Journal of Controlled Release 223 (2016) 85–98

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Journal of Controlled Release

journal homepage: www.elsevier.com/locate/jconrel

Review article

Drug delivery and drug targeting with parenteral lipid

nanoemulsions — A review
Karl Hörmann a, Andreas Zimmer b,⁎
a
Fresenius Kabi Austria GmbH, Hafnerstraße 36, A-8055 Graz, Austria
b
Karl-Franzens-University of Graz, Institute of Pharmaceutical Sciences, Department of Pharmaceutical Technology, Member of BioTechMed Graz, Universitätsplatz 1, A-8010 Graz, Austria

a r t i c l e i n f o a b s t r a c t

Article history: Lipid nanosized emulsions or nanoemulsions (NE) are oil in water dispersions with an oil droplet size of about
Received 16 November 2015 200 nm. This size of oil droplets dispersed in a continuous water phase is a prerequisite for the parenteral, namely
Accepted 12 December 2015 intravenous administration. Many parenteral nutrition and drug emulsions on the market confirm the safe use of
Available online 14 December 2015
NE over years.
Parenteral emulsions loaded with APIs (active pharmaceutical ingredients) are considered as drug delivery sys-
Keywords:
Emulsion
tems (DDS). DDS focuses on the regulation of the in vivo dynamics, such as absorption, distribution, metabolism,
Nanoemulsion and extended bioavailability, thereby improving the effectiveness and the safety of the drugs. Using an emulsion
Intravenous as a DDS, or through the use of surface diversification of the dispersed oil droplets of emulsions, a targeted
Parenteral increase of the API concentration in some parts of the human body can be achieved. This review focuses on NE
Drug targeting similar to the marketed once with no or only low amount of additional surfactants beside the emulsifier from
Drug delivery system a manufacturing point of view (technique, used raw materials).
Manufacturing © 2015 Elsevier B.V. All rights reserved.
Materials
Ingredients

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
86
2. Classification of parenteral nanoemulsions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86
3. Nanoemulsions for nutrition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86
3.1. Biological fate of nanoemulsions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
3.2. Modification of the pharmacological profile of drugs owing to treatment with lipid nanoemulsions . . . . . . . . . . . . . . . . . . . . 87
4. Nanoemulsions as matrix for lipophilic APIs (drug delivery systems) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
4.1. Targeting liver and MPS (mononuclear phagocyte systems) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
4.2. Targets other than the liver . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
4.3. Droplet size . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90
5. Nanoemulsion with cationic surface charge . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
6. NE with PEGylation on droplet surface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
6.1. Opsonization and phagocytosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
7. NE with PEGylation on droplet surface with targeting ligand . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
7.1. Peptide mediated targeting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
7.2. Antibody mediated targeting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94
7.3. Sugar ligands and albumin as targeting moieties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96
8. The concept of differential protein adsorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96
9. Summary/conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96

⁎ Corresponding author.
E-mail address: andreas.zimmer@uni-graz.at (A. Zimmer).

