating lung cancer [215].

r
u
Gemcitabine-HCl is an effective drug for lung cancer therapy. However, it is highly metabolized by plasma enzymes resulting in toxicity and

Jo
reduced efficacy. Gandhi et al published in 2015 the successful pulmonary delivery (by intratracheal instillation) of Gemcitabine-LIP using a dry
powder inhaler (LDPI) [216]. Indeed, data from cell studies as well as from vivo studies showed sustained release of the drug with better cellular
uptake and increased toxicity towards A549 cells, whereas the pulmonary pharmacokinetics resulted in lower lung tissues toxicity of the LIP
compared to free drug. This study proved that LIP drugs can be deposited throughout the lung via dry powder inhalers, a strategy that may be used
for safe treatment of lung cancer.

Another report using the spray-dried powder technology involved the incorporation of Vincristine (VCR), a bis-indole alkaloid that is a widely
used as a chemotherapeutic agent in LIP and delivery by a dry powder inhaler. Improved release but decreased half-life of the drug in the lung
epithelium was demonstrated after pulmonary delivery, as well increased toxicity in A549 cells (compared to control fornulations) [217].

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Another approach for treatment of lung cancer is the use of LIP for gene delivery to the lungs, for localized delivery of the oligonucleotides to the
site of disease (lungs and airways). Wu et al used anti-SP-C-conjugated lipoplexes as LIP gene carriers, delivered to the lungs through intranasal
instillation. The targeted lipoplexes exhibited the characteristics required for effective treatment of pulmonary diseases that are caused by
disregulation of ATII cells, such as lung cancer. Specifically, the lipoplexes efficiently targeted ATII cells, and were retained by them for at least
48 hours, while they did not accumulate in other lung cells or viscera. The expression of mature miR-486 was approximately 4-fold higher in

o f
ATII cells than the whole lung, 48 h after treatment with anti-SP-C-conjugated lipoplexes (incorporating miR-486) [218]. In the same direction,
four more recent studies used cationic LIP to deliver specific shRNAs and siRNAs directly to the lungs for cancer treatment [219-222]. In all the

o
studies intratracheal delivery of antisense oligonucleotides against cancer related genes was applied. Specifically, intratracheal administration of

r
p
siMcl1 in nanolipoplexes significantly silenced Mcl1 mRNA and protein levels in lung tissue and additionally significantly reduced the metastatic

-
tumor nodules in the lungs [221]. In another report, treatment of mice with shRNA against Annexin A2 in LIP, significantly reduced the the tumor

e
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burden and more specifically the lung tumors derived from H1650 SP cells [219]. Moreover, neutral and cationic LIPs were used to deliver DOX
together with antisense oligonucleotides (ASO) and siRNA intratracheally to the lungs and the local delivery was compared to systemic delivery.

a l
The study highlighted local intratracheal delivery of LIP drugs or siRNA advantages, for lung cancer treatment [220]. Of note, another team
synthesized a more efficient and safe new cationic lipid, particularly 6-lauroxyhexyl lysinate (LHLN), and demonstrated that it can be used for

r n
effective intratracheal instillation of genes to the lungs [222].

u
A promising strategy to improve the therapy of pulmonary infections, mainly tuberculosis, is the administration of antibiotics directly to the lung

Jo
by nebulization. The latter delivery method results in higher drug concentrations at the infection sites and alsoreduces systemic side effects. For
poorly soluble drugs, as rifampicin, LIPs were found to improve lung delivery. Targeted aerosol delivery of antituberculous drugs to alveolar
macrophages of the lung, is efficient for treating local tuberculosis infections [223,224]. Patil et al proved the enhanced retention and efficiency of
intratracheally delivered rifampicin-LIP, suggesting that LIP deliver the drugs directly and much faster to macrophages compared to free drugs
[211].

In lungs (as in many other organs) the macrophages are key players in ischemia–reperfusion injury. Intratracheal instilled LIP encapsulating
Dichloromethylene diphosphonate (Clodronate – CL) deplete the local macrophages [225]. In several studies involving the treatment of bacterial
lung infections, such as Aspergillosis or Pneumonia, different types of drugs, such as amphotericin B, Ciprofloxacin and Andrographolide were

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successfully incorporated in LIP. All studies showed sustained release, as well as increased retention and efficacy of the drugs in the diseased lung
epithelium after intratracheal instillation in mice or rats [226,227,228].

Tacrolimus-LIP were delivere to the lundgs with the help of a dry powder inhaler for the prevention of lung refractory rejection following lung
transplantation.The LIP realized prolonged retention at the site, reducing the risk of acute rejection as well as chronic rejection [229].

Two studies exploited the use of LIP for the treatment of asthma in vivo, in a guinea pig asthma model. Procaterol hydrochloride (PRO), a short-

f
acting pulmonary b2-agonist, was encapsulated in LIP and delivered to the lungs. LIP realized increased lung retention of the drug [230]. In

o
o
addition, encapsulation of Secretory Leukocyte Protease Inhibitor (rSLPI) into DOPS LIP and its intratracheal instillation increased the lung

r
residence time and reduced the clearance of the drug, while also improving its stability, [231].

p
-
Many studies focused on developing LIP for prologed lung retention. Modified LIP (ssLIP), which incorporated polyvinyl alcohol (with a

e
r
hydrophobic anchor (PVA-R), were labeled with Indocyanine green (ICG), and evaluated for their in vivo distribution and retention after

P
pulmonary administration to rats, by non-invasive in vivo imaging (IVIS imaging system). Compared with unmodified LIP, ssLIP realized long-

a l
term retention in the lungs. Moreover, the PVA-R modification was found to prevent the rapid elimination of ssLIP by macrophages, prolonging
their retention in the lungs [232]. The same group also showed that by modifying the surface of submicron-sized LIP with Chitosan

r n
oligosaccharide (oligoCS) , their adherence to lung tissues was increased and caused opening of the tight junctions, leading thus to enhanced drug
absorption [233].

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In another study, N-acetylcysteine (NAC)-loaded chitosan (CH)-coated-LIP showed a good deposition and retention in the lungs after being
administered intratracheally [234].

