Drug delivery and drug targeting with parenteral lipid nanoemulsions — A
review

Karl Hörmann, Andreas Zimmer

PII: S0168-3659(15)30268-6
DOI: doi: 10.1016/j.jconrel.2015.12.016
Reference: COREL 8015

To appear in: Journal of Controlled Release

Received date: 16 November 2015

Accepted date: 12 December 2015

Please cite this article as: Karl Hörmann, Andreas Zimmer, Drug delivery and drug
targeting with parenteral lipid nanoemulsions — A review, Journal of Controlled Release
(2015), doi: 10.1016/j.jconrel.2015.12.016

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 ACCEPTED MANUSCRIPT

Drug delivery and drug targeting with parenteral lipid

nanoemulsions – a review
Karl Hörmann1 and Andreas Zimmer2*

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1. Fresenius Kabi Austria GmbH, Hafnerstraße 36, A-8055 Graz, Austria
2. Karl-Franzens-University of Graz, Institute of Pharmaceutical Sciences, Department of
Pharmaceutical Technology, Universitätsplatz 1, A-8010 Graz, Austria
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* Corresponding Author: Univ.-Prof. Dr. Andreas Zimmer, Ph.D. andreas.zimmer@uni-graz.at

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Table of contents

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1 Abstract ........................................................................................................................................... 3

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2 Introduction ..................................................................................................................................... 3
3 Classification of parenteral nanoemulsions .................................................................................... 3

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4 Nanoemulsions for nutrition ........................................................................................................... 4

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4.1 Biological fate of nanoemulsions ............................................................................................ 6
4.2 Modification of the pharmacological profile of drugs owing to treatment with lipid

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nanoemulsions .................................................................................................................................... 8
5 Nanoemulsions as matrix for lipophilic APIs (drug delivery systems) ............................................. 8
5.1 Targeting liver and MPS (mononuclear phagocyte systems) ................................................ 11
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5.2 Targets other than the liver................................................................................................... 11
5.3 Droplet size ............................................................................................................................ 14
6 Nanoemulsion with cationic surface charge ................................................................................. 15
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7 NE with PEGylation on droplet surface ......................................................................................... 16

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7.1 Opsonization and Phagocytosis ............................................................................................. 19

8 NE with PEGylation on droplet surface with targeting ligand ....................................................... 21
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8.1 Peptide mediated targeting .................................................................................................. 21

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8.2 Antibody mediated targeting ................................................................................................ 24

8.3 Sugar ligands and albumin as targeting moieties.................................................................. 24
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9 The Concept of Differential Protein Adsorption ........................................................................... 25

10 Summary/Conclusion ................................................................................................................ 26
11 Bibliography............................................................................................................................... 28

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1 Abstract

Lipid nanosized emulsions or nanoemulsions (NE) are oil in water dispersions with an oil droplet size
of about 200 nm. This size of oil droplets dispersed in a continuous water phase is a prerequisite for
the parenteral, namely intravenous administration. Many parenteral nutrition and drug emulsions on

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the market confirm the safe use of NE over years.
Parenteral emulsions loaded with APIs (active pharmaceutical ingredients) are considered as drug

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delivery systems (DDS). DDS focuses on the regulation of the in vivo dynamics, such as absorption,
distribution, metabolism, and extended bioavailability, thereby improving the effectiveness and the

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safety of the drugs. Using an emulsion as a DDS, or through the use of surface diversification of the

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dispersed oil droplets of emulsions, a targeted increase of the API concentration in some parts of the
human body can be achieved. This review focuses on NE similar to the marketed once with no or only
low amount of additional surfactants beside the emulsifier from a manufacturing point of view

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(technique, used raw materials).

Keywords:
Emulsion, nanoemulsion, intravenous, parenteral, drug targeting, drug delivery system,
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manufacturing, materials, ingredients

2 Introduction
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The aim of new research in this field is to achieve an improved drug delivery and drug targeting by
modifying existing and developing new emulsions. Intravenous fat, also known as oil or lipid,
emulsions have been in medical use for over 5 decades (e.g. Intralipid, approved in Europe in 1962).
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Parenteral nutrition emulsions supply critical ill patients, which cannot be fed orally, with
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triglycerides. These triglycerides are metabolized further to (essential) fatty acids in the human body.
The accepted regulatory status of the raw materials used for NEs and the safe metabolism is one of
the advantages of NEs. Another important advantage is the capability of dissolving not water soluble
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APIs. Many novel therapeutic agents have poor or no water solubility. Formulating these substances
as lipid NE can be a very smart way to overcome this hurdle and make the effect of these substances
possible only.
All NEs in this review are intended for intravenous administration just like the marketed products. As
an exception, Mendes et al. [1] reported an NE composed of approximately 47.8% phospholipids,
48.0% cholesteryl oleate, 1.9% free cholesterol and 2.3% triacylglycerols administered intralesionally
to patients with breast cancer 12 h prior to surgery directly into or near the tumor. Radioactive
labeling with 14C-cholesteryl oleate showed that peritumoral injection has the best concentration
effect. Further examples of intratumoral and intramuscular injections are given in the review on lipid
carrier systems by Hashida et al. [2].

3 Classification of parenteral nanoemulsions

This review focuses on o/w (oil in water) emulsions. There is also some current research ongoing
with non-aqueous and/or oil in oil emulsions; for example a study [3] used castor oil in silicone oil

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emulsions. This strategy can be used as a DDS for lipophilic and/or hydrolytically unstable drugs for
intramuscular injection. The release of the drug is substantially slowed down when using such a DDS.

For intravenous applications of emulsions, the dispersed oil droplets must be below the size of the
smallest blood vessels to avoid embolisms. The smallest blood vessels are present in the lung with

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diameters down to 5µm. This is in accordance with one of the parameters noted in the American

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Pharmacopeia, USP<729>, PFAT5. The percentage of fat droplets greater than 5 microns must not
exceed 0.05% of intravenous applied emulsions [4].

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The mean droplet size is, therefore, in the submicron area, about 200 – 400 nm and these emulsions
are categorized as nanoemulsions (NEs), as well as nanosized emulsions, submicron emulsions,

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miniemulsions, fine dispersed emulsions, etc. [5].
The energy required to produce a NE can be generated by a mechanical device (high-energy
emulsification) or from the chemical potential of the components, most usually surfactants (low-

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energy emulsification). There are a number of processes for high-energy emulsification, i.e. high-
pressure homogenization (HPH), ultrasonication and microfluidization. Though this review is focusing
on high-energy emulsification as used in the pharmaceutical industry for intravenous emulsions, the
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low-energy emulsification processes are phase inversion temperature (PIT), phase inversion
composition (PIC) and solvent diffusion [6].

During the microfluidization process the liquid is pumped through an interaction chamber at high
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pressure. The liquid in this chamber is then divided into two streams, which are brought together
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with ultrahigh velocities [7]. High-pressure homogenization, using a homogenizer from companies
such as GEA, APV, etc., transfer the liquid using high-pressure from plunger pumps through a small
gap. Droplet size is reduced by high-shear and cavitation forces more so then by high-speed collisions
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with other droplets which is the main force for microfluidization.

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Based on a classification of NEs from Tamilvanan et al. [8], parenteral NEs can also be classified as:

– NE for nutrition (phospholipid based)

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– NE as matrix for APIs (drug delivery systems)

– NE with cationic surface charge
– NE with PEGylation on droplet surface
– NE with PEGylation on droplet surface with targeting ligand

4 Nanoemulsions for nutrition

The first attempt to administer oil parenterally was at the end of the 17th century when English
naturalist William Courten administered olive oil intravenously to a dog (1 g olive oil/kg dog). The dog
went into severe respiratory distress and died, probably from lung emboli [9]. Injection of pure oil is
lethal.
After further efforts in the following hundreds of years, the first safe intravenous fat emulsion was
developed by Arvid Wretlind and approved in Europe in 1962, and it is still marketed as Intralipid
[10,11]. It is an emulsion used for parenteral nutrition and contains 10 or 20% soya bean oil, 1.2% egg
phospholipids as emulsifier/surfactant, 2.5% glycerin for tonicity and sodium hydroxide for pH
adjustment (no further ingredients).

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NEs for nutritional purposes have no active pharmaceutical ingredient (API). However, to understand
the NE system it is worth having a look at this first marketed NE. The first successfully developed
emulsions for parenteral/intravenous use were developed to meet the nutritional needs of critically
ill patients. Formerly, only glucose and amino acid solutions were available. These NEs provide the
patient with high caloric triglycerides and are an enormous benefit for patients who must be fed
parenterally.