http://dx.doi.org/10.1016/j.jconrel.2015.12.016
0168-3659/© 2015 Elsevier B.V. All rights reserved.
86 K. Hörmann, A. Zimmer / Journal of Controlled Release 223 (2016) 85–98
1. Introduction
The aim of new research in this field is to achieve an improved
drug delivery and drug targeting by modifying existing and develop-
ing new emulsions. Intravenous fat, also known as oil or lipid, emul-
sions have been in medical use for over 5 decades (e.g. Intralipid,
approved in Europe in 1962). Parenteral nutrition emulsions supply
critical ill patients, which cannot be fed orally, with 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 advan-
tages of NEs. Another important advantage is the capability of
dissolving not water soluble 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
14
C-cholesteryl oleate showed that peritumoral injection has the best
concentration effect. Further examples of intratumoral and intramuscu-
lar injections are given in the review on lipid carrier systems by Hashida
et al. [2].
2. Classification of parenteral nanoemulsions
This review focuses on o/w (oil in water) emulsions (Fig. 1). 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 emul-
sions. This strategy can be used as a DDS for lipophilic and/or hydrolyt-
ically 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 diameters down
to 5 μm. This is in accordance with one of the parameters noted in the
American Pharmacopeia, USPb729N, PFAT5. The percentage of fat drop-
lets greater than 5 μm must not exceed 0.05% of intravenous applied
emulsions [4].
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,
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
Fig. 1. Oil in water emulsion, stabilized through phospolipids on the surface with dissolved
drug molecules (red dots), adapted from [4].
potential of the components, most usually surfactants (low-energy
emulsification). There are a number of processes for high-energy emul-
sification, 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 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 pressure. The liquid in
this chamber is then divided into two streams, which are brought
together with ultrahigh velocities [7]. High-pressure homo-
genization, 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 with
other droplets which is the main force for microfluidization.
Based on a classification of NEs from Tamilvanan et al. [8], parenteral
NEs can also be classified as:
– NE for nutrition (phospholipid based)
– 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.
3. 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 ap-
proved 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).
NEs for nutritional purposes have no active pharmaceutical ingredi-
ent (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.
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 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 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 struc-
tured triglycerides as an oil source, in which long and medium chain tri-
glycerides 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
(docosahexaenoic acid).
To stabilize the fine dispersed oil droplets in the continuous water
phase an amphiphilic emulsifier, which is located at the interface
 K. Hörmann, A. Zimmer / Journal of Controlled Release 223 (2016) 85–98
between oil and water, is required due to their lipophilic and hydrophil-
ic molecule section.
Most of the NEs currently on the market use egg yolk phospholipids
as an emulsifier with the main chemical substance being phosphatidyl-
choline. In a recent review on lecithin based NEs [12], the peculiarities of
lecithin as an emulsifier were presented. The seldom used soybean
phosphatidylcholine is reported to be less prone to hydrolysis than
egg yolk PC during shelf life [13]. The combination of egg yolk phospho-
lipids and synthetic surfactants are often used in research to further sta-
bilize 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% 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.
3.1. Biological fate of nanoemulsions
An interesting question is, where the “life” of oil droplets of intrave-
nous emulsions ends and if this can be used for drug targeting. Accord-
ing to Rossi [14], oil droplets of an intravenous emulsion are 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 Fig. 2) are about 75–1000 nm in size
and metabolized in the blood stream, which results in a release of free
fatty acids from the triglycerides. The remnants of the chylomicrons
(30–50 nm) are cleared in the liver by low- density lipoprotein
receptors.
There are many parameters that influence the biological fate of
emulsions' oil droplets in the human body, Table 1 lists a number
of these. (For interpretation of the references to color in this figure
legend, the reader is referred to the web version of this article.)
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-labeled with 1α,2α (n)-3 H
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.
Fig. 2. Schematic chylomicron structure with ApoA, ApoB, ApoC, ApoE (apolipoproteins);
T (triacylglycerol); C (cholesterol); green (phospholipids), from [15].
87
3.2. Modification of the pharmacological profile of drugs owing to treat-
ment 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 pre-
treatment with Intralipid to increase the blood half-time of nano- and
micron-sized superparamagnetic iron-oxide particles. The proposed
mechanism behind 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 inexpensive NE.
Assink et al. [19] showed that after a life-threatening intoxication
with verapamil, a calcium channel blocker, for which there is no
antidote, a treatment with a lipid NE is beneficial.
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 addi-
tional cardiotonic effect of a lipid NE. This effect couples to accelerate
movement of the toxin 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.
Another study with bupivacaine also underlined the enhanced elim-
ination 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.
4. Nanoemulsions as matrix for lipophilic APIs (drug delivery
systems)
The incorporation of lipophilic drugs in o/w emulsion is a smart way
to overcome solubility problems. 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 (Table 2).
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 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 the administered drug in
the blood stream and subsequently further metabolized. If the lipophi-
licity 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 for lipophilicity, should be N 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 activ-
ity 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 aforemen-
tioned 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
88 K. Hörmann, A. Zimmer / Journal of Controlled Release 223 (2016) 85–98

Table 1
Parameters influencing the metabolism as lipoproteins, the capture by the mononuclear phagocyte system (MPS) and the elimination from the blood circulation of NEs after parenteral
administration, adapted from [16].