Interestingly, when the surface of LIP was modified with carbopol (CP) (a mucoadhesive polymer), and also with wheat germ agglutinin (a ligand
with affinity towards alveolar epithelial cells), the LIP demonstrated increased bioadhesion to lung epithelia and enhanced interaction with A549
lung epithelial cells in vitro. The efficacy of calcitonin (which was encapsulated in the former modified LIP as a model peptide drug), was
increased and prolonged, while no signs any of toxicity were observed [235].

The effect of the surface modification of LIP with mannose, and their particle size, on their uptake by alveolar macrophages (AMs) was studied.
After pulmonary administration to rats, the uptake of non-modified LIP by AMs increased as the LIP size increased (in the range 100–2000 nm).

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The mannosylated-LIP (1000 nm) uptake (in-vitro and in-vivo) by rat AMs was significantly higher than non-modified LIP (of same size). It is
concluded that the uptake of LIP by rat AMs is affected by the LIP size and increases when the LIP surface is modified by mannose [236].

In another recent study, the encapsulation of the glucocorticoid drug Fluticazole proprionate (FTZ) in LIP resulted in 20 and 30 times higher
accumulation in the lungs in comparison with free FTZ, 30 min and 3h post-administration (by inhalation), respectively [237].

Summarizing the above reports, it is evident that the pulmonary delivery of LIP drugs is a very promising strategy to treat localized pathologies,

f
particularly due to the high potential to achieve long retention of LIP drugs at the site (which can be prolonged by use of polymer coated LIP) and

o
o
furthermore to target specific cell types (such as alveolar macrophages). The safety profile of LIP drugs, following pulmonary delivery and the
decreased systemic side-effects, are additional advantages.

p r
3.7.2 Intrapleural Administration (IPA) of LIP drugs

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In Table S7B, the recent cases of local administration of LIP-drugs in the pleural cavity, are reported. Intrapleural administration of compounds is
a thoracic cavity targeted, innovative therapeutic strategy for mesothelioma, secondary pleural infections or malignant pleural effusion formation.

a l
These kinds of diseases are difficult to approach through systemic or even IPA of free drugs because the concentration of the drug that finally

r n
reaches the site is very low and ineffective. Thus, intrapleural injection could be defined as a route for direct drug delivery that minimizes potential
for systemic toxicity. However, the injection is particularly invasive (Figure 11) and not absolutely toxicity-free since high doses of drugs in the

u
pleural cavity have been demonstrated to cause pleural adhesion (in several cases). Thereby, a potential way to sustain a gradual release of drugs in

Jo
the pleural cavity, avoiding the presence of high and potentially toxic drug concentrations, could be by topical injection of LIP drugs [238].

In the following paragraphs we review the most important recent (2010-to-date) cases for preclinical studied LIP-drugs intended for IPA, after
evaluating the corresponding hits of Table 1. From the total ten hits of Table 1, four references were not considered since the two were targeting
mediastinal nodes through intrapleural injection, one was a clinical case of using free antibiotics intrapleurally and the other one used intravenous
administration.

Malignant pleural mesothelioma (MPM) is an aggressive tumor that disseminates in the pleural cavity. is The currently approved treatment for
MPM is a combination of Pemetrexed (PMX) and Cisplatin; however the response rate is low. Numerous LIP-PMX formulations (differing in

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their lipid compositions) weredeveloped by Ando et al, and evaluated their in vivo therapeutic effect on an orthotopic tumor mouse model of
MPM. Chol-free cationic PMX-LIP (dramatically decreased the size of pleural cavity tumors, while free PMX was minimally effective. The
enhanced anti-tumor efficacy of LIP was credited, on their prolonged pleural cavity retention (due to electrostatic interaction with negative-charge
membranes of tumor cells), and to the faster release of the drug from the “fluid” liposomal membranes after intrapleural administration [239].
Moreover, Lila et al working in the same direction with PMX exploited the RNA interference (RNAi) technology to substantially enhance the

o f
therapeutic efficacy of PMX. Specifically, they incorporated in cationic LIP the non-viral anti-thymidylate synthase RNAi to guide the
downregulation of thymidylate synthase (TS) in human malignant mesothelioma. TS shRNA lipoplex effectively enhanced the in vitro cell growth

o
inhibition as well as efficiently combated the progression of orthotopic thoracic tumors and as a result prolonged the survival of mice, by treatment

r
p
with PMX and also downregulation of TS [240].

e -
On the other hand, Ando et al have recently introduced an intelligent RNA expression device (iRed), comprising the minimum essential

P r
components needed to transcribe short hairpin RNA (shRNA) in cells. To study the usefulness of topically delivered iRed , they engineered iRed
encoding luciferase shRNA (Luc iRed) and complexed with cationic LIP (Luc iRed/LIP-complexes) that were intrapleurally injected into an

a l
orthotopic mesothelioma mouse model. Luc iRed/lLIP-complexes markedly suppressed the the luciferase marker gene expression in pleurally
disseminated mesothelioma cells while it did not induce any innate immune stimulation [241].

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Little is currently known about the retention of LIP drugs at the site, after topical administration in physiological and in pleural inflamed

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conditions, such as malignant pleural effusion formation. Malignant pleural effusion (MPE) is the accumulation of fluid in the pleural cavity due to
malignancies affecting the lungs, pleura and mediastinal lymph nodes. Marazioti et al [238] studied the retention of different compositions of LIP
in the pleural cavity following intrapleural injection, and how this could be modulated by LIP properties and disease progression. Interestingly,
smaller LIP size and PEG-coating significantly increase the residence time in the pleural cavity. More than 20% LIP-DiR was retained 24 d post-
injection, indicating high potential towards localized diseases. Furthermore, high LIP-DiR signals in tumors of MPE mice suggested high tumor
targeting of IPA LIP, which was not affected by the presence of effusion [238].

Hocking et al compared the local biodistribution and pharmacokinetics as well as the safety profile of curcumin-LIP following IPA and
intravenous administration. Curcumin-LIP were found to be safe when administered directly into the pleural cavity, a strategy that may bea viable
alternative to i.v. infusion, for the treatment of patients with pleural-based tumors [242].