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As regards oil sources, soybean oil is very often used due to its high content of essential C18 fatty
acids like linoleic acid, and therefore called long chain triglycerides (LCT). There are some concerns

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that a high content of ω-6 polyunsaturated fatty acids may lead to negative side effects in prolonged
administration, such as immunosuppressive actions (MPS impaired function) and inhibition of

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lymphocytes.
To overcome these possible negative side effects, NEs were developed with a 1:1 mixture of LCT and
medium chain triglycerides (MCT; from coconut oil). Other commercially marketed NEs use

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structured triglycerides as an oil source, in which long and medium chain triglycerides are bound to
the same glycerol backbone, olive oil and, fish oil. Fish oil is especially interesting owing to its high
content of ω-3 polyunsaturated fatty acids, i.e. EPA (eicosapentaenoic acid) and DHA
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(docosahexaenoic acid).

To stabilize the fine dispersed oil droplets in the continuous water phase an amphiphilic emulsifier,
which is located at the interface between oil and water, is required due to their lipophilic and
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hydrophilic molecule section.

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Most of the NEs currently on the market use egg yolk phospholipids as an emulsifier with the main
chemical substance being phosphatidylcholine. In a recent review on lecithin based NEs [12], the
peculiarities of lecithin as an emulsifier were presented. The seldom used soybean
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phosphatidylcholine is reported to be less prone to hydrolysis than egg yolk PC during shelf life [13].
The combination of egg yolk phospholipids and synthetic surfactants are often used in research to
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further stabilize critical NEs, e.g. pegylated phosphatidylethanolamine (PEG-PE), Pluronic® F68, etc.

For the adjustment of tonicity almost all marketed NEs use glycerin in a typical amount of 2.2 – 2.5 %
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to achieve an osmolality in the same range as that of blood - around 300 mOsm/kg.

Among the other ingredients of approved NEs are free fatty acids (oleic acid) as co-emulsifier,
stabilizers such as EDTA, and antioxidants such as α-tocopherol.

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Figure 1
Oil in water emulsion, stabilized through phospolipids on the surface with dissolved drug molecules
(red dots), adapted from [4].
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4.1 Biological fate of nanoemulsions

An interesting question is, where the “life” of oil droplets of intravenous emulsions ends and if this
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can be used for drug targeting. According to Rossi [14], oil droplets of an intravenous emulsion are
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cleared either by the mononuclear phagocyte systems (MPS; e.g. Kupffer cells of the liver and splenic
macrophages) or metabolized as endogenous chylomicrons. Chylomicrons (see
Figure 2) are about 75-1000 nm in size and metabolized in the blood stream, which results in a
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release of free fatty acids from the triglycerides. The remnants of the chylomicrons (30–50 nm) are
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cleared in the liver by low- density lipoprotein receptors.

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Figure 2

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Schematic chylomicron structure with ApoA, ApoB, ApoC, ApoE (apolipoproteins); T (triacylglycerol);
C (cholesterol); green (phospholipids), from [15].

There are many parameters that influence the biological fate of emulsions’ oil droplets in the human
body, Table 1 lists a number of these.

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Table 1
Parameters influencing the metabolism as lipoproteins, the capture by the mononuclear phagocyte

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system (MPS) and the elimination from the blood circulation of NEs after parenteral administration,
adapted from [16].

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Factors Metabolism as lipoprotein Capture by MPS Elimination from the
blood circulation
Poor Extensive Poor Extensive Slow Rapid
Particle size Large Small Small Large Small Large

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Emulsifier DPPC EYPC DPPC DSPC DPPC EYPC
DSPC - SM - SM DSPC
SM - - - - -
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Co- Poloxamers - Poloxamers - Poloxamers -
emulsifier HCO-60 - HCO-60 - HCO-60 -
PEG - PEG - PEG -
Polysorbates - Polysorbates - Polysorbates -
Solutol - Solutol - Solutol -
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- - - - Cholesterol -
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- - - - CO -
Cationic lipid SA/OA - SA/OA - SA/OA -
Oil phase LCT MCT - - LCT MCT
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- - - - SLS SLM
Opsonization - - Low Large Low Large
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Instability of - - - - Low Large

oil droplets
in blood
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stream
Overloading Large Small
of MPS
DPPC, dipalmitoylphosphatidylcholine; DSPC, distearoylphosphatidylcholine; SM, sphingomyelin; EYPC, egg yolk
phosphatidylcholine; HCO-60, polyoxyethylene-(60)-hydrogenated castor oil; PEG-PE,
phosphotidylethanolamine derivative with polyethylene glycol; CO, cholesteryl oleate; SA, stearylamine; OA,
oleylamine; LCT, long chain triglycerides; MCT, medium chain triglycerides; SLS, structured lipid with short
chain fatty acids; C8–C10; SLM, structured lipid with medium chain fatty acids, C4.

The impact of long chain ω-3 polyunsaturated fatty acids (PUFA) on the metabolism was studied by
Ton et al. [17]. Soybean oil (LCT), MCT/LCT/ω-3 (5:4:1, wt/wt/wt), and MCT/ω-3 (8:2, wt/wt)
emulsions were prepared and radio-labelled with 1α,2α (n)-3H cholesteryl oleoyl ether. In a mice in
vivo study they found the MCT/ω-3 (8:2, wt/wt) NE to be cleared more efficiently from blood and
that ω-3 PUFA seems to be directed more towards extrahepatic tissues.

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4.2 Modification of the pharmacological profile of drugs owing to

treatment with lipid nanoemulsions
There are some studies in which a marketed NE is used to alter the pharmacological profile of a drug
or diagnostic. Liu et al. [18] used a pretreatment with Intralipid to increase the blood half-time of
nano- and micron-sized superparamagnetic iron-oxide particles. The proposed mechanism behind

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this is, that due to the pre-treatment with a lipid NE, the MPS capacity for uptake is decreased, which
means that other drugs vehicles can benefit from a pre-treatment with a well-established and

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inexpensive NE.
Assink et al. [19] showed that after a life-threatening intoxication with verapamil, a calcium channel

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blocker, for which there is no antidote, a treatment with a lipid NE is beneficial.

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A further study, carried out by Fettiplace et al. [20], used bupivacaine as an intoxication model drug.
Therein, they reported on a proposed mechanism of detoxification via transient drug scavenging and
an additional cardiotonic effect of a lipid NE. This effect couples to accelerate movement of the toxin

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away from organs influenced by the toxin. In vivo and in silico models of toxicity were combined and
the contribution of the multi-modal activity, i.e. scavenging and cardiotonic actions, of lipid NEs was
tested.
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Another study with bupivacaine also underlined the enhanced elimination effect of lipid NE
administration and examined the localization of the drug after enhanced clearance by the NE [21]. In
a rat study they revealed that concentration of bupivacaine in the NE group was lower in heart,
brain, lung, kidney, and spleen compared to the control group, but higher in the liver.
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5 Nanoemulsions as matrix for lipophilic APIs (drug delivery

systems)
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The incorporation of lipophilic drugs in o/w emulsion is a smart way to overcome solubility problems.
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However, the commercial use of pharmaceutical drug NEs is limited to very few products; see the
non-exhaustive list below regarding the commercially available NE drug delivery systems.
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Table 2
Marketed Nanoemulsions as drug delivery systems, adapted from [22].

Drug Marketed name Manufacturer Market Indication

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Mitsubishi Tanabe Vasodilator, platelet

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Alprostadil palmitate Liple® Japan
Pharma inhibitor

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The Medicines Calcium channel
Clevidipine Cleviprex® US
Company blocker

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Dexamethasone- Mitsubishi Tanabe
Limethasone® Japan Rheumatoid arthritis
palmitate Pharma

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Diazepam Diazemuls® Actavis Nordic Europe etc. Sedative

Etomidate Etomidat-Lipuro® Braun Melsungen Europe Anesthetic

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Nonsteroidal
Flurbiprofen axetil Lipfen® Green Cross Japan
analgesic
Propofol Diprivan® Astra Zeneca Worldwide Anesthetic

Vitamins A, D, E, K Vitalipid® Fresenius Kabi Europe etc. Parenteral nutrition

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A great deal of research is ongoing in this field, mainly due to the fact that many of the newly
discovered drugs have a low solubility in water. The drug’s lipophilicity incorporated in a lipid NE is an
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important factor here for biodistribution. If the lipophilicity is low, the drug may be released quite
quickly from the oil droplet to the blood stream, where sink conditions exist due to the dilution of
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the administered drug in the blood stream and subsequently further metabolized. If the lipophilicity
is high, the drug may be retained in the oil droplet, so the oil droplet can function as a drug delivery
system or aid in drug targeting. Takino et al. reported, that the log P, used as measuring parameter
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for lipophilicity, should be > 9 to prevent fast release of the drug to blood serum [23]. However,
there are many other authors proposing other log P limits, both lower and higher. Nevertheless, a
good strategy for increasing circulation time and consequently changing the biodistribution of a drug
may be the increase of the drug’s lipophilicity by esterification with a fatty acid such as oleate,
palmitate, valerate, etc.
Ganta et al. [24] developed a NE with the cancer treatment drug chlorambucil and evaluated the
pharmacokinetics and anticancer activity in mice. The NE consists of soybean oil (10%w/v) as the lipid
phase, and egg phosphatidylcholine (1.8% w/v) and cholesterol (0.2% w/v) as the surfactant/co-
surfactant and 0.2% w/v chlorambucil. All these ingredients were dissolved in chloroform and formed
a lipid film after evaporation of the chloroform. After addition of the aqueous phase containing
glycerol (2.21%) for tonicity the emulsion was formed using high-energy ultrasonication. The NE
produced in the aforementioned manner, with a particle size of 182.7 ± 0.8 nm, was administered
intravenously to C57 BL/6 male mice. The NE loaded with chlorambucil showed improved
pharmacokinetics compared to chlorambucil solution, e.g. AUC was roughly doubled (32.4 ± 0.1
µg/ml h vs. 16.9 ± 0.1 µg/ml h) and growth suppression rate of the colon-38 adenocarcinoma in the
mouse was significantly greater.