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

Emulsifier DPPC EYPC DPPC DSPC DPPC EYPC
DSPC – SM – SM DSPC
SM – – – – –
Co-emulsifier Poloxamers – Poloxamers – Poloxamers –
HCO-60 – HCO-60 – HCO-60 –
PEG – PEG – PEG –
Polysorbates – Polysorbates – Polysorbates –
Solutol – Solutol – Solutol –
– – – – Cholesterol –
– – – – CO –
Cationic lipid SA/OA – SA/OA – SA/OA –
Oil phase LCT MCT – – LCT MCT
– – – – SLS SLM
Opsonization – – Low Large Low Large
Instability of oil droplets in blood stream – – – – Low Large
Overloading of MPS Large Small

DPPC, dipalmitoylphosphatidylcholine; DSPC, distearoylphosphatidylcholine; SM, sphingomyelin; EYPC, egg yolk phosphatidylcholine; HCO-60, polyoxyethylene-(60)-hydrogenated cas-
tor 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.

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 adenocarcino-
ma in the mouse was significantly greater.
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.
An example of overcoming poor water solubility while also enhanc-
ing chemical stability is reported by Zhao et al. [26]. In this study,
Cheliensisin A, a novel anticancer drug derived from a natural source,
was formulated in a lipid NE. Interestingly, to overcome any stability is-
sues, the lipid emulsion was lyophilized after filtration through a 0.8 μm
filter and finally gamma sterilized. As a lipid 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
to 1000 ml with double distilled water the cryoprotection agent sucrose
was added (10% w/w).
Due to the formulation as a NE, the IC50 (50% inhibitory concentra-
tion) decreased to at least one third for several cancer cell types, most
cell types showed a remarkably lower decrease. The in vivo 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 solu-
bility in long chain triglycerides like soybean oil and MCT oil. Tocopherol
or its derivatives (tocol emulsions) can be used as another oil 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 com-
posed of tocopherol succinate, soybean oil, MCT oil and oleic acid in
the oil phase, whereas oleic acid formed a lipophilic ionpair with
clarithromycin and increased the 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.
The non-steroidal anti-inflammatory drug (NSAID) diclofenac as a
water based solution has a short half-time of 1–2 h, 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] devel-
oped 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

Table 2
Marketed nanoemulsions as drug delivery systems, adapted from [22].