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Recently, the clinical pharmacokinetics of Paclitaxel-LIP after a single intrapleural infusion were studied, in nine patients with advanced non
small-cell lung cancer (NSCLC) having malignant pleural effusions,. The data offer opportunities to design personalized therapeutic dosage
regimens for such patients, based on the pharmacokinetic parameters determined [243].

Judging from the encouraging results of the recent studies discussed above, direct intrapleural injection of LIP drugs seems to be a promising
strategy for delivery of conventional and advanced therapeutics (such as RNAi), for the treatment of localized diseased cuxh as MPM and MPE.

3.8 Vaginal and Uterine Delivery of LIP drugs

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r o
As a route for drug administration, vaginal drug delivery (VDD) has some advantages, such as avoidance of the first-pass liver metabolism and

- p
gastrointestinal tract adverse reactions which are possible after oral administration. Also it is not associated with any pain or discomfort (as for
instance intramuscular injection). Moreover, VDD could preferential deliver high levels of drug to the endometrial tissue by first uterine-pass

r e
effect [244,245], and as a result much greater topical therapeutic effects may be achieved [246].

P
In Table S8, five of the reported hits of Table 1 were not included since they involved models to predict vaginal permeation, or did not involve

l
a
vaginal delivery or LIP, or were review articles. More details about the reports for vaginal delivery of LIP drugs follow:

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In order to develop efficient localized therapy of Sertaconazole nitrate for the treatment of vaginal candidiasis the drug was loaded in cationic LIP

u
containing dimethyldidodecylammonium bromide (DDAB), coated with pectin (0.05%, 0.1% and 0.2%) to increase their mucoadhesive properties,

Jo
and the LIP were finally incorporated into a gel [247] . An ex vivo permeation study showed that the mucoadhesive liposomal gel increased
Sertaconazole tissue retention and reduced drug tissue penetration, compared with a conventional gel. Also, the efficacy of Sertaconazole
mucoadhesive LIP gel for treating rats with vaginal candidiasis was evaluated (in vivo) and it was shown that DDAB demonstrated enhanced
anticandidal activity in the mucoadhesive LIP gel in comparison with Sertaconazole conventional gel.

Vaginally delivered sildenafil citrate is known to efficiently prepare the uterine lining for successful embryo implantation during the procedure for
in vitro fertilization. Sildenafil-loaded LIP which were also coated with bioadhesive polymers were prepared, as carriers with potential for
enhanced vaginal retention and improved drug permeation [248]. The effectiveness of coating of the LIP with chitosan and HPMC was confirmed
by the increases measured in both, size and zeta potential of LIP. The prolonged release of sildenafil, and finally the improvement in their

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mucoadhesive properties were also confirmed by in vitro studies. The optimized formulation, was the one coated with chitosan (HPMC was also
evaluated) which showed greatest permeation of sildenafil through vaginal mucus. Vaginal application of the optimal chitosan-coated LIP in
female rats showed a clear dilatation and congestion of endometrial blood vessels and an increase in the thickness of the endometrial layer,
compared with the control groups.

Hydrogel-core and lipid-shell nanoparticles (nanolipogels) were developed for the vaginal delivery of Maraviroc (MVC) and Tenofovir disoproxil

o f
fumarate (TDF), two anti-retro-viral (ARV) drugs with different mechanisms of action, currently used in the treatment of HIV. After the LIPs
were formulated they were subjected under UV-light for polymerization and gel formation. The formulations were tester for their antiviral activity,

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in vitro and in vivo [249]. The results showed that the vaginal administration of LIP formulations, in a murine model, lead to antiviral activity

p
against HIV-1 BaL in cervico-vaginal lavages. The nanolipogel carriers are thus promising for the vaginal delivery of ARV drugs, for HIV

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e
prevention and/or treatment.

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Fibrauretine (FN) has a significant inhibitory effect on many pathogenic microbes especially fungus and a favorable anti-Trichomonas vaginalis
activity [250]. It also possesses ability to enhance the immunity of organism, so it has been approved for therapy of female reproductive system

a l
diseases. The ability of special types of deformable LIP to deliver FN across the vagina epithelia (in vitro) and its biodistribution after vaginal

r n
delivery was studied [251]. Results showed that propylene glycol-embodying deformable LIP could enhance FN penetration into the vaginal
mucosa and that a higher fraction of administrated FN remains in the vaginal tissue (compared to free drug administration), avoiding the

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absorption of high amount of drug into the systemic circulation. Safety evaluation studies showed that the FN loaded propylene glycol-embodying
deformable LIP are additionally non-irritant and well tolerated in vivo.

Chitosan-coated LIPs (for increased mucoadhesion) were loaded with Clotrimazole as a safe treatment for vaginal infections, especially during
pregnancy [252]. In such cases, it is of great importance that the drug is retained in the vaginal epithelia and not absorbed systemically, for
minimizing side effects. The results proved the prolonged release of the drugs from LIP (coated or not), and showed that the lowest chitosan
content (0.1%) realized optimal mucoadhesion. Furthermore, coated LIP assured increased Clotrimazole tissue retention and reduced drug
absorption, proving their potential for vaginal delivery of antimicrobials to treat localized infections.

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With the aim to develop therapeutic anti-HSV-2 agents, the most potent PEI found before [253], was combined with LIP (200 nm) with or without
oleic acid and the optimal formulation (PEI-LIP) was evaluated after intravaginal delivery in mice (mouse genital herpes model). PEI-LIPs
showed higher antiviral activity post-infection than pre-infection (in vivo). PEI-LIPs with PEI having less primary amine residues, exhibited more
potent therapeutic antiviral activity than acyclovir and PEI alone, without acute lesion appearance or any signs of toxicity pre- or postinfection.
LIP concentrations were important for the effectiveness of PEI-LIPs. This finding suggests that PEI -LIPs formulated with PEI that has slightly

of PEI may contribute to the inhibitory efficiency against viral infection [254].
o f
decreased numbers of primary amines, may be an effective vaginally administrated antiviral drug, and that secondary and tertiary amine residues

r o
Octylglycerol (OG)-LIP were developed as a vaginal microbicides [255]. LIP gels with varying OG /lipid ratios, viscosity increasing agents, and

p
OG concentrations were formulated. The OG-LIP with the optimal OG/lipid ratio (optimized based on the results of in vitro screening studies) had

