Nanomedicine

Lipid-Based Nanostructures for Drug

Delivery and Monitoring
Liposomes
Small artificial vesicles of spherical shape made using
cholesterol and natural nontoxic phospholipids.
Promising systems for drug delivery.
Liposome properties differ considerably with lipid
composition, surface charge, size, and the method of
preparation
Lipid-Based Nanomedicines for
Cancer
The liposome is the oldest and the most
common type of lipid-based nanostructure
used for biomedical applications.
Advantage:
Can contain hydrophobic drugs at the lipid bilayer
hydrophilic drugs inside the lipid bilayer
cationic liposomes electrostatically bind anionic
nucleic acids to their surface
Diagram of a bilaminar liposome. The hydrophobic region traps drugs in the
central core when the liposomes are prepared. The outer surface can be
functionalized with ligands for active targeting or PEGylated. Liposomes can
vary in the number of lipid bilayers they possess and can be classified into
three categories: (i) multilamellar vesicles, (ii) large unilamellar vesicles and
(iii) small unilamellar vesicles.
 The liposome bilayer can be composed of either synthetic or
natural phospholipids.

The predominant physical and chemical properties of a liposome

are based on the net properties of the constituent phospholipids
permeability
charge density
steric hindrance

The process of liposome formation is spontaneous because the

amphiphilic phospholipids selfassociate into bilayers.

Drug loading into liposomes can be achieved through

liposome formation in an aqueous solution saturated with soluble
drug;
the use of organic solvents and solvent exchange mechanisms;
the use of lipophilic drugs;
pH gradient methods;
Liposomes can target specific tissues through
both active and passive targeting strategies
At sites of pathology where the
endothelium layer is inflamed,
mediators such as bradykinin,
vascular endothelium growth
factor and prostaglandins increase
the endothelial permeability.
Liposomes extravasate through the
gaps between cells and enter the
interstitial fluid.
Active targeting is achieved by
conjugating ligands to the
liposome that bind to a specific
target cell receptor, leading to
internalization or release of the
drug.
Passive targeting can be mediated
by internalization or local high-
concentration release of the drug
Certain advantages
Potential of liposomes to combat the increasing problem of multidrug
resistance (MDR) acquired by cancers.
Proposed mechanisms underlying MDR at the cellular level include:
(i) increased metabolism of drugs due to increased enzyme expression,
especially of glutathione S-transferase;
(ii) drug transporters and efflux proteins ;
(iii) point mutations in proteins that are therapeutic or drug targets.

PEG liposomal doxorubicin (Doxil1) was tested on colon cancer (C26)

cells and their doxorubicin-resistant (MDR) subclone, which overexpresses
P-gp efflux pumps. PEG liposomal doxorubicin had anti-tumour effects on
both doxorubicin-resistant and non- doxorubicin-resistant C26 cells

PEGylated liposomes may be used to reduce clearance by the MPS,

increasing the circulation half-life.
Schematic diagram of three different approaches to engineer liposomenanoparticle hybrids.
Hydrophobic nanoparticles embedded in the lipid bilayer (left); hydrophilic nanoparticles
encapsulated in the aqueous core (right); and nanoparticles chemically conjugated or
physically adsorbed/complexed to the liposome surface (bottom
TMAG, N-(R-trimethylammonio-acetyl)-didodecyl-D-glutamate chloride; DLPC, dilauroylphosphatidylcholine; DOPE,
dioleoylphosphatidylethanolamine; SPC, soy phosphatidylcholine; PS, phosphatidylserine; DPPC, dipalmitoylphosphatidylcholine; DSPE-
PEG2000, distearoylphosphatidylethanolamine polyethylene glycol of MW 2000; EYPC, egg yolk phosphatidylcholine; NIR, near infrared;
DOTAP, 1,2-dioleoyl-3-trimethylammonium-propane; DOPC, dioleoylphosphocholine; DOPS, dioleoylphosphatidylserine; DHP,
dihexadecylphosphate; DODAB, dioctadecyldimethylammonium bromide; DODAC, dioctadecyldimethylammonium chloride; PC,
phosphatidylcholine; DMPC, dimyristoylphosphatidylcholine; Chol, cholesterol; DSPC, distearoylphosphatidylcholine; DPPG,
dipalmitoylphosphatidylglycerol; DC-Chol, 3 [N-(N,N-dimethylaminoethane)-carbamoyl] cholesterol; SOPC,1-stearoyl-2-
oleoylphosphatidylcholine; Y2O3,Er3, rare earth dopped ceramic; DDAB, dimethyl-dioctadecyl ammonium bromide; SPIO,
superparamagnetic iron oxides; TOPO, trioctylphosphine oxide; CdSe, cadmium selenide
Liposome Nanoparticle Hybrids for Theranostic
Applications
Liposome Viral Nanoparticle Hybrids:
Viruses are being explored as gene therapy vectors and in nanomedicine.
Adenovirus (Ad) and adeno-associated virus (AAV) are being extensively
studied.
Despite their high gene transfer and expression efficacy, Ad and AAV suffer
from a variety of issues that have precluded their widespread clinical
translation:
rapid blood clearance (requiring multiple administrations).
tissue toxicity (liver in the case of Ad).
activation of severe and complex immune responses.
Strategies to prolong their blood circulation and reduce their
immunogenicity and toxicity include,
genetic modifications of viral capsids
chemical conjugation of hydrophilic polymers eg PEG
an alternative approach :engineering a liposome virus hybrid
system
An alternative approach by engineering a
liposome virus hybrid system