Drug Marketed name Manufacturer Market Indication

Alprostadil palmitate Liple® Mitsubishi Tanabe Pharma Japan Vasodilator, platelet inhibitor
Clevidipine Cleviprex® The Medicines Company US Calcium channel blocker
Dexamethasone-palmitate Limethasone® Mitsubishi Tanabe Pharma Japan Rheumatoid arthritis
Diazepam Diazemuls® Actavis Nordic Europe etc. Sedative
Etomidate Etomidat-Lipuro® Braun Melsungen Europe Anesthetic
Flurbiprofen axetil Lipfen® Green Cross Japan Nonsteroidal analgesic
Propofol Diprivan® Astra Zeneca Worldwide Anesthetic
Vitamins A, D, E, K Vitalipid® Fresenius Kabi Europe etc. Parenteral nutrition
 K. Hörmann, A. Zimmer / Journal of Controlled Release 223 (2016) 85–98
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 positive-
ly charged NE in negatively charged cell membranes.
A poorly water-soluble anti-cancer substance isolated from the tra-
ditional Chinese herb magnolia, Honokiol, was studied as both a solu-
tion 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 plasma clearance reduced. The tissue dis-
tribution results in tumor-burdened mice indicated that the NE has bet-
ter targeting properties for lung, brain and tumor tissues than a
Honokiol solution.
4.1. 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 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 of 2.5. The
chylomicron-like emulsion was prepared from olive oil, phosphatidyl-
choline (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 emulsion.
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%), soy-
bean oil (4%), soybean lecithin (4%) and 91% water and was found to be
isotonic. In comparison with a 2-AP solution, the 2-AP emulsion showed
significantly faster clearance of the blood stream and higher accumula-
tion in the organs, especially the liver (about double the amount com-
pared to solution).
An NE loaded with SQ641, which is the most potent analogue of
capuramycin antibiotics and has a bactericidal effect against Mycobacte-
rium tuberculosis, was reported to target lung and spleen in a tuberculo-
sis 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.
4.2. 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% phosphatidyl-
ethanolamine), Tween 80 and poloxamer 188 were used as surfactants.
The approximately 200 nm sized NE was prepared by high-pressure
89
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 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 homoge-
nization technique and had a droplet size of 150 ± 0.78 nm. The lipid
phase was formed by soybean lecithin S75 (1% w/v) in 15 ml MCT oil,
isoflurane was added just before homogenization with the water
phase. The water phase consisted of polysorbate 80 (2% w/v), sorbitol
(2% v/v) and 64.5 ml water.
Within preclinical evaluation, the isoflurane emulsion showed
significantly decreased necessary doses compared to inhaled isoflurane
at the same levels of clinical biomarkers. However, minor 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 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 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 homogenization.
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 LDL-receptors for the development
of a NE drug delivery system. Cholesteryl ester of carborane, cholesteryl
1,12-dicarba-closo-dodecaborane-1-carboxylate (BCH) was incorporat-
ed as drug in the NE, which can be used for boron neutron capture ther-
apy of cancers. The NE was produced as follows: all lipid fractions were
dissolved in chloroform and mixed together in a ratio (w/w) of:
triolein:egg phosphatidylcholine:lysophosphatidylcholine:cholesterol
oleate:cholesterol:BCH, 70:22.7:2.3:3.0:2.0:2.0, respectively. After re-
moval 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 electrophore-
sis. Additionally, a sufficient uptake of BCH for cancer therapy was
achieved, or at minimum an absorption on rat 9 l 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 Dauno-
rubicin; moreover, the anti-cancer effects, namely tumor growth inhibi-
tion 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 radio-
active labeled 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
90 K. Hörmann, A. Zimmer / Journal of Controlled Release 223 (2016) 85–98
effects. Consequently, this drug delivery system appears to be a promis-
ing cardiovascular therapy approach minus toxicity effects.
The same NE was also used in a study in eight patients with gyneco-
logic cancers [40]. The NE was prepared from 40 mg cholesteryl oleate,
20 mg egg phosphaditylcholine, 1 mg triolein and 0.5 mg 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 as an ethanolic solution.
After sonification the liquid was centrifuged to remove unbound pacli-
taxel oleate. A double labeling was performed using 3H-paclitaxel oleate
and 14C-cholesteryl ester. 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 al-
most identical increase of concentration in the cancer tissue of 3.6
times (compared with commercial paclitaxel) and 3.5 times for the cho-
lesterol rich NE confirm that the whole drug delivery system is incorpo-
rated by cancer cells, supported by overexpression of low-density
lipoprotein receptors which internalize 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/lec-
ithin 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 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 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 in-
creased the brain availability 2.37 times. However, the tissue ranking
was still lung N liver N spleen N brain N kidney N heart.
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% squalene as the lipid phase, respectively. To obtain
a positive charge for all three categories of nanoparticles, Forestall, a cat-
ionic 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 in-
cluded 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 anti-
cancer drug Doxorubicin was developed with a diameter of approx.
200 nm, where focus was put on the utilization of raw materials
which are already approved for intravenous use and a production pro-
cess which is easy to scale-up (high-pressure homogenization). To im-
prove 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 (300 mg DOX-OA)
was dissolved in 200 ml ethanol containing 600 mg lipoid E80, 50 mg
vitamin E, and 400 mg soybean oil. The solvent was eliminated with a
rotary evaporator resulting in a thin lipid layer. With 50 ml water this