-
e
greater efficacy than conventional gel formulations and maintained their efficacy for at least 2 months. The OG-LIP demonstrated activity against

P r
Neisseria gonorrhoeae, HSV-1, HSV-2 and HIV-1 (in vitro), and did not affect the innate vaginal flora. No toxicity was observed for the LIP
formulation in an ex vivo human ectocervical tissue model, and also in vivo (in a macaque safety model). Furthermore, minimal toxicity was

and N. gonorrhoeae.
a l
observed to lactobacilli in vitro and in vivo. The optimal OG LIP formulation is a promising microbicide product with efficacy against HSV, HIV

r n
In order to identify LIP that are potentially suitable as intravaginal microbicides to prevent HIV-1 infection, a wide range of natural and synthetic

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lipids with varying physicochemical properties, were used for LIP formation [256]. LIP with most favorable balance between decreasing HIV
virus counts and cytotoxicity were selected; the therapeutic index of the LIP was found to improve as their cardiolipin content and their degree of
unsaturation was increased. After intravaginal instillation the tissue reaction to these formulations was benign, and they were proven to have high
potential as microbicides for prevention of HIV infection.

A novel alginate scaffold system containing PEGylated lipoplexes (PLAS) that are inert in mucus, was developed for sustained vaginal retention
of lipoplexes and facilitation of siRNA/ oligonucleotide delivery to the vaginal epithelium [257]. PLAS entrapped about 50% of PEGylated
lipoplexes, releasing them gradually in vitro and in vivo. After intravaginal delivery in mice, a six-fold increased uptake of siRNA by the vaginal
epithelium was demonstrated by PLAS, compared to control transfection systems. A siLamin A/C-containing PLAS system achieved significant in

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vivo knockdown of Lamin A/C level in vaginal tissues. In general the results confirm the potential of the PLAS system for sustained delivery of
siRNA or other oligonucleotides to the vaginal epithelium [257].

The uterus is an organ that is directly accessible via the transvaginal route; however the cases reported for using LIP for drug or gene delivery to
the uterus, even in animal models, are limited. From the 13 hits in Pub med when searching by “uterine” and “administration” and “LIP” and “in
vivo”, only one was actually about LIP delivery directly to the uterus, while others involved systemic delivery of uterine-targeted LIP or vaginal

o f
administration. In the one case, an optimized LIP formulation of hepatitis B virus envelope L-protein was delivered locally to the uterus of a
mouse. TAT peptide was used as targeting ligand, and results showed that gene transfer using TAT-LIP was 5000- or 18-fold more efficient than

r o
naked DNAs or cationic LIP, respectively. TAT-LIP were rapidly eliminated from uterus and had no effect on the pregnancy rate or fetal growth,

p
proving the potential of local uterine injection of LIP for gene delivery to treat the uterine endometrium [258].

-
r e
The promising results of the recent studies conserning the vaginal (or uterine) delivery of various types of polymer-coated (for enhanced retention
at the site and decreased system absorption) and/or targeted LIP-drugs, indicate that such strategies should be further exploited in future studies.

3.10 Other routes

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n a
r
In addition to the routes described and analyzed above, several other routes for drug administration are being evaluated for potential advantages of

u
using LIP instead of free drugs. Some routes are particularly invasive, such as: Intratracheal installation with the help of an endotracheal tube,

Jo
which is used for the delivery of lung surfactants in premature neonates, and intraventricular/intrathecal route which is a known strategy for
bypassing the BBB that has been studied extensively in preclinical research and in clinical trials [259]. Between less invasive localized routes,
studies concerning LIP-drug administration locally to the inner ear (cochlea) have been recently reviewed [260]. In any case, we should stress the
fact that the current review is not extensive and all the potential routes for localized administration of LIP have not been fully covered.

4. Locally delivered LIP in Clinical use and testing

4.1. Locally delivered LIP products

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Numerous LIP drugs have been in clinical use for many years as therapeutics for different pathologies, the high majority being indicated for
treatment of different types of cancer, as recently reviewed [25-27, 261].

Between the many formulations included in the relevant lists provided in recent review articles [25, 27, 261]and with the addition of the recently
approved formulation of siRNA targeting transthyretin [261, 262] as well as the just approved mRNA vaccines for COVID-19 vaccination [263], a
total of 30 LIP products are listed in Table 2 (Part A and Part B). Between the 30 LIP products, 2 generic products of Doxil are included [261],

o f
and also 3 lung surfactant products which are administered intracheally to treat Respiratory Distress Syndrome (RDS) in premature infants [25],
although we are not quite sure if theu can be considered as LIP. Furthermore 6 of the 30 LIP products are LIP vaccines; four of which could be

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considered are more “conventional” LIP-vaccines (Epaxal, Inflexal, Shingrix and Mosquirix), while the two are the recently approved m-RNA
vaccines for Covid-19 (BNT162b2 (Biontech/Pfizer) and mRNA-1273 (Moderna)) [263].

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r e
From the 30 products which are included in the cumulative list of Table 2, only 10 products are intended to be administered by routes other than
intravascular, intramuscular or subcutaneous, and may thus be considered as “localized” delivered therapeutics (Table 10, Part B).

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When the administration routes of the ten LIP products are considered, it is observed that almost all products (with the exception of the Estradiol

a
topical emulsion Estrasorb™), involve very invasive routes requiring assistance of specialized personnel for their administration. This makes

n
r
sence since as mentioned in the introduction, when such demanding drug administration routes are required; the use of carriers that can deliver

u
large amounts of drugs is preffered (Figure 1, Case 2). Furthermore, most of them are injectable formulations having sterility and pyrogen-free

Jo
requirements.

Between, the latter LIP products for localized administration some have not been approved by FDA and/or EMA, one is not for human use, and (as
mentioned above) the three lung surfactancts may not justify as LIP products, according to some recent reports [27, 261].