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Commercial docetaxel injections contain ethanol and Tween 80, whereas the latter is known to cause
hypersensitivity. An alternative formulation of docetaxel in a NE [25] was reported to have the same
anticancer efficiency while showing less toxicity. MCT oil containing 3% (w/v) oleic acid was used as a
lipid phase to dissolve docetaxel. The aqueous phase consisted of glycerin 2.5% (w/v) and the
surfactants lecithin and poloxamer 188 at 1.3% (w/v) each. Both phases were heated, mixed
thoroughly and high-pressure homogenization, resulting in a NE with a mean droplet size of 169 nm.

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An example of overcoming poor water solubility while also enhancing chemical stability is reported
by Zhao et al. [26]. In this study, Cheliensisin A, a novel anticancer drug derived from a natural

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source, was formulated in a lipid NE. Interestingly, to overcome any stability issues, the lipid
emulsion was lyophilized after filtration through a 0.8 µm filter and finally gamma sterilized. As a lipid

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phase 200.0 g medium chain triglycerides were chosen, in which 2.0 g Cheliensisin A, 40.0 g soybean
phospholipids and 0.3 g vitamin E were dissolved. The lipid phase was mixed with 600 ml double
distilled water by a high-shear mixer, followed by high-pressure homogenization. After final dilution

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to 1000 ml with double distilled water the cyroprotection agent sucrose was added (10% w/w).
Due to the formulation as a NE, the IC50 (50% inhibitory concentration) decreased to at least one
third for several cancer cell types, most cell types showed a remarkably lower decrease. The in vivo
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activity was studied in pulmonary metastasis of colon cancer-bearing BALB/c mice model, showing
significantly increased life span and anti-tumor activity compared with a Cheliensisin A injection
solution consisting of cremophor:ethanol:phosphate-buffered saline PBS (1:1:5, v/v/v).
Clarithromycin is a semisynthetic antibacterial drug with low solubility in long chain triglycerides like
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soybean oil and MCT oil. Tocopherol or its derivatives (tocol emulsions) can be used as another oil
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source. In the formulation optimization study of Li et al. [27] tocopherol succinate is used to
formulate clarithromycin in a less painful injectable NE. This was confirmed with a rabbit ear vein
irritation test. The NE was composed of tocopherol succinate, soybean oil, MCT oil and oleic acid in
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the oil phase, whereas oleic acid formed a lipophilic ionpair with clarithromycin and increased the
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drug loading of the NE. In the water phase glycerin was included for tonicity, poloxamer 188 as a
non-ionic surfactant for additional stability and NaOH for pH adjustment. Both phases were mixed
using an Ultraturrax, followed by high-pressure homogenization.
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The non-steroidal anti-inflammatory drug (NSAID) Diclofenac as a water based solution has a short
half-time of 1 – 2 hours, which results in a number of injections per day in the case of, for example,
the treatment of arthritic conditions. The formulation of diclofenac as an acid in lieu of the sodium
salt in a lipid NE can be a solution to overcoming pain at the injection site and also reduced dosing.
Ramreddy et al. [28] developed a 200 – 250 nm NE composed of 100 mg soya bean oil, 12 mg egg
lecithin, 2.5 mg diclofenac acid, α-tocopherol acetate 2 mg, cholesterol 0 to 3 mg, oleic acid 2.5 mg
or, alternatively, stearyl amine 3 mg (to form negatively or positively charged NEs), 22.5 mg glycerin
in double distilled water up to 1 ml. The oil phase and the water phase, including glycerin, were
mixed at 70°C and then sonicated. Incorporated diclofenac acid NEs improved the pharmacokinetics
in rats owing to an increase in AUC, elimination half-life and MRT in comparison to the commercial
diclofenac solution Voveran®. The use of cholesterol should form a more rigid surface of the oil
droplet than when egg lecithin is used alone. However, the diclofenac levels in blood serum were
lower for cholesterol containing NEs, which may be attributed to enhanced uptake of oil droplets
through LDL receptors in tissues like liver, kidney, brain, spleen, etc. Positively charged NEs showed
lower blood serum levels, also when compared with the plain NE without cholesterol and stearyl
amine. This may be explained by an increased uptake of positively charged NE in negatively charged

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cell membranes.
A poorly water-soluble anti-cancer substance isolated from the traditional Chinese herb magnolia,
Honokiol, was studied as both a solution and an NE [29]. The NE was prepared with high-pressure
homogenization and consisted of soybean lecithin, Synperonic F68 and glycerol as the water phase
and soybean oil with dissolved Honokiol as the lipid phase. Here again, the half-life of the 187 nm NE
was substantially longer than that of the solution (8.0 min. versus 4.3 min.), AUC was greater and

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plasma clearance reduced. The tissue distribution results in tumor-burdened mice indicated that the

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NE has better targeting properties for lung, brain and tumor tissues than a Honokiol solution.

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5.1 Targeting liver and MPS (mononuclear phagocyte systems)
Through incorporation of lipophilic drugs in oil droplets of an o/w emulsion the distribution of the
drugs in the human body can be influenced. Using emulsions as drug delivery systems can increase

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the concentration of the drug in liver and/or MPS enormously.
A study with primaquine [30], a drug used to treat various stages of parasitic malaria, showed that
incorporation of the drug in an emulsion, versus the solution, enhanced the liver uptake by a factor
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of 2.5. The chylomicron-like emulsion was prepared from olive oil, phosphatidylcholine (chicken
eggs), lyso-phosphatidylcholine, cholesterol, and cholesteryl oleate by thin lipid layer hydration,
followed by vortexing and sonication. The API primaquine bisphosphate was complexed with Sodium
lauryl sulfate and egg phosphatidic acid respectively and co-dissolved with the lipid fraction of the
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emulsion.
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A new 2-(allylthio)pyrazine (2-AP)-loaded NE, a drug which has hepatoprotective effects against
toxicants, was developed by Jang et al. [31], varying oil to drug ratio, oil to lecithin ratio and tested
some additional co-surfactants. The resulting NE consisted of 2-AP (1%), soybean oil (4 %), soybean
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lecithin (4%) and 91% water and was found to be isotonic. In comparison with a 2-AP solution, the 2-
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AP emulsion showed significantly faster clearance of the blood stream and higher accumulation in
the organs, especially the liver (about double the amount compared to solution).

An NE loaded with SQ641, which is the most potent analogue of capuramycin antibiotics and has a
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bactericidal effect against mycobacterium tuberculosis, was reported to target lung and spleen in a
tuberculosis mouse model [32]. Interestingly, SQ641 itself was not active in vivo which may be
caused by its low water solubility. The NE was made of phospholipid Phosal 53 MCT (Lipoid GmbH,
Ludwigshafen, Germany) using α-tocopheryl polyethylene glycol 1000 succinate (TPGS) as surfactant.

5.2 Targets other than the liver

NEs could also be used as drug delivery systems to target tissues in the body other than lung and
MPS. A cancer treatment was proposed by Alayoubi et.al., using tocotrienol rich fraction (TRF) of
vitamin E [33] and TRF and simvastatin [34]. In a later study, a viscous 70/30 blend of TRF and
medium chain triglycerides (MCT, Miglyol 812) was used as a lipid phase of the NE, in which
simvastatin was dissolved at 9% w/w loading in some formulations. Phospolipids (Lipoid E80S,
composed of 64–79% phosphatidylcholine and 12–18% phosphatidylethanolamine), Tween 80 and
poloxamer 188 were used as surfactants. The approximately 200 nm sized NE was prepared by high-
pressure homogenization and had a surface potential of −45 mV. The anticancer activity of these NEs
was tested on human mammary tumor cells. The IC50 of NE against the human mammary tumor
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cells MCF-7 and MDA-MB-231 with only TRF was 14 and 7 μM. The IC50 decreased to 10 μM and
4.8 μM for those NEs containing additional simvastatin.