lipid film was rehydrated and vortexed rigorously. After high-pressure
homogenization the DOX-OA NE was obtained. A pharmacokinetic
study was conducted in mice showing significantly higher AUC and cir-
culation time in blood than DOX solution and a positively altered tissue
distribution. The DOX concentration in the heart was found to be lower
than 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
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 Poloxamers.
4.3. Droplet size
Size is an important factor for the biodistribution of the oil droplets
in the blood stream. As discussed before under biological fate of NEs,
the oil droplets of intravenous emulsion (which are around 200 nm)
are captured by the MPS quiet fast. Smaller oil droplets, or general nano-
particles, stay longer in the blood circulation because they are not recog-
nized by the MPS. Besides that, the enhanced permeation retention
(EPR) effect is found and used in cancer research (Fig. 3). Tumor tissue
is fast growing and has therefore defective vascular architecture. This
and impaired lymphatic drainage let nanoparticles diffuse into tumor
tissue and let them concentrate there.
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 commer-
cial emulsions to 25–50 nm of LNS.
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 macro-
phages in a more pronounced way than smaller particles.
For example, Qi et al. [48] studied lipid emulsions made of different
oils (LCT, LCT/MCT, LCT/MCT/fish oil, fish oil) and saw a significant dif-
ference (p b 0.01) between large- and small-sized emulsions containing
the same oil mixture. Unfortunately, the exact sizes were not measured;
they were compared with gel chromatography. The difference in drop-
let size was achieved by oil to egg yolk phosphatidylcholine 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 cov-
ered by egg lecithin molecules, the change in biodistribution here is only
influenced by the size of the oil droplets.
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 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 ob-
tained 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
 K. Hörmann, A. Zimmer / Journal of Controlled Release 223 (2016) 85–98
Fig. 3. Schematic illustration of the enhanced permeation and retention effect (EPR), from [46].
mice and in vitro cell culture tests revealed the advantageous properties
of 100 nm curcumin NE despite curcumin NE with 50 nm or 200 nm.
For drug delivery systems, an increased circulation time is often re-
quired to develop their desired 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 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.
5. 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 cation-
ic NEs as oligonucleotide delivery systems for the treatment of Plasmo-
dium falciparum topoisomerase II (Malaria). Oligonucleotide alone
show increased degradation in the body and their ability to enter infect-
ed 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-
trimethylammonium-propane (DOTAP). Different +/− charge ratios
were adjusted by adding different amounts of negatively charged oligo-
nucleotide, which formed together with the positively charged NE ion
pairs. Complexes with a high charge ration (+6/−1) and/or the cation-
ic NE alone caused increased hemolysis. This is attributed to the pres-
ence 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 hemolysis.
6. NE with PEGylation on droplet surface
To achieve targets in the human body other than MPS or liver with
NE, an extension of blood 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
91
mechanisms to increase the half-time blood circulation time of NEs
the enhancement of hydrophilicity is due to:
- 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 addition of other surfac-
tants or stabilizers. After an incubation time (24 h under gentle mixing
is 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 surface of the NE [51]. This elegant method is used
e.g. for incorporating PEG2000-DSPE into an existing NE.
POEs are often used in the form of triblock-copolymers, marketed as
Poloxamers or Pluronics. In 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 to-
wards 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-copolymers
and their interface-interactions was provided by Torcello-Gómez et al.
[52].
Of the abovementioned mechanisms the PEGylation is the most
commonly used. PEGylation simply refers to the coverage of a particle
or oil droplet surface by the covalently grafting, entrapping, or
adsorbing of PEG chains (Fig. 5). 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 resulting in a
high hydrodynamic volume, non-toxic and non-immunogenic, FDA ap-
proved 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 num-
ber of points to consider. The molecular weight of the PEG chain must be
2000 or greater, as indicated by most studies for polymeric nanoparti-
cles. Further, the PEG-chain density on the particle surface and the con-
formation 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,
92 K. Hörmann, A. Zimmer / Journal of Controlled Release 223 (2016) 85–98
Table 3
Physicochemical characteristics of polyoxyethylene (Pluronics and Poloxamers), adapted from [52].
Pluronic Poloxamer MW CMC at 37 °C POE units POP units
L61 P181 2000 1.1 ∗ 10−4 2 30
P85 P235 4600 6.5 ∗ 10−5 26 40
F68 P188 8400 4.8 ∗ 10−4 75 29
F127 P407 12,600 2.8 ∗ 10−6 100 65
Pluronic, tradename; Poloxamer, generic term; MW, molecular weight; CMC, critical micelle concentration in mol/l; POE, polyoxyethylene; POP, polyoxypropylene; HLB, hydrophilic-li-
pophilic balance.
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 con-
formation. 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, different mix-
tures 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 divid-
ed 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 the circulation time of NEs best, which
was accompanied by a decrease in liver accumulation. PEG-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 in-
creased hydrophilicity of the oil droplet surface. Further, the formation
of a steric barrier may also contribute to this effect.
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 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 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.