In more detail, Lipotalon®, is only authorized in Germany (as concluded by the list of nationally authorised medicinal products of EMA2).
Futhermore Arikayce®, an amikacin LIP product intended for treatment of patients with persistent mycobacterium avium (MAC)-infections, was
approved (accelerated approval) by FDA in 2018 (the first antibiotic containing liposome formulation which consists of a liposome dispersion for

2
https://www.ema.europa.eu/en/documents/psusa/dexamethasone-apart-centrally-authorised-products-list-nationally-authorised-medicinal-
products/00000973/201601_en.pdf

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pulmonary delivery with the assistance of a nebulizer); however is has not yet been approved by EMA (received orphan drug designation) [261].
Additionally, one of the bupivacaine LIP products, Nocita® is only for animal use.

It should be also mentioned that Exparel® , the bupivacaine LIP for human use, that was abroved by FDA in 2011 and recenty approved by EMA
(November 2020), consists of large multivecicular liposomes (about 25 um), so this product could not be considered as a nanomedicine, as
mentioned recently [261]. Nevertheless, it is logical to use large LIP for the specific delivery route, since larger construction in the microparticle
range are known to provide more extended retention after intra-articular injection (Figure 6) [20].

o f
o
Finally, it is of interest to note that the marketing of two of localized delivered LIP products of Table 2, DepoCyte and DepoDur have been
discontinued due to commercial reasons by the marketing authorisation holder, Pacira Ltd.

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4.2 Recently approved LIP products of nucleic acid drugs

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Before discussing the localized delivered LIP products which are currently under clinical testing, and although out of the basic scope of this review
(since the specific products are not intended for localized delivery) it is noteworthy to emphasize the recent success with the approval of LIP (or

a l
more precisely Lipid-NP (LNP)) products that encapsulate short interfering RNA (siRNA) , or messenger-RNA (mRNA), since we believe that
this success will potentially lead to clinical development of similar localized-delivered LIP products.

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Indeed, it is of great importance that the LIP (or LNP) technology has been successfully extended to deliver nucleic acid-based drugs, such as short

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interfering RNA (siRNA) and messenger RNA (mRNA). As already mentioned in section 2, nucleic acids (NAs) and oligonucleotides in general,
face very serious problems regarding their delivery, due to their extremely low stability (they are readily broken down within minutes, in
biological fluids), and their inherent inability to permeate through membranes in order to accumulate in target tissues and finally penetrate into
target cells (if they magane to reach the desired tissues), due to their large molecular size and high hydrophilic nature. Furthermore, the carriers of
NA drugs are additionally required to facilitate the intracellular delivery of these macromolecules into their target cells, and also their early
lysosomal escape, so they can be rescued from the harch intralysosomal environment.

Additionally, the manufacturing techniques developed for LIP formulations of small molecule drugs could not be applied to NAs because of their
large size and high negative charge.

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As recently explained, the delivery problems of siRNAs have been sucesfully resolved by development of novel types of ionizable lipids
[264]which made it possible to overcome the toxicity associated with LIP containing permanently positively charged lipids due to immune
activation and cytotoxicity. LNP structures that efficient encapsulate siRNA, and furthermore provided all the additional attributed required for its
delivery (displaying low surface charge at physiological pH and being relatively non-toxic and non-immunogenic), were thus developed. A
readily scalable manufacturing process was used for rapid mixing of lipids (in ethanol) with siRNA in (in pH (pH 4) aqueous media) [262].

o f
As LNP-mediated siRNA therapeutic Onpattro® (patisiran) advanced towards approval [262], it was natural that the mRNA delivery field whould
adopt the existing technology [263]. After identifying the required properties of novel lipid materials for efficient mRNA delivery, such materials

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were customized leading to the rapid development and approval of mRNA vaccines, which were already under clinical evaluation before the

p
COVID 19 pandemic. More details about Onpattro® and BNT162b2 & mRNA-1273 vaccine development can be found in [262] and [263],

-
e
respectively.

4.3 Clinical Studies of locally delivered LIP

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a l
Numerous LIP formulations (for localized delivery) are listed in completed or currently on-going clinical trials, as concluded after searching in the
database www.clinicaltrials.org. The number of LIP products involving delivery by the various topical/localized delivery routes (investigated in

n
this review) under clinical testing in the last 10 years were initially found by searching in the database under the Key words “Liposome” and

r
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“<route>”. As seen in Table 3, this search gave a total of 478 hits; after initial screening of the hits, 127 trials testing LIP products and considering

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localized delivery routes were identified. Some of them are mentioned below, according to the route of delivery.

Pulmonary delivery is the localized route with the most clinical trials for LIP products. Several trials involve fungal infections (from
mycobacterium, pseudomonas aeruginosa, m. abscessus etc.), complications from lung transplantation, lung cancer, invasive pulmonary
aspergillosis and bronchiolitis. Other trials examined LIP formulations for pulmonary arterial hypertension, cystic or non-cystic fibrosis, and
bronchiectasis and rib trauma.

The majority of LIP formulations were inhaled aerosols, such as inhaled lipid formulations of amphotericin B (Ambisome or Abelcet) for allergic
bronchopulmonary aspergillosis (NCT02273661, NCT04267497, etc.), pulmonary mucormycosis (NCT04502381) and lung transplantation
(NCT00177710, NCT00177684).

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Aerolised LIPs encapsulating 9-nitro-20(S)-Camptothecin (L9NC), were tested as a treatment of lung cancer corpus uteri (NCT00277082), and
other lung diseases (NCT00250068).

Inhaled Amikacin-LIPs (Arikace) were tested for cystic fibrosis (NCT00777296, NCT03905642, NCT01316276, etc.) and fungal infections
(NCT03038178, NCT01315236, NCT02344004, etc). Inhaled Cyclosporine LIPs were tested for bronchiolitis obliterans and lung transplantation
disorders (NCT03657342, NCT03656926, etc.). Ciprofloxacin LIPs were evaluated for non-cystic fibrosis and bronchiectasis (NCT02104245) and
inhaled Cisplatin LIPs for metastatic osteosarcoma (NCT00102531).

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In other trials, inhaled Doxorubicin LIPs were examined for lung and metastatic cancer (NCT00082472, NCT00004930, and NCT00020124).