As an alternative to inhalation, an NE as drug delivery system can be used for isoflurane, a

halogenated ether administered for general anesthesia [35]. The NE was produced with the high-
pressure homogenization technique and had a droplet size of 150±0.78 nm. The lipid phase was

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formed by soybean lecithin S75 (1 % w/v) in 15 ml MCT oil, isoflurane was added just before

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homogenization with the water phase. The water phase consisted of polysorbate 80 (2 % w/v),
sorbitol (2 % v/v) and 64.5 mL water.

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Within preclinical evaluation, the isoflurane emulsion showed significantly decreased necessary
doses compared to inhaled isoflurane at the same levels of clinical biomarkers. However, minor

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adverse effects like tachypnea, edema, and erythema were recognized when isoflurane-loaded
nanoemulsions were administered.
Chen et al.[36] prepared a disulfiram-loaded lipid emulsion in his study. Incorporating the cancer

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drug disulfiram in a lipid emulsion seems to minimize the instability of the drug in blood. The
optimization of the NE formulation finally ended in the following composition resulting in better
stability during autoclaving and storage under heat stress: 10% (w/v) MCT oil, 0.6% (w/v) soybean
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phosphatidylcholine S100, 0.6% (w/v) Pluronic F68, 0.2% (w/v) oleic acid, 2.25% (w/v) glycerin, and
0.5% (w/v) disulfiram. Interestingly, in this study CO2 and a buffer were used to maintain a stable pH
value at the optimal range of 4.5 to 5, whereas in most NEs on the market sodium hydroxide is used
to adjust the pH. The emulsion was prepared using a high-shear mixer and high-pressure
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homogenization.
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Tumor cells grow rapidly and, consequently, have a great need for cholesterol to build new cell
membranes. Natural carriers of cholesterol-esters in the blood are low-density and high-density
lipoproteins (LDL and HDL). Shawer et al. [37] built on the known theory of an increased number of
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LDL-receptors for the development of a NE drug delivery system. Cholesteryl ester of carborane,
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cholesteryl 1,12-dicarba-closo-dodecaborane-1-carboxylate (BCH) was incorporated as drug in the

NE, which can be used for boron neutron capture therapy of cancers. The NE was produced as
follows: all lipid fractions were dissolved in chloroform and mixed together in a ratio (w/w) of:
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triolein : egg phosphatidylcholine : lysophosphatidylcholine : cholesterol oleate : cholesterol : BCH,

70 : 22.7 : 2.3 : 3.0 : 2.0 : 2.0, respectively. After removal of the solvent, the water phase was added
and the mixture was sonicated to obtain the final NE. In in-vitro studies, the interaction of this
developed NE with human lipoprotein was shown by electrophoresis. Additionally, a sufficient
uptake of BCH for cancer therapy was achieved, or at minimum an absorption on rat 9L glioma cells.
Another example of the reduction of toxic effect by incorporating a drug into a NE is described by
[38]. In this study, the cancer treatment substance N-oleyl-daunorubicin was formulated in a
cholesterol-rich NE that binds to low-density lipoprotein receptors. The maximum tolerated dose was
found to be 65-fold higher than commercial Daunorubicin; moreover, the anti-cancer effects, namely
tumor growth inhibition and survival rates, were better. This is also a further example of increasing
the lipophilicity of a molecule by addition of a fatty acid ester. Oleyl is derived from oleic acid
C18H34O2, which is classified as a monounsaturated omega-9 fatty acid, abbreviated with a lipid
number of 18:1 cis-9.
A cholesterol rich NE comprising paclitaxel oleate was used for the treatment of atherosclerosis in
rabbits [39]. In this study some of the rabbits were fed a 1% cholesterol diet, which caused
atherosclerotic lesions. In the aortic arch of cholesterol-fed rabbits, the uptake of radioactive labelled

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NE with 14C-cholesteryl oleate was double that observed in animals fed only regular feed. The
treatment with NE with paclitaxel oleate reduced the lesion areas of cholesterol-fed animals by 60%
and inhibited smooth muscle cell proliferation, without showing toxic effects. Consequently, this
drug delivery system appears to be a promising cardiovascular therapy approach minus toxicity
effects.
The same NE was also used in a study in eight patients with gynecologic cancers [40]. The NE was

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prepared from 40 mg cholesteryl oleate, 20 mg egg phosphaditylcholine, 1 mg triolein and 0.5 mg

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cholesterol as the lipid phase, which was sonificated in water and homogenized in a two-step process
with KBr to obtain a cholesterol rich NE. Interestingly, paclitaxel oleate was added to the finished NE

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as an ethanolic solution. After sonification the liquid was centrifuged to remove unbound paclitaxel
oleate. A double labeling was performed using 3H- paclitaxel oleate and 14C-cholesteryl ester.

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Comparing blood decay curves of both labels, which are in principle identical, showed that the drug
paclitaxel oleate stays within the NE drug delivery system until the end. Further, an almost identical

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increase of concentration in the cancer tissue of 3.6 times (compared with commercial paclitaxel)
and 3.5 times for the cholesterol rich NE confirm that the whole drug delivery system is incorporated
by cancer cells, supported by overexpression of low-density lipoprotein receptors which internalize
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cholesterol containing NE oil droplets.
A new NE, incorporating the anti-epileptic drug carbamazepine for a drug delivery to the brain, was
developed by [41]. 1-O-alkylglycerol/lecithin blends were used as emulsifiers, e.g. 0.25% w/w soy
lecithin and 1.25 %w/w 1-O-decylglycerol. Soybean oil (10% w/w) as the lipid phase dissolved the
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emulsifier and 0.1% w/w carbamazepine. After adding the water phase containing glycerol (2.5
%w/w), the emulsion was formed by high-pressure homogenization. In a tissue distribution study in
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mice showed a significant uptake in all tissues for all NEs tested. 1-O-alkylglycerols are reported to
improve the brain delivery, which was best shown using the NE containing 1-O-decylglycerol, which
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increased the brain availability 2.37 times. However, the tissue ranking was still lung > liver > spleen >
brain > kidney > heart.
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A study which also targeted the brain was carried out by Wen et al. [42], which compared solid lipid
nanoparticles (SLN), nanostructured lipid carriers (NLC), and NEs. These three categories where
produced by using 100% cetyl palmitate, different ratios of cetyl palmitate and squalene and 100%
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squalene as the lipid phase, respectively. To obtain a positive charge for all three categories of
nanoparticles, Forestall, a cationic surfactant, was used. As a dye, sulforhodamine B was
incorporated for imaging. An in vivo study in rats investigating the accumulation of the dye in the
brain revealed that the retention time was most prolonged, from 20 to 50 min, using NEs. It seems
that due to the flexibility of NE’s oil droplets, compared to more rigid nanostructured lipid carriers
and solid lipid nanoparticles, the uptake into the brain overcoming the brain blood barrier is
enhanced.
A breviscapine, also known as scutellarin, a substance from a traditional Chinese herb, lipid emulsion
was evaluated in an in vivo pharmacokinetics study in rats [43]. The lipid phase consisted of soybean
oil (10.0%), oleic acid (0.9%), lecithin (1.5%), and poloxamer 188 (0.4%). The latter two were used as
surfactants. The water phase included glycerol (2.5%). In comparison to the commercial product,
breviscapine injection, the NE has about 1.5 fold higher AUC. In a further study dealing with
breviscapine NE, the AUC could be increased more than 14 times [44]. The main exposure of
breviscapine was found in blood plasma and lungs.
In the study carried out by Zhang et al. [45], a lipid NE with the anticancer drug Doxorubicin was
developed with a diameter of approx. 200 nm, where focus was put on the utilization of raw

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materials which are already approved for intravenous use and a production process which is easy to
scale-up (high-pressure homogenization). To improve the poor lipophilicity of Doxorubicin (logP
−1.32 for the free base) an ionic complex was formed with oleic acid (DOX-OA, logP of 1.81), which
led to an increase of entrapment efficacy from about 30% (DOX alone) to over 90% for DOX-OA. This
complex (300mg DOX-OA) was dissolved in 200ml ethanol containing 600mg lipoid E80, 50mg
vitamin E, and 400mg soybean oil. The solvent was eliminated with a rotary evaporator resulting in a

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thin lipid layer. With 50 ml water this lipid film was rehydrated and vortexed rigorously. After high-

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pressure homogenization the DOX-OA NE was obtained. A pharmacokinetic study was conducted in
mice showing significantly higher AUC and circulation time in blood than DOX solution and a

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positively altered tissue distribution. The DOX concentration in the heart was found to be lower than

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for the DOX solution. The dosage of DOX is limited due to its known cardiotoxic side effects.
Interestingly, the release pattern of the DOX-OA NE was biphasic with a “burst” release in the
beginning and a sustained release until end. This may be explained by DOX-OA, which is located in

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the oil droplet core but also at the surface within the egg phospholipids layer. Compared to literature
data of PEGylated liposome the DOX-OA NE showed shorter circulation time. This may be related to
the above-mentioned “burst” release portion and the absence of stealth agents like PEG and
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poloxamers.