Desphande et al. [57] engineered an omega-3 polyunsaturated fatty
acid (PUFA) emulsion 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. 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, 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
HLB Administration (in vivo studies)
3 Subcutaneous, oral
16 Intravenous, intraperitoneal, subcutaneous, ocular
29 Parenteral, ocular, percutaneous, topical, oral, rectal
22 Intravenous, intramuscular, subcutaneous, percutaneous, oral, ocular, buccal, rectal, topical
added as surfactant. DOTAP, placed on the surface of the NE contributes
to the cationic charge of the whole vehicle, which should ease the trans-
fer 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 com-
pared 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
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 ingredi-
ents 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.
In a newer study [60], Paclitaxel was used without derivatization
into a prodrug. In a pharmacokinetic 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 weight ratio of
160:120:300:100:1.2. For the manufacturing of the PEGylated NE an ad-
ditional 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 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 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.
 K. Hörmann, A. Zimmer / Journal of Controlled Release 223 (2016) 85–98
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].
Prior to application, the vial containing Paclitaxel was diluted with the
emulsion to obtain a 1 mg/ml 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 of the two-vial formulation
seem to be attributed to a greater amount of Paclitaxel in the phospho-
lipid 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 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 triglycer-
ides in 200 ml dichloromethane. 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, respective-
ly). Further, LBT-OA-PEG-NE achieved high concentration in the lung,
whereas lower concentration was observed in heart, liver and kidney.
6.1. 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 min [63]. Prior to the uptake of the MPS blood proteins, known as op-
sonins (e.g., immunoglobulin γ, complement factors and fibrinogen),
attach to the surface of foreign particles (Fig. 4).
If dysopsonins (e.g. serum albumin) attach to the particle surface,
the MPS uptake is minimized and blood circulation time is consequently
prolonged. Opsonization depends on a number of factors:
• 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 lowest for non-charged particles, which means
nanoparticles or nano oil droplets with a low zeta potential [51].
Fig. 4. Opsonization and phagocytosis of oil droplets, from [11].
93
• Hydrophilicity: The blood serum proteins adhere easier on hydropho-
bic surfaces. A hydrophilic surface, which is realized e.g. with PEG
chains and poloxamer 188, suppresses the attachment of the opsonins
on the surface [54].
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 coen-
zyme Q10 loaded NE TPGS-NE showed better drug targeting properties
to heart tissue compared to a lecithin stabilized NE [65]. The NE was pre-
pared with hot high-pressure 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 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 en-
dogenous antioxidant coenzyme Q10 in the heart 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 N liver N kidney.
7. 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.
7.1. Peptide mediated targeting
Many different moieties can be used as targeting ligands. The advan-
tage 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.
Jarzyna et al. [67] reported on NEs with three different mean diam-
eters (30, 60, and 95 nm), which had incorporated oleic acid coated
iron oxide (Fe2O3) nanocrystals 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 emul-
sifier 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 chloroform. The
mixture was added, dropwise, to the heated water phase. The resulting
crude 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 and NIRF
imaging.
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 angiogenesis of cancer tissue
(Fig. 6). 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 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 revealed significant
94 K. Hörmann, A. Zimmer / Journal of Controlled Release 223 (2016) 85–98
Fig. 5. PEGylation avoids opsonization and can prolong circulation time in the blood, from [64].
accumulation in different colon cancer mouse models. The RGD ligand
provided specific interaction with ανβ3 integrin expressing cell
types. Mice treated with prednisolone acetate valerate emulsions,
RGD-prednisolone acetate valerate emulsion and prednisolone acetate
valerate + FeO emulsion, showed a significantly inhibited tumor
growth compared to blank NEs, FeO emulsions, saline and free prednis-
olone acetate valerate (all at a dose of 10 mg prednisolone acetate val-
erate per kg).
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 (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 (Fig. 7).
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 fi-
brin 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
Fig. 6. Schematic depiction of a multifunctional nanoemulsion, from [68].
study, the in vitro studies revealed that positive charges of the oil drop-
lets led to more rapid opsonization and, consequently, swifter uptake in
the MPS. Even the addition of PEG2000-DSPE for increase of the reten-
tion 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.
7.2. Antibody mediated targeting
Despite the disadvantage of an antibody's large size, there was some
interesting research with NEs. A NE stabilized with poly(ethylene gly-
col)-modified phosphatidylethanolamine (PEG-PE) as a drug carrier
was described by [72]. The anti-B-cell lymphoma monoclonal antibody
LL2 was successfully 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 monoclo-
nal antibody to three types of Burkitt's lymphoma cell lines, Raji, Ramos
and Daudi. This indicates that 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 poly-
sorbate 80 in a weight ratio of 80.2:1:0.4. For a sub-study 2 to 8 mol%
PEG2000-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 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.
 K. Hörmann, A. Zimmer / Journal of Controlled Release 223 (2016) 85–98 95