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Also, intercostal Bupivacaine LIP was examined for the management of blunt chest wall trauma (NCT03906617).

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Finally pleural delivery of Cisplatin LIsP alone (NCT00004033) or combined with low dose of Gemcitabine (NCT01869023) were studied for

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treatment of malignant mesothelioma.

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Several clinical trials involve the Intra-articular or peri-articular administration of LIP drugs. These trials examine the clinical effect of LIP
formulations of local anesthetics, such as Bupivacaine (Exparel) and Ropivacaine (TLC599). Some of the evaluated therapeutic indications are

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total knee arthroplasty (NCT02223364, NCT04086186, NCT02299349, etc.), hip arthroplasty (NCT02543801, NCT02242201), osteoarthritis

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(NCT02473198, NCT03754049, NCT02777749, etc.) and pain management (NCT02808728, NCT04003506, and NCT03001453).

The therapeutic strategy (in most cases) is designed to compare the effect of direct intra-articular injection into joints or the effect of a peri-

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articular injection around the joints (that offers analgesia through peripheral nerve blocks (femoral nerve block or adductor canal block)), with the
analgesia realized after opioid administration.In other words they do not compare localized delivery with systemic delivery, of with local delivery
of the free drug.
The majority of clinical trials of LIP products for Ocular delivery, aim to treat dry eye diseases. LIP Sirolimus, Crosslinked Hyaluronic Acid with
LIP, and Crocin and tear substitutes (Ectoin® Eye Spray – Colloidal versus Liposomal eye spray Tears Again® (NCT03519815), Liposic versus
Tears Naturale Forte (NCT02992392, NCT03211351), Lamelleye Dry Eye Drops versus Optive Plus (NCT03052140)), are some of the products
under clinical evaluation for dry eye treatment.

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Other indications evaluated include ocular hypertension, meibomian gland dysfunction, ocular infections, glaucoma, blepharitis, intraocular
retinoblastoma and postoperative pain. Subconjunctival injection of Latanoprost LIPs were proposed for ocular hypertension and open-angle
glaucoma (NCT01987323 and NCT02466399 respectively). The effectiveness of Ozonized-oil LIPs was examined for ocular infections after
cataract surgery (NCT04087733). In another clinical trial, liposomal Vincristine sulphate, Carboplatin, and Etoposide were examined for treating
patients who had undergone surgery for newly diagnosed retinoblastoma (NCT00335738).

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Other clinical trials evaluate Nasal delivery of LIPs. Topical disorders such as allergic rhinitis, viruses (influenza, Covid 19), cystic fibrosis or
tracheostomy complications were examined. In particular, the efficacy of Lactoferrin LIPs was recently examined in COVID-19 patients with

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mild-to moderate disease and in COVID-19 asymptomatic patients. Also, the safety and tolerability of Lactoferrin LIPs for oral and intra-nasal
delivery was studied (NCT04475120).

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For influenza, a vaccine given as a nasal spray may improve compliance and local immunity. One particular study aimed to examine the safety and
nasal and humoral responses to the nasal vaccine compared to the commercial muscular vaccine (NCT00197301).

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In another study, the efficacy and safety of lipid-mediated transfer of the cystic fibrosis transmembrane conductance regulator gene to nasal

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epithelium in patients with cystic fibrosis was evaluated (NCT00004806).

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As far tracheostomy complications are concerned LipoAerosol© Inhalation to Maintain the Functional Integrity of the Tracheo-bronchial System
in Patients after Tracheostomy was compared with physiologic saline inhalation (NCT02157129).

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Several clinical trials have studied the effect of Intratumoral (IT) injection of LIP in different types of cancer, such as head and neck cancer,
advanced or metastatic cancer, liver cancer and solid tumors. A Phase I trial, studied the effectiveness of gene therapy (Intatumoral EGFR
Antisense DNA and LIP) in treating patients with advanced head and neck cancer (NCT00009841). In another study the efficacy of interleukin-2
gene versus Methotrexate LIP are compared (NCT00006033). In a Phase I safety trial for advanced or metastatic cancer, bifunctional shRNA-
STMN1 (pbi-shRNA™STMN1) BIV-(bilamellar invaginated vesicle)-lipoplexes (LP), pbi-shRNA™ STMN1 LP, were administered by a single
IT injection (NCT01505153). Another study involved the proof of concept for low temperature sensitive liposome (LTSL) (ThermoDox®)
activated by mild hyperthermia using focused ultrasound (FUS), to achieve high IT Doxorubicin concentrations (NCT02181075).

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Dermal disorders, such as cutaneous leishmaniasis, vitiligo, cutaneous T-cell lymphoma and facial acne vulgaris have been the subjects of interest
for several clinical trials involving LIP. The majority of drug formulations were in the form of LIP gels and creams. A cream with Meglumine
antimonate (Glucantime)-LIPs and Paromomycin-LIPs was tested for treatment of cutaneous leishmaniasis (NCT01050777). In another trial, a LIP
gel with 3% Amphotericin B was studied for the same disease (NCT01845727). Lithium LIPs were tested for vitiligo (NCT04171427), while
micro needle array-Doxorubicin (MNA-D) was examined for the therapy of cutaneous T-cell lymphoma (NCT02192021). Finally, Retinyl
Palmitate-loaded Ethosomes were studied for Acne Vulgaris (NCT04080869).

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Concerning Dental administration and Intraoral injections of LIPs, local anesthesia and pain management strategies have been clinically

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investigated. In more detail, LIP formulations of several local anesthetics such as Bupivacaine (NCT02517905), Prilocaine (NCT01073371),

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Ropivacaine (NCT01307969, NCT01054547), Lidocaine (NCT01425840) and Mepivacaine (NCT01032798), were administered as injectable

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solutions or gels. These formulations were examined for maxillary infiltration anesthesia or topical anesthesia in the palatal or buccal mucosa.