5.3 Droplet size

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Figure 3
Schematic illustration of the enhanced permeation and retention effect (EPR), from [46].

Seki et al. [47] reported on artificial lipoprotein-like particles, lipid nano-sphere (LNS®). These LNS are
25–50 nm in diameter. They are composed of soybean oil and egg lecithin like commercially available
emulsions for parenteral nutrition (e.g. Intralipid). The soybean oil and egg lecithin ratio differs as
follows, for LNS the ratio is 1:1, whereas in commercial emulsions the ratio is 1:0.12. Additionally, the
high-pressure homogenization for this LNS is done at 1000 bar, whereas the NE used for comparison
is carried out at 500 bar. It seems that this is the reason for the enormous size reduction of 200 – 300
nm of commercial emulsions to 25- 50 nm of LNS.

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The biological destination of oil droplets from parenteral nutrition emulsion with diameters of 200 –
300 nm is, as mentioned above, the MPS and liver. Further, these droplets are cleared from blood
circulation quite rapidly. LNS, on the other hand, showed much higher levels in the blood (AUC three
times higher than NEs with 200-300 nm) and much lower levels in the liver and spleen. Some other
studies describe larger particles or oil droplets being eliminated from circulation by macrophages in a
more pronounced way than smaller particles.

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For example, Qi et al. [48] studied lipid emulsions made of different oils (LCT, LCT/MCT, LCT/MCT/fish

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oil, fish oil) and saw a significant difference (p < 0.01) between large- and small-sized emulsions
containing the same oil mixture. Unfortunately, the exact sizes were not measured; they were

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compared with gel chromatography. The difference in droplet size was achieved by oil to egg yolk

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phosphatidlycholine weight ratio of 4:1 for the large-sized emulsions and 1:1 for the small-sized
emulsions. As the surface of the oil droplets in both formulations is covered by egg lecithin
molecules, the change in biodistribution here is only influenced by the size of the oil droplets.

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An interesting in vitro and in vivo study about the influence of the droplet size on the anti-allergic
and anti-inflammatory activities of a curcumin NE was conducted by Onodera et. al. [49]. The tested
curcumin NE was prepared by a thin-film hydration method. Soybean oil and HEPC were dissolved in
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the organic solvent chloroform, as curcumin and Tween-80 separately. All three solutions were
mixed and dried afterwards by rotary evaporation and vacuum desiccation. The so obtained dry thin
film was hydrated with distilled water and sonicated at 25 - 55°C till the desired oil droplet size of 50
nm, 100 nm and 200 nm was obtained. Remarkable, both in vivo tests in orally fed mice and in vitro
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cell culture tests revealed the advantageous properties of 100 nm curcumin NE despite curcumin NE
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with 50 nm or 200 nm.

For drug delivery systems, an increased circulation time is often required to develop their desired
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capabilities, e.g. to store an API within the lipid core for a certain time period and maintenance of a
(constant) release from the oil droplet to the blood and surrounding tissue. Or, for the altered
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biodistribution, especially when the MPS like liver or spleen is not the primary target, there is some
time needed to achieve an accumulation effect in the desired parts of the body. Particle and/or oil
droplet size is an important factor; additional influencing parameters are listed in Table 1.
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6 Nanoemulsion with cationic surface charge

Cationic surface charges on nanoparticles, in general, are supposed to support the transfer of such
nanoparticles through negatively charged bio-membranes. However, in the last few years, research
with these cationic NEs has diminished. Bruxel et al. [50] investigated cationic NEs as oligonucleotide
delivery systems for the treatment of Plasmodium falciparum topoisomerase II (Malaria).
Oligonucleotide alone show increased degradation in the body and their ability to enter infected red
blood cells is very low due to their high molecular weight and hydrophilicity.
The NE composed of 8 % MCT oil, 2 % egg lecithin, 2.25 % glycerol, and water to 100 % was produced
by spontaneous emulsification. A clear ethanolic solution of the lipid phase was slowly added to the
water phase. After removal of the solvent ethanol under 50 °C with reduced pressure the emulsion
was formed. The positive surface charge of the NE was realized by adding 0.132 % 1,2-dioleoyl-3-

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trimethylammonium-propane (DOTAP). Different +/− charge ratios were adjusted by adding different
amounts of negatively charged oligonucleotide, which formed together with the positively charged
NE ion pairs. Complexes with a high charge ration (+6/-1) and/or the cationic NE alone caused
increased hemolysis. This is attributed to the presence of more cationic charges, which can interact
with the red blood cells. Parasite growth and reinfection capacity was evaluated in vitro, whereas the
best results were obtained with the highest charge ration (+4/-1), which showed no increased

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hemolysis.

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7 NE with PEGylation on droplet surface

To achieve targets in the human body other than MPS or liver with NE, an extension of blood

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circulation time is required. This also means that clearance rate is decreased, which may prevent
toxic effects due to less accumulation in the clearance tissues, too. As regards important mechanisms
to increase the half-time blood circulation time of NEs the enhancement of hydrophilicity is due to:
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- polyoxyethylene (POE)
- or polyethylene glycol (PEG) chains on the surface.
The surface can be modified by adding an emulsifier with such POE or PEG chains in the
manufacturing process. Alternatively, the surface of an existing NE can be modified by the simple
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addition of other surfactants or stabilizers. After an incubation time (24 h under gentle mixing is
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often used) the additional surfactant is admixed to the existing NE. The addition or substitution of
surfactant molecules follows the Nernst equation. According to Nernst, partitioning coefficients
between the water phase and the stabilizer layer of the NE of all surfactants in the system forms the
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surface of the NE [51]. This elegant method is used e.g. for incorporating PEG2000-DSPE into an
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existing NE.

POEs are often used in the form of triblock-copolymeres, marketed as Poloxamers or Pluronics. In
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Table 3 commonly used surfactants from this group are listed with their most important
characteristics and main application areas. According to the schematic structure below, it is obvious
that the inner, hydrophobic part will be located at the surface of the oil core, whereas both
hydrophilic POE chains will orientate towards the continuous water phase. By the thus increased
hydrophilicity, in comparison to the most used EYP of the oil droplet, the elimination rate of the NE is
decreased. A recent review on triblock-copolymeres and their interface-interactions was provided by
Torcello-Gómez et al. [52].

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Table 3

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Physicochemical characteristics of polyoxyethylene (Pluronics and Poloxamers), adapted from [52].

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CMC at POE POP
Pluronic Poloxamer MW HLB Administration (in vivo studies)
37°C units units

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-4
L61 P181 2000 1.1 * 10 2 30 3 Subcutaneous, oral

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-5 Intravenous, intraperitoneal,
P85 P235 4600 6.5 * 10 26 40 16
subcutaneous, ocular
-4 Parenteral, ocular, percutaneous, topical,
F68 P188 8400 4.8 * 10 75 29 29
oral, rectal

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Intravenous, intramuscular,
-6
F127 P407 12600 2.8 * 10 100 65 22 subcutaneous, percutaneous, oral,
ocular, buccal, rectal, topical
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Pluronic, tradename; Poloxamer, generic term; MW, molecular weight; CMC, critical micelle concentration in
mol/L; POE, polyoxyethylene; POP, polyoxypropylene; HLB, hydrophilic-lipophilic balance
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Of the abovementioned mechanisms the PEGylation is the most commonly used. PEGylation simply
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refers to the coverage of a particle or oil droplet surface by the covalently grafting, entrapping, or
adsorbing of PEG chains. The first PEGylated drug was developed in 1970 to make use of the
advantages of PEG: very good solubility in hydrophilic and hydrophobic phases, high hydration
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resulting in a high hydrodynamic volume, non-toxic and non-immunogenic, FDA approved material
for internal use, and available in different molecular weights with low polydispersity [53].