The Ad virions were entirely encapsulated

within liposomes, resulting in construction of
artificial (lipid bilayer) viral envelopes by self-
assembly.
Viral particles could be enveloped with
cationic, zwitterionic, and PEGylated lipid
bilayer envelopes.
 Recombinant adenovirus (Ad) is a powerful tool in gene therapy. However, the ability to
deliver Ad by systemic administration is limited due to rapid clearance from blood
circulation, transfection of nontarget tissues, toxicity, and immunogenicity. To address
these limitations, an artificially enveloped Ad vector was prepared by self-assembly of lipid
bilayers around the Ad capsid. The physicochemical and structural features of the
enveloped Ad vector can be altered according to the type of lipid used without the need
for genetic modification or conjugation chemistry.

Dimyristoyl phosphatidylcholine (DMPC); cholesterol (Chol); DOTAP [(1,2-dioleoyloxypropyl)-N,N,N-

trimethylammonium chloride]; 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-
2000] (DSPE-PEG2000)
Artificial envelop of Ad decreases the immunogenicity and hepatic affinity of naked Ad in
vivo. Unfortunately, this also resulted in a significant reduction of gene expression,
attributed to poor endosomal release of the Ad from its artificial lipid envelope.
Artificial envelopment of Ad within pH-sensitive DOPE:CHEMS
bilayers.
The artificially enveloped viral vectors exhibited good stability at physiological
pH but immediately collapsed and released naked Ad virions at pH 5.5
Artificial envelopment of Ad within pH-sensitive DOPE:CHEMS
bilayers.
The artificially enveloped viral vectors exhibited good stability at physiological
pH but immediately collapsed and released naked Ad virions at pH 5.5

Cationic lipid envelopes dramatically

reduced the transfection capability of
Ad in vitro (Figure 2E) due to their
failure to escape the endosomal
compartments following endocytosis
(Figure 2D, middle).
In contrast, PEGylated lipid envelopes
prolonged the Ad blood circulation and
reduced their liver gene expression and
toxicity following systemic
administration, allowing for passive
targeting to solid tumors
 These liposome virus hybrid systems showed similar levels of gene expression as those of
naked Ad (Figure 2E) in vitro.
Intracellular trafficking of fluorescently labeled Ad confirmed that both Ad and pH
sensitive enveloped Ad successfully escaped the endosomes and trafficked to the
perinuclear region, in contrast to Ad enveloped in cationic envelopes where clear
endosomal localization was observed (Figure 2D).
The high level of gene expression of pH-sensitive lipid enveloped Ad was also maintained
in vivo following intra-tumoral injection.
 This hybrid system has been designed with
potential theranostic capabilities, whereby the
encapsulated virion is the (gene) therapeutic
component and imaging probes can be
incorporated either on the viral capsid or on
the lipid bilayer wrapping it.
Liposome Quantum Dot Hybrids for Cancer
Theranostics

L-QD hybrids allows hydrophobic QD

nanoparticles to be used in aqueous (i.e.,
biological) environments, while their
incorporation in the acyl environment of the lipid
bilayer significantly enhanced QD optical stability
during storage and exposure to UV light.
 (D) Serum stability of L-QD-Dox hybrids incubated in 50% mouse serum. QD were embedded in
EPC:Chol:DSPE-PEG2000 and DSPC:Chol:DSPE-PEG2000 liposomes. Dox was loaded using the pH-gradient
technique and Dox release was assessed by measuring Dox fluorescence. (E)
Cytotoxicity of L-QD-Dox hybrids. MCF-7 cells were incubated with free Dox, L-QD-Dox, and L-QD hybrids,
and cell viability was assessed using MTT
assay

Dox-loaded L-QD (L-QD-Dox) hybrids were uptaken by cells and were able to release
Dox intracellularly, with significant enhancement in the cytotoxicity, almost to the
same level as the free drug (Figure 3E).
Solid lipid NPs
Solid lipid NPs, also referred to as lipospheres or solid lipid nanospheres,
are solid lipids at human physiological temperature and have a diameter
of 501000 nm.
They can be formed from a range of lipids, including mono-, di- and
triglycerides, fatty acids, waxes and combinations thereof.
produced by using solid lipid in an oil-in-water emulsion
stabilized by surfactants
are biodegradable and biocompatible; low toxicity.