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Clinical trials for Vaginal delivery include the delivery of antibiotic creams, injections of local anesthetics or sub urethral sling pudendal block, for
several therapeutic applications. Vaginally delivered LIP formulations were particularly examined for postoperative pain after vaginal

a l
hysterectomy (NCT03907033) or other uro-gynecologic surgeries (NCT02287246, NCT03875664, and NCT04032327), pelvic organ prolapse

(NCT01293643).
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(NCT03664986, NCT02449915, and NCT02890199), urinary incontinence (NCT02296099), bacterial vaginosis and candidiasis vulvovaginal

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Finally, for uterine disorders, uterus delivery for treatment of ovarian and endometrial cancer (NCT01970722, NCT02262455, and NCT0575952)
was evaluated. The strategy involved i.p. injections of LIP anticancer drugs and Transversus Abdominal Plane (TAP) or pudendal block using
local LIP-anesthetics for the management of acute pain (NCT02289079, NCT02519023).

From all the above mentioned clinical studies, it becomes evident that localized delivered LIP drugs are currently under intensive investigation;
between the studied drugs several are NA-drugs.

5. Conclusions and Future Perspectives

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As concluded from the previous parts, localized delivery of LIP drugs offers the advantage of overcoming specific BBs for treatment of very
diverse pathologies. In many cases preclinical and clinical studies have indicated high therapeutic advantages of such app roaches. Particularly
nose-to-brain delivery after nasal administration of LIP-gels, transdermal delivery of LIP drugs in cases of drugs with short biological half-life
and/or low aqueous solubility, and direct delivery in confined body areas (such as tumors, lungs, intrapleural cavity and others) where high
concentration of free-drugs may be toxic and/or drug retention at the site is difficult to achieve (problems that can easily be solved using LIP
formulations), are of high therapeutic interest and should be further exploited.

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We hope that we succeeded to highlight the potential of localized delivered liposomal formulations in the current report, and encourage research

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on this topic, that in addition to overcoming the obstacles of BBs are also potential solutions to the current bottlenecks for the translation of
actively-targeted LIP.

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It is interesting that some LIP products are currently approved for localized delivery of drugs, nevertheless only one of these LIP products is

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intended for pulmorary delivery and one other for dermal administration, while none are currently approved for ocular, nasal, intratumoral or
vaginal delivery Table 2). Futhermore, no LIP vaccine is currently delivered by oromucosal, sublingual or intranasal route despite the ongoing

a
preclinical research, in this area. Judging from the results of many advanced pre-clinical studies we believe that in the near future several new

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localized delivered LIP products will soon be developed.

Despite the fact that between the LIP drug products currently marketed, a very small number of them is indicated for what can be considered as

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“localized” drug delivery, the high number of such LIP products under clinical testing in the last decade spears hope that more LIP products
indicated for localized delivery will be available for clinical use in the near future, bringing with them significant therapeutic advantages.

Finally, the recent success of approvals of a siRNA-incorporating LIP therepeutic product, and also (very recently) two mRNA vaccines for
Covid-19, will most probably lead to clinical development of similar localized-delivered LIP products, some of which are already under clinical
investigation.

Acknowledgements

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M.K., A.M. and S.G.A. acknowledges the support by the project: "Development of Innovative Neuroprotective Neurogenerative Synthetic Micro-
Neurotrophins" (MIS-5032840) which is implemented under the Special Service of the Operational Program Competitiveness Entrepreneurship
and Innovation. The project is funded by the Operational Programme "Competitiveness, Entrepreneurship and Innovation" (NSRF 2014-2020) and
co-financed by Greece and the European Union (European Regional Development Fund); V.N. and SGA acknowledge the support by the project:
"Preclinical development of INNOvative FORmulations of antibiotics for intraocular administration for the treatment / prevention of postoperative

o f
endophthalmIitis" (MIS- 5031792) which is implemented under the Special Service of the Operational Program Competitiveness Entrepreneurship
and Innovation. The project is funded by the Operational Programme "Competitiveness, Entrepreneurship and Innovation" (NSRF 2014-2020) and

o
co-financed by Greece and the European Union (European Regional Development Fund); GK, FG and SGA acknowledge the support by the

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project: “Development of Innovative Nanocarriers of Insulin-like Peptides with prolonged action”, which is co-funded by the Management

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Authority of Western Greece Region and the Public Investment Programme of Greece.

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Supplementary data

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Supplementary material

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Table 1. Numbers of hits in Pub MED (from 2010 –up to date) for the search: A]“ROUTE”& “LIPOSOME” + (“DELIVERY” OR “ADMINISTRATION”) +(“TOPICAL” OR
“LOCAL”); B] SEARCH A & “IN VIVO”, as well asother terms used (with the corresponding # of hits), and final number of reports analyzed for each route.

ROUTE Search A Search B Other terms as route Analyzed

Intraarticular 18 3 Joints (22) 9

Periarticular (2)
Intranasal 15 11 Inhalation (13) 26
- {“topical” or “local”} (42)
Intra-tumor 59 27 - 15
Skin 578 152 Dermal (32) 41
Transdermal (94)
Ocular 102 33 Intraocular (14) 35

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Intavitreous (5)
- {“topical” or “local”} (71)
Oral 203 34 Oromucosal (13) 38
Dental (7)
- {“topical” or “local”} (326)
Pulmonary 169 46 Lung (41) 25
Pleural (1)
Vaginal 40 7 Microbicides (91) 9
- {“topical” or “local”} (15)
Uterine 17 4 - 1

Table 2. Currently available Liposomal drug products

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ro
Table 2 - Part A: LIP products For Systemic Delivery
# Product Adm. Active Agent Indication Company Ref

-p
1 AmBisome i.v. Amphotericin B Fungal Infections; Leishmaniasis Astellas Pharma /Gilead 25, 27, 261
2 Abelcet& i.v. Amphotericin B Invasive severe Fungal Infections Leadiant, Teva 25, 27, 261
3 Amphotec i.v. Amphotericin B Severe Fungal Infections Ben Venue Lab. 27

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4 DaunoXome i.v. Daunorubicin AIDS- Related Kaposi‟s Sarcoma Galen 25, 27, 261
5 Doxil/Caelyx i.v. Doxorubicin Ovarian cancer; Breast cancer; Kaposi‟s Jansen 25, 27, 261
Sarcoma
6

7
Doxorubicin
Hydrochloride Liposomes
Lipodox
i.v.

i.v.
Doxorubicin (generic)