To be successful in prolonging the circulation time there are a number of points to consider. The
molecular weight of the PEG chain must be 2000 or greater, as indicated by most studies for
polymeric nanoparticles. Further, the PEG-chain density on the particle surface and the conformation
can contribute to the stealth effect. An optimal surface coverage is achieved when there are no gaps
in the PEG-chain coverage of the particle and the density is relatively low to allow the single PEG-
chains free movement. This leads to a “mushroom” configuration, where parts of the PEG-chain are
freely moveable, which is a low energy conformation. Attachment of opsonins would compress these
PEG-chains, reducing their freedom to move and increasing their energy conformation. This energy
change produces an opposing repulsive force, which can be higher than the attractive forces
between opsonin and particle. At higher PEG-chain density only a “brush” configuration is possible
due to limited space available between the PEG-chains [54].

The biodistribution of NEs coated with phosphatidylethanolamine derivatives with three different
molecular weight PEGs was studied by Liu and Liu [55]. Castor oil was used as the lipid phase,

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different mixtures of surfactant were added to the oil in weight ratio 1.5:0.6, whereas the 0.6
consists of egg yolk phosphatidylcholine to 100% or it was divided into 0.2 phosphatidylcholine and
0.4 PEG-PE with different chain lengths. The NEs, with about 200 nm average diameter, were
prepared by dissolving the lipids in chloroform. After removal of the solvent, the lipid film was
resuspended in PBS, pH 7.4, vortexed and subsequently homogenized with a Tissue-Tearor.
Among the tested molecular weights of PEG (1000, 2000 and 5000), the PEG-2000 was able to extend

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the circulation time of NEs best, which was accompanied by a decrease in liver accumulation. PEG-

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5000 was at the same level, whereas PEG-1000 was at the level of the NE with PC only. The most
likely reason for the prolonged circulation time is the increased hydrophilicity of the oil droplet

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surface. Further, the formation of a steric barrier may also contribute to this effect.

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Alayoubi et al. [56] investigated the effect of poloxamer or 1,2-distearoyl-sn-glycero-3-
phosphoethanolamine-N-[amino(polyethylene glycol)2000] (PEG2000-DSPE) surfaces of tocotrienol-
rich fraction’s NE. Though the PEG molecules amount was equimolar in both NEs, the different

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observed effects were found to be determined by different steric confirmation of the PEG chains.
PEG2000-DSPE has more dense brush confirmation than poloxamer and is believed to have more
repellent action against other hydrophobic surfaces, e.g. red blood cells and blood proteins like
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immunoglobulin G (IgG). Accordingly, this PEG2000-DSPE NE has a 7-fold increased half-life time after
i.v. injection in rats. Poloxamer NEs, however, showed higher uptake and lower IC50 against MCF-7
tumor cells in vitro.
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Desphande et al. [57] engineered an omega-3 polyunsaturated fatty acid (PUFA) emulsion
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incorporating 17β-estradiol and C6-ceramide for possible treatment of arterial restenosis. All three
substances were reported to have, in principle, a positive effect on restenosis. However, extensive
protein binding and their high lipophilicity hampered the substances in reaching their cellular targets.
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A NE tailored as a drug delivery system for omega-3 PUFA, 17β-estradiol and C6-ceramide, which
delivers the three substances specific to endothelial cells and arterial vascular smooth muscle cells,
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was achieved in this report. Flaxseed oil rich in omega-3 PUFAs was used as an oil phase, and egg
phosphatidylcholine (Lipoid 80, DOTAP and PEG2000-DSPE were added as surfactant. DOTAP, placed
on the surface of the NE contributes to the cationic charge of the whole vehicle, which should ease
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the transfer through the membrane of the endothelial cells. Also placed on the surface is PEG2000-
DSPE, which may help to prolong the circulation time in the vasculature. The NE was produced via
microfluidization technique.

Improving the chlorambucil NE in the previous study [24], Ganta et al. also decided to add PEG2000-
DSPE to the chlorambucil NE to enhance the circulation time in the body and, at the same time, the
pharmacokinetics and tissue distribution [58]. The chlorambucil NE with PEG2000-DSPE showed
improved pharmacokinetics when given intravenously to C57 B/6 mice compared to plain
chlorambucil NE or chlorambucil solution. For example, the AUC was 1.7-fold higher compared to NE
or 2.7-fold higher compared to the solution. The half-time was increased 1.3-fold compared to NE
and 7.6-fold compared to solution.
Paclitaxel, one of the most commonly used anti-cancer agents, was used in another study for
incorporation in an NE with a particle size of 50 nm [59]. The commercially available formulation
consists of Cremorphor EL and ethanol (50:50 % v/v) due to the low water solubility of paclitaxel. To
increase the solubility of paclitaxel in lipid emulsions, a prodrug, paclitaxel oleate was used. Besides
eliminating the toxic effects of the Cremorphor EL/ethanol mixture, the NE with paclitaxel oleate

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demonstrated significantly greater AUC, higher Cmax and lower Vss in a pharmacokinetic study in
rabbits. The NE was composed of 5.6 µmol triolein, 6.4 µmol egg phosphatidylcholine, 1.52 µmol
polysorbate 80 (poly-oxyethylenesorbitan monooleate), 0.36 µmol PEG-PE, and 120 nmol paclitaxel
oleate (or unesterified paclitaxel). All these ingredients were dispersed in a solvent and mixed
together. After evaporation of the solvent, the lipid fraction was lyophilized. PBS was added as the
water phase, the mixture was vortexed and then sonicated.

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In a newer study [60], Paclitaxel was used without derivatization into a prodrug. In a pharmacokinetic

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study in mice, same sized liposomes and NE (around 100 nm) were prepared. The emulsion consisted
of egg yolk phosphatidylcholine, Tween 80, Tricaprylin (C8) and Tricaproin (C6), and Paclitaxel in a

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weight ratio of 160 : 120 : 300 : 100 : 1.2. For the manufacturing of the PEGylated NE an additional

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PEG-DSPE was added to the mixture (5 mol%). The lipid phase was dissolved in chloroform and
mixed. The dried oil phase was mixed with the water phase including 2.25% (w/v) glycerol.
Afterwards, the mixture was sonificated. The outcome of this study was that the NE released the

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Paclitaxel in the blood circulation very rapidly, even using a PEGylated NE, while the liposome
formulation performed quite well. This poor performance of the NE may be contributed to the only
slightly lipophilic drug (log p 4.7), which can be related to a biofate more similar to water based
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solution and/or less probably the usage of triglyceride with relatively short chains (C6, C8) which may
also contribute to a faster clearance of the blood stream due to faster lipid metabolism.

An interesting approach using a two vial formulation of Paclitaxel (25 mg/mL) in PEG400 solution and
a commercially available 20% emulsion (LCT : MCT, 1 : 1, w/w) was investigated by Jing et al. [61].
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Prior to application, the vial containing Paclitaxel was diluted with the emulsion to obtain a 1 mg/mL
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Paclitaxel emulsion. In comparison to a conventional Paclitaxel-loaded NE [59], this two-vial

formulation showed a lower clearance by MPS and higher accumulation in tumors. Moreover, the
anti-tumor efficiency was increased in A2780 and Bcap-37 bearing mice. These advantageous effects
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of the two-vial formulation seem to be attributed to a greater amount of Paclitaxel in the

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phospholipid layer and a smaller proportion in the oil core than a conventional NE, where Paclitaxel
is dissolved in the oil core.
To overcome the extremely short half-life of lycobetaine in blood, an anti-cancer drug which shows
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significant cytotoxic activity against at least Lewis lung carcinoma, a lipid NE was developed [62]. A
lycobetaine ion pair complex with oleic acid (LBT-OA) was formed by co-precipitation method, a
strategy that has already been reported to increase the lipophilicity of a drug [45]. The NE was
prepared by a lipid film hydration, high-pressure homogenization method. LBT–OA, formed from 100
mg lycobetaine and 250 g oleic acid, was dissolved with 800 mg phospholipids (lipoid 80) and 500 µL
structured triglycerides in 200 mL dichlormethane. The solvent was removed by rotary evaporation.
The obtained lipid film was re-suspended in 50 ml water, followed by strong vortexing and high-
pressure homogenization. In contrast to the described LBT-OA-NE, the LBT-OA-PEG-NE was produced
in the same way, but using 760 mg lipoid E80 and 150 mg PEG2000-DSPE instead of 600 mg lipoid
E80.
The biodistribution and in vivo pharmacokinetics was studied in rats. A remarkable increase of AUC
could be achieved by LBT-OA-PEG-NE compared with LBT-OA-NE and lycobetaine solution (3452.09
mg/L*minute, 1208.16 mg/L*minute and 112.99 mg/L*minute, respectively). Further, LBT-OA-PEG-
NE achieved high concentration in the lung, whereas lower concentration was observed in heart,
liver and kidney.