SLNs form a strongly lipophilic matrix into which drugs can be loaded for
subsequent release.
The principal factors affecting drug loading into the SLN matrix are:
(i) the solubility of the drug in lipid;
(ii) the chemical and physical properties of the lipid or mixture of lipids;
(iii) the crystalline characteristics of the lipid(s) at biological temperature;
(iv) the polymorphic form of the lipid(s) used.

Use of a heterogeneous lipid mixture promotes an imperfect crystalline

structure with larger gaps for superior drug loading
 SLNs have been investigated for the delivery of various
anticancer drugs, with promising results in preclinical mouse
trials specifically showing that SLNs might help to overcome
MDR in cancers

Benefits of SLNs in doxorubicin delivery.

The cytotoxicity of free doxorubicin,
doxorubicin-loaded SLNs and unloaded
SLNs at different concentrations towards
HT-29 colorectal cancer cells after 72-h
exposure is shown. Doxorubicin-loaded
SLNs showed the highest toxicity, offering
more potent treatment than conventional
free doxorubicin. Unloaded SLNs did not
induce any significant toxicity, which
confirms that they are a safe carrier in
vitro.
Methods of liposome preparation

All the methods of preparing the liposomes

involve four basic stages:
1. Drying down lipids from organic solvent.
2. Dispersing the lipid in aqueous media.
3. Purifying the resultant liposome.
4. Analyzing the final product.
Method of liposome preparation and drug
loading
The following methods are used for the
preparation of liposome:
1. Passive loading techniques
2. Active loading technique.

Passive loading techniques include three different

methods:
1. Mechanical dispersion method.
2. Solvent dispersion method.
3. Detergent removal method (removal of
nonencapsulated material
Mechanical dispersion method

The following are types of mechanical dispersion

methods:
1.1. Sonication.
1.2. French pressure cell: extrusion.
1.3. Freeze-thawed liposomes.
1.4. Lipid film hydration by hand shaking, non-hand
shaking or freeze drying.
1.5. Micro-emulsification.
1.6. Membrane extrusion.
1.7. Dried reconstituted vesicles
1.1 Sonication
Probe sonication.
The tip of a sonicator is directly engrossed into the
liposome dispersion.
The energy input into lipid dispersion is very high
(heating)
Lipids can be de-esterified.
Metal from tip may slough off and pollute the solution.

Bath sonication.
Controlling the temperature of the lipid dispersion is
easier.
Material being sonicated can be protected in a sterile
vessel.
1.2 French pressure cell
French pressure cell involves the extrusion of MLV
through a small orifice.
Involves gentle handling of unstable materials,
proteins not significantly affected as in sonication.
The resulting liposomes are rather larger than
sonicated SUVs.
Disadvantage: working volumes are comparatively
small (about 50 mL as the maximum)
1.3 Freeze-thawed liposomes

SUVs are rapidly frozen and

thawed slowly. The short-lived sonication
disperses
aggregated materials to LUV. The creation of
unilamellar vesicles is as a result of the fusion of
SUV
throughout the processes of freezing and thawing
[26-28]. This type of synthesis is strongly
inhibited by
Advantages of nanomedicines
biodistribution can be controlled by the change of the
nanoparticle properties
stealth type nanoparticles are able to maintain an
effective blood concentration by escaping the removal
carried out by the reticuloendothelial system
high potency hydrophobic compounds can be
dispersed in water and delivered systemically by
nanoparticles
nanoparticles can enhance the level of signal detection
for the more sensitive detection of disease.
Strategies of lipid-based theranostic
nanomedicines for cancer
Lipid-Based Nanomedicines for
Cancer
The liposome is the oldest and the most
common type of lipid-based nanostructure
used for biomedical applications.
Advantage:
Can contain hydrophobic drugs at the lipid bilayer
hydrophilic drugs inside the lipid bilayer
cationic liposomes electrostatically bind anionic
nucleic acids to their surface
A Potent Expression System for the Enhancement of Liposome-
Mediated Gene Transduction
(A) Plasmid DNAs. (B) Diagram of cationic
multilamellar vesicle (MLV) liposomes
containing plasmid DNA and the HMG-1,2
protein. (C) Optimization of the liposome dose
for the maximum transfection activity. Mice
were intraperitoneally administered liposomes
on day 21, including the luciferase-expressing
pcagluc, and the luciferase activity in tumor
tissues was examined on day 23. (D)
Monitoring of the reporter gene expression in
each tissue. Mice were administered the
liposomes (3.8 107 mol) containing
pcagluc. (E) Treatment in a metastatic breast
cancer model. Mice were intraperitoneally
given INF- (4000 U: white arrows) and the
liposomes (3.8 107 mol) containing
pcagTNF- (blue arrows). (C-E) MCF-7 cells
(7.5 106) were intraperitoneally inoculated
into the mice on day 0. Mice were
intraperitoneally given liposomes constructed
with a cationic lipid (10 mol) including the
plasmid DNA (300 g) and the HMG-1,2
protein (96 g).