Doxorubicin (generic)

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Ovarian cancer

Ovarian cancer
Dr Reddy‟s

Sun Pharma
261

261

a
8 Myocet i.v. Doxorubicin Advanced Breast cancer Zeneus/Cephalon Inc;Elan Pjarm.; 25, 27, 261
Sopherion, Teva
9 Marqibo i.v. Vincristine

r n Acute lymphoblastic leukemia Talon Therapeutics; Acrotech

CASI/China
27, 261

u
10 Mepact i.v. Miramurtide High-grade, resectable, non-metastatic Takeda Pharmaceuticals; 27, 261
osteosarcoma

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11 Visudyne i.v. Verteporfin Age-related Macular degeneration; Choroidal Novartis; Bausch + Lomp; 25, 27, 261
neovascularisation
12 Onivyde i.v. Irotecan Combination with 5-FU and leucovorin; Marrimack Pharm. Inc; Ipsen 27, 261
Metastatin pancreas adenocarcinoma Biopharma;
13 Vyxeos i.v. Daunorubicin/Cytarabin Acute myeloid leukemia Jazz Pharma 261
14 Onpattro& i.v. siRNA targeting transthyretin Transthyretin induced amyloidosis (hATTR) Alnylam Pharmaceuticals 261
15 Epaxal i.m. Inactivated Hepatitis A virus Hepatitis A Crucell, Berna Biotech, Janssen 25, 27, 261
Cilag;
16 Inflexal V i.m. Inactivated hemaglutinine of Influenza Crucell, Berna Biotech 25, 27
Influenza virus a & B
17 Shingrix i.m. Glycoprotein E based vaccine Shringles (Varicella zoster) GSK 261
18 Mosquirix i.m. RTS,S antigaen based xaccine Malaria GSK 261
19 BNT162b2& i.m An mRNA targeting a membrane COVID-19 (SARS-CoV-2) Pfizer/BioNTech 262
20 mRNA-1273& i.m. anchored pre-fusion stabilized spike COVID-19 (SARS-CoV-2) Moderna 262
protein of the virus
& Strickly speaking not conventional liposomes structures

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Table 2- Part B. LIP Products Delivered by local/topical routes

# Product Adm. Active Agent Indication Company Ref
21 Arikayce Inhalation (with Amikacin Mycobacterium avium complex (MAC) Insmed Netherlands B.V. (orphan drug status) 261
nebulizer) lung infection
22 Estrasorb™ Topical emulsion Estradiol hemihydrate Menopausal Hot flushes; Novavax 25
USP
23 Exparel® i.v and local Bupivacaine Postsurgical regional analgesia; Pain Pacira Pharmaceuticals, Inc. 27, 261
infiltrations management
24 Nocita local infiltration inj. Bupivacaine Local Anesthetic; nerve block (cats) Aratana/Elanco (animal application) 261

25
26
DepoDur™
DepoCyte®
(dogs)
Epidural
Spinal; Epidural
Morphine sulfate
Cytarabine
Pain management; Analgesia;
Neoplastic meningitis

o f
Skye Pharma Inc.; discontinued [261];
SkyPharma Inc.; EKR Therapeutics; Pacira;
25, 27, 261
25, 27, 261

o
discontinued [261]

r
27 Alveofact Intratracheally by Bovactant Respiratory Distress Syndrome (RDS) BoehringerIngelheim GmbH 25
instillation through a (bovine lung lavage) (hyaline membrane disease) in premature
28

29
Survanta®

Curosurf®
end-hole catheter Beractant (bovine lung
homogenate)
Poractant alfa (porcine
infants

- p Abbott Laboratories

ChiesiFarmaceuticiSpA
25

25

30 Lipotalon®* Intra articular;

infiltration
lung homogenate)
Dexamethasone 21-
palmitate
r e
Rheumatic disorders; "tennis elbow" MerckleRecordati GmbH 17

*Available only in Germany

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n a
Table 3. Number of LIP products involving topical/localized delivery routes under clinical testing in the last 10 years after screening the hits of

r
the searches using as Key words “Liposome” and “<route> “ in Clinical trial database.

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Route Accepted hits/ Total hits
Intra-articular 6/7
Peri-articular 17/26
Intranasal 2/2
Nasal 5/16
Intratumoral 4/6
Dermal 8/27
Transdermal 0/1
Ocular 16/205
Oral administration:
Intraoral 1/1
Dental 5/6
Sublingual -

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oromucosal -
Pulmonary:
Lung 45/113
Pleural 2/8
Vaginal 10/12
Uterine 6/48
TOTAL 127 / 478

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Figure 1. Local Drug administration: Potential cases depending on route of administration and site of action. Advantages and challenges

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Figure 2.Advantages of DDS for localized Drug Administration

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Figure 3. Structure of Liposomes - Advantages and Disadvantages as DDS for local delivery of drugs, compared to other types of nanocarriers.
(Partly adapted from Ref. 14, after Permission from Copyright Clearance Center, Elsevier)

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Figure 4. Liposome types depending on their Physicochemical Properties and their in vivo fate of application

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Figure 5. Methodology of hit selection

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Figure 6. Intra-articular delivery of Liposomal Drugs (Adapted from Ref 20, after Permission from Copyright Clearance Center, Elsevier)

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Figure 7. Routes for Brain Targeting following Intranasal Administration; Direct targeting of the brain via olfactory and trigeminal neural
pathways bypassing the BBB, of Nose-to-Brain delivery. (From Ref. 16, after Permission from Copyright Clearance Center, Elsevier)

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Figure 8. A: Structure of Skin; B: Possible Routes of Drug transport across the skin; C: Structure of the most important types of „Elastic”
liposomes, in comparison to conventional liposomes. (Adapted from Ref. 91 after Permission from Copyright Clearance Center, Elsevier)

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Figure 9. Ocular Drug Delivery and related Barriers for Delivery of Drugs (Influenced from Ref. 134, after Permission from Copyright Clearance
Center, Elsevier)

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Figure 10. Oral delivery of LIP drugs; Barriers and Strategies

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Figure 11. Pulmonary Delivery of LIP drugs; Advantages, Challenges and Strategies.

Graphical abstract

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