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7.1 Opsonization and Phagocytosis

A critical characteristic of both drug delivery systems and drug targeting systems is the ability to
avoid the fast uptake through the MPS out of the blood stream. Opsonization and the consequent
clearance of the particles by the liver macrophages occurs very quickly, typically up to 90% of the
injected drug is taken up by the liver within 5 minutes [63]. Prior to the uptake of the MPS blood
proteins, known as opsonins (e.g., immunoglobulin γ, complement factors and fibrinogen), attach to

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the surface of foreign particles.

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Figure 4
Opsonization and phagocytosis of oil droplets, from [11].

If dysopsonins (e.g. serum albumin) attach to the particle surface, the MPS uptake is minimized and
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blood circulation time is consequently prolonged. Opsonization depends on a number of factors:

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 Size: Large particles are more quickly cleared from the blood stream by macrophages than
small particles.
 Charge: Clearance is highest for positively charged particles, lower for negative ones and
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lowest for non-charged particles, which means nanoparticles or nano oil droplets with a low
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zeta potential [51].

 Hydrophilicity: The blood serum proteins adhere easier on hydrophobic surfaces. A
hydrophilic surface, which is realized e.g. with PEG chains and poloxamer 188, suppresses the
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attachment of the opsonins on the surface [54].

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Figure 5
PEGylation avoids opsonization and can prolong circulation time in the blood, from [64].
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A successful drug targeting to the heart in an in vivo study in rats was shown by using TPGS (D-alpha-
tocopheryl polyethylene glycol 1000 succinate), a derivate of the natural vitamin E. In a study with a
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coenzyme Q10 loaded NE TPGS-NE showed better drug targeting properties to heart tissue
compared to a lecithin stabilized NE [65]. The NE was prepared with hot high-pressure
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homogenization, whereas 3 g coenzyme Q10 and 3 g TPGS or lecithin were dissolved in 3 g of

caprylic/capric triglyceride which together form the lipid phase. The lipid phase was added to 100 ml
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of a 45 % w/w solution of glycerol in water and mixed to form the pre-emulsion at 60°C. The NE was
homogenized at 1200 bar with 6 cycles, ending up in about 50 nm mean oil droplet size. The TPGS-NE
showed a 2.8 times higher concentration of the endogenous antioxidant coenzyme Q10 in the heart
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compared to lecithin stabilized NE five minutes after intravenous administration to rats. Higher
concentration levels were maintained after 90 min., in summary NE-TPGS had a 2.3-fold greater AUC.
The tissues distribution rank order was heart > liver > kidney.

8 NE with PEGylation on droplet surface with targeting ligand

To improve the specificity of drug targeting systems, many studies were carried out with a targeting
ligand on the surface of nanoparticles (“active targeting”). To the best knowledge of this author, only
these few studies of NEs with targeting ligands have been reported so far.

8.1 Peptide mediated targeting

Many different moieties can be used as targeting ligands. The advantage of peptides, in addition to
e.g. antibodies, is their size. Antibodies themselves are in the size range of nanoparticles (10 * 15
nm). In his review, Raha et al. [66] focused on peptides used for cancer targeting on nanoparticles in
general and gave a comprehensive overview.

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Jarzyna et al. [67] reported on NEs with three different mean diameters (30, 60, and 95 nm), which
had incorporated oleic acid coated iron oxide (Fe2O3) nanocyrstals for magnetic resonance imaging
(MRI) and a fluorescent dye (Cy5.5 coupled on PEG2000-DSPE) for near infrared (NIRF) imaging.
Soybean oil was used as a lipid core, Distearoyl-sn-glycero-3-phosphocholine (DSPC) was used as the
emulsifier and, additionally, PEG2000-DSPE and Cy5.5 coupled on PEG2000-DSPE were added for
surface modification. The NE was produced as follows: the lipophilic components were dissolved in

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chloroform. The mixture was added, dropwise, to the heated water phase. The resulting crude

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emulsion was homogenized by sonication. Within this in vivo study, the accumulation of this
multimodal imaging NE in subcutaneous human EW7 tumors in nude mice could be shown with MRI

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and NIRF imaging.

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Based on this work, Gianella et al. [68] reported a study using this platform to incorporate the cancer
treatment drug prednisolone acetate, which is provided as valerate ester to increase lipophilicity.
Further, the PEG2000-DSPE was coupled to αvβ3-specific RGD-peptides to enhance targeting

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angiogenesis of cancer tissue. RGD (arginylglycylaspartic acid) is a tripeptide and is composed of L-
arginine, glycine, and L-aspartic acid. This platform technology is considered as “theranostic” by
some. Theranostics are a combination of therapy and diagnostics using nanotechnology. To achieve
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this, imaging and therapeutic ingredients are combined in one nanoparticle such as oil droplets of
NEs. Interestingly, the addition of further material or functionalities does not result in a change in
diameter (all around 50 nm) or polydispersity compared to the blank NE. This small size facilitates
long circulation time in the blood as well as the uptake in cancer cells. MRI and NIRF imaging
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revealed significant accumulation in different colon cancer mouse models. The RGD ligand provided
specific interaction with ανβ3 integrin expressing cell types. Mice treated with prednisolone acetate
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valerate emulsions, RGD-prednisolone acetate valerate emulsion and prednisolone acetate valerate
+FeO emulsion, showed a significantly inhibited tumor growth compared to blank NEs, FeO
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emulsions, saline and free prednisolone acetate valerate (all at a dose of 10 mg prednisolone acetate
valerate per kg).
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Figure 6
Schematic depiction of a multifunctional nanoemulsion, from [68].

The effect of the density of PEG-chains on the surface of the abovementioned “theranostic” NE was
investigated by Hak et al. [69]. This could be a critical parameter defining the circulation time within
the body. The PEG density was varied from 5 to 50 mol%. At low PEG density the PEG chains are
more flexible, they organize in what is known as a mushroom configuration. At higher PEG density

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(more than 10%, which is commonly used for such applications), the distance between the PEG
chains is low, which transforms the PEG units to a brush configuration and may hinder the targeting
ligands RGD peptides in finding their targets. This steric configuration seems to determine the
targeting efficiency and specificity, because, interestingly, the best results are achieved at low PEG
surface density.

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Figure 7
Nanoemulsions with different PEG2000-DSPE content and mushroom/brush configuration indicated,
from [69].

For drug targeting, the most widely used peptides are still RGD peptides (integrin-targeted), which
were the first tumor-targeting peptide discovered [70].
For targeting atherosclerosis, Deshpande et al. [71] developed a NE which used CREKA-peptide
(Cysteine– Arginine–Glutamic acid–Lysine–Alanine) as the targeting moiety. CREKA seems to target
the fibrin clots present in atherosclerotic lesions selectively. In his previous study [57], a NE made of
flaxseed-oil containing a high level of omega-3-fatty acids, in which 17β-estradiol was successfully
incorporated. DOTAP was used on the surface of the emulsion for a positive charge to enhance the
transfer and uptake in endothelial cells. In this new study, the in vitro studies revealed that positive
charges of the oil droplets led to more rapid opsonization and, consequently, swifter uptake in the
MPS. Even the addition of PEG2000-DSPE for increase of the retention period in the blood stream
could not negate this effect. The NE system with the CREKA-PEG2000-DSPE conjugate on the surface
reached AUClast in blood of 263.8 ± 21.81 min*% injected dose/ml, whereas the DOTAP NE 20.2 ±
1.86 min*% injected dose/ml and the 17β-estradiol solution 44.9 ± 1.24 min*% injected dose/ml.

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8.2 Antibody mediated targeting

Despite the disadvantage of an antibody’s large size, there was some interesting research with NEs. A

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NE stabilized with poly(ethylene glycol)-modified phosphatidylethanolamine (PEG-PE) as a drug
carrier was described by [72]. The anti-B-cell lymphoma monoclonal antibody LL2 was successfully

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coupled to the surface, which was tested with a rat monoclonal anti-idiotype antibody, WN. Direct
cellular ELISA revealed similar binding of emulsion-LL2 complexes compared to free monoclonal

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antibody to three types of Burkitt's lymphoma cell lines, Raji, Ramos and Daudi. This indicates that

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the NE coupled with LL2 has the potential as a useful drug delivery system to B-cell malignancies. The
basic NE was prepared of triolein, Dipalmitoylphosphatidylcholine (DPPC) and polysorbate 80 in a
weight ratio of 80.2 : 1 : 0.4. For a sub-study 2 to 8 mol% PEG2000-

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Dipalmitoylphosphatidylethanolamine (DPPE, related to DPPC) was also added to the oil phase,
which showed no effect on coupling efficiency. After removal of the solvent the lipid film was re-
suspended with phosphate buffered saline (PBS), vortexed and sonicated. As a linking partner for the
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antibody conjugation 2 mol% DSPE-PEG-VS (related to DPPC) was included, whereas VS indicate the
reactive terminus of the PEG chain, a vinylsulfone group.
A cationic NE with conjugated monoclonal antibody AMB8LK, which should target over-expressing H-
ferritin in tumors, was reported by Goldstein et al. [73]. The NE consists of MCT (5 %w/w), poloxamer
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188 (1 %w/w), glycerol (2.25 %w/w), lipoid E80 (1 %w/w), stearylamine (0.25 %w/w), α-tocopherol
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(0.01 %w/w), and water (up to 100 %w/w). It is worth noting that in this particular study the coupling
of the ligand is made with cross linkers graft over stearylamine molecules on the surface of the oil
droplets, and not over DSPE-PEG moieties as commonly used. In this in vitro study the NE with the
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monoclonal antibody AMB8LK achieved a 50% increase in cell uptake compared to cationic NE
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without conjugated monoclonal antibody.

In a newer study [74], an NE loaded with paclitaxel palmitate and anti-HER2 monoclonal antibody
(Herceptin) as a targeting ligand was assessed. NE composition except the new covalent linked
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monoclonal antibody was the same as above. The water and oil phases were mixed separately and
heated to 70°C. After unifying the two phases, the emulsion was produced via microfluidization
process. The paclitaxel palmitate NE inhibits the tumor growth in mice significantly more than a
paclitaxel palmitate solution or the base cationic NE.
With Monoclonal antibody 34A the lung can be targeted, because 34A specifically binds to
pulmonary endothelial cell surface in mice. As a drug delivery system a long-circulating NE was
studied by Song et al. [75] composed of castor oil, phosphatidylcholine and DSPE-PEG coupled with
the antibody (antibody to lipid ratio 2:1 w/w). After injection into the mouse tail vein 30% of the
injected dose was found in the lung 30 minutes after administration. A kinetic study showed that
after 5 minutes the highest amount was found in the lung which then decreased. However, after 4
hours there were still 50% of the maximum bound emulsions present in the lung. The lipid phase of
the NE was composed of 1.5 mg castor oil, 0.2 mg of phosphatidylcholine, 0.4 mg of DSPE-PEG-
COOH, which was all dissolved in chloroform. After re-suspension in 0.5 ml MES buffer (pH 5.5) the
suspension was vortexed and homogenized resulting in oil droplets of about 200 nm.

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8.3 Sugar ligands and albumin as targeting moieties

Drug targeting can also be realized using sugar recognition mechanisms. Ishida et al. [76] investigated
galactosylated emulsions which are intended to target asialoglycoprotein receptor on hepatocytes in
the liver. The ligand chosen was cholesten-5-yloxy-N- (4-((1-imino-2-D-
thiogalactosylethyl)amino)butly)formamide (Gal- C4-Chol). The lipid mixture containing soybean oil,
egg phosphatidylcholine and Gal-C4-Chol (weight ratio 70 : 25 : 5) was dissolved in chloroform. After

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vacuum desiccation, the lipid phase was re-suspended in the water phase followed by sonification.

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Radiolabeling of the emulsions was realized by addition of 3H markers to the finished NE. The in vivo
tests in mice revealed a faster clearance in the blood and 3.2-fold increased accumulation in the liver

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compared to a bare NE without Gal- C4-Chol.

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Mannosylated NE incorporated with cholesten-5-yloxy-N-(4-((1-imino-2-d-
thiomannosylethyl)amino)alkyl) formamide (Man-C4-Chol) were tested in in vivo studies with mice
[77,78]. The lipid phase consisted of soybean oil (70 %w/w), egg phosphatidylcholine (25 %w/w), and

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cholesterol or Man-C4-Chol at various weight ratios and was dissolved in chloroform. After the
removal of chloroform, PBS was added as the water phase. The mixture was then sonificated.
Compared with the bare NE and the mannosylated NE with 2% Man, the mannosylated NE with 5%
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and 7% achieved rapid elimination from the blood circulation and were mainly accumulated in the
liver, whereas non-parenchymal cells were targeted. This strategy could be used to treat liver related
diseases like liver fibrosis.
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An optimized Diclofenac containing NE was developed, where albumin was used as a targeting ligand
for inflamed areas [79]. Albumin, due to its non-toxicity and biodegradability, is the perfect ligand
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targeting tissues found at areas of inflammation (and tumors). There are hypoalbuminemia albondin
receptors overexpressed for receptor-mediated endocytosis of albumin. After several formulation
trials the stability optimized NE was composed of (related to 1ml final NE): 2.5 mg diclofenac acid,
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100 mg soybean oil, 12.0 mg egg phosphatidylcholine, 2.5 mg α-tocopherol acetate, 3.0 mg
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cholesterol, 22 mg glycerin, 1.0 mg bovine serum albumin and 3.0 mg stearylamine. The NE was
prepared at 70°C, without solvent, using the sonification method. Afterwards, the albumin was
coupled to the NH2-groups of stearlyamine on the surface of the NE with the aid of 1-Ethyl-3-(3-
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dіmethylamino) propyl carbodiimide during an incubation time of 2 hours, which led to an increase in
average diameter from approx. 200 nm to 250 nm - 300 nm. In a pharmacokinetic study in rats the
therapeutic availability of the drug at the site of inflammation (granuloma air pouch fluid) was 2.89
times that of drug solution and better than other NEs containing diclofenac without albumin as the
targeting ligand. Furthermore, the drug concentration ratio of inflamed areas to blood serum was
analyzed above one at all times, which indicates a good targeting potential of the ligand albumin.

9 The Concept of Differential Protein Adsorption

It must be recognized that in many in vivo studies there is an unpredictable change in

pharmacokinetics when compared to the in vitro studies. In the blood the drug, incorporated in a
nanoparticle or NE, comes into contact with more than thousands of proteins, a few hundred in
higher concentrations. Some of the proteins absorb on the surface of the NE (protein absorption
pattern). These proteins can determine the organ distribution, e.g. apolipoprotein C III is known to be

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responsible for the inhibition of MPS uptake, especially in the liver. Introducing PEG chains to the
surface of NEs leads to an increased albumin absorption, a known dysopsonin, which leads to less
cognition by the MPS [51]. Generally, a change in emulsifier or co-surfactant may alter the protein
absorption pattern and hence, can influence the distribution of the drug in the body.
It may be useful to evaluate the differential protein adsorption right in the beginning of the
development of a new NE (see Figure 8) to get a better prediction of the distribution in the body.

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Figure 8
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Schematic diagram of the differential protein adsorption concept, from [51].

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10 Summary/Conclusion

NEs for intravenous use are an interesting system for drug delivery systems and for drug targeting.
The following advantages can be summarized so far:
 Ease of manufacturing compared to other nanoparticle systems.
 Oil droplet inside as drug reservoir, higher drug loading as e.g. liposomes [80].
Even liposome (much smaller loading capacity) can deliver ten thousands of anticancer drug
molecules directly into target cells [81].
 NEs are a perfect delivery system for high lipophilic drugs. If the drugs lipophilicity itself is not
high enough, there are often easy ways to increase the lipophilicity through esterification
with long chain fatty acids.
 Provides lipophilic drugs in a water based formulation, avoiding ingredients with unwanted
side effects.
 Due to the water-based formulation NE products have a low viscosity.
 In contrast to other nanocarriers, parenteral o/w nanoemulsions have an accepted
regulatory status. Many ingredients are already used for parenteral nutrition emulsions and
drug emulsions.

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 Accumulation in the human body of the drug delivery system NE itself or its ingredients is no
issue due to the metabolism of these ingredients.
 Long shelf life compared to other nanoparticle systems.
 Lipid emulsions prevent drug adsorption onto plastic infusion sets and allow the stabilization
of labile drugs against enzymatic degradation or oxidation. The therapeutic advantages
include the elimination of irritation and toxicity associated in contrast to other dosage forms

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[11].
 Can be a protection against hydrolysis and oxidation, when the drug is incorporated into the

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oil core of emulsions, e.g. protection of the lacton ring of Hydroxycamptothecin in NE [82].

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As a possible disadvantage of NEs, the relatively short circulation time in the body has to be
mentioned here, which is related to the safe (and fast) metabolism of NEs in the human body. With

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size reduction of the oil droplets and/or PEGlation of the surface the half-life time can be increased
efficiently, but still remains short in comparison with other, i.e. solid, nanoparticles. Nevertheless,
NEs as a drug delivery system or system for drug targeting is seen as a very promising strategy to

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overcome limitations of conventional drug systems in respect of biodistribution, avoidance of
adverse effects and in developing new possibilities for an efficient treatment of many diseases.
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