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

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 188
(1%w/w), glycerol (2.25%w/w), lipoid E80 (1%w/w), stearylamine
(0.25%w/w), α-tocopherol (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 sur-
face of the oil droplets, and not over DSPE-PEG moieties as commonly
used. In this in vitro study the NE with the monoclonal antibody
AMB8LK achieved a 50% increase in cell uptake compared to cationic
NE 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 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 min after administration. A kinetic study
showed that after 5 min the highest amount was found in the lung
which then decreased. However, after 4 h 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 phosphatidyl-
choline, 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.

Fig. 8. Schematic diagram of the differential protein adsorption concept, from [51].
96 K. Hörmann, A. Zimmer / Journal of Controlled Release 223 (2016) 85–98
7.3. Sugar ligands and albumin as targeting moieties
Drug targeting can also be realized using sugar recognition mecha-
nisms. 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)butyl)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
vacuum desiccation, the lipid phase was re-suspended in the water
phase followed by sonification. 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 compared to a bare NE without Gal- C4-Chol.
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 phosphati-
dylcholine (25%w/w), and 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% 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.
An optimized Diclofenac containing NE was developed, where albu-
min was used as a targeting ligand for inflamed areas [79]. Albumin, due
to its non-toxicity and biodegradability, is the perfect ligand targeting
tissues found at areas of inflammation (and tumors). There are hypoal-
buminemia albondin receptors overexpressed for receptor-mediated
endocytosis of albumin. After several formulation trials the stability op-
timized NE was composed of (related to 1 ml final NE): 2.5 mg
diclofenac acid, 100 mg soybean oil, 12.0 mg egg phosphatidylcholine,
2.5 mg α-tocopherol acetate, 3.0 mg cholesterol, 22 mg glycerin,
1.0 mg bovine serum albumin and 3.0 mg stearylamine. The NE was pre-
pared at 70 °C, without solvent, using the sonification method. After-
wards, the albumin was coupled to the NH2-groups of stearyl amine
on the surface of the NE with the aid of 1-Ethyl-3-(3-dіmethylamino)
propyl carbodiimide during an incubation time of 2 h, 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 contain-
ing diclofenac without albumin as the targeting ligand. Furthermore,
the drug concentration ratio of inflamed areas to blood serum was ana-
lyzed above one at all times, which indicates a good targeting potential
of the ligand albumin.
8. 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 nanoparti-
cle or NE, comes into contact with more than thousands of proteins,
a few hundred in higher concentrations. Some of the proteins ab-
sorb 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 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 Fig. 8) to get a
better prediction of the distribution in the body.
9. 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.
• 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 [11].
• Can be a protection against hydrolysis and oxidation, when the
drug is incorporated into the oil core of emulsions, e.g. protection
of the lacton ring of Hydroxycamptothecin in NE [82].
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 size reduc-
tion of the oil droplets and/or PEGylation 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 are seen as a very promising
strategy to overcome limitations of conventional drug systems in re-
spect of biodistribution, avoidance of adverse effects and in developing
new possibilities for an efficient treatment of many diseases.
References
[1] S. Mendes, S.R. Graziani, T.S. Vitório, A.F. Padoveze, R. Hegg, S.P. Bydlowski, et al., Up-
take by breast carcinoma of a lipidic nanoemulsion after intralesional injection into
the patients: a new strategy for neoadjuvant chemotherapy, Gynecol. Oncol. 112
(2009) 400–404, http://dx.doi.org/10.1016/j.ygyno.2008.10.018.
[2] M. Hashida, S. Kawakami, F. Yamashita, Lipid carrier systems for targeted drug and
gene delivery, Chem. Pharm. Bull. (Tokyo) 53 (2005) 871–880, http://dx.doi.org/10.
1248/cpb.53.871.
[3] O. Suitthimeathegorn, J.A. Turton, H. Mizuuchi, A.T. Florence, Intramuscular absorption
and biodistribution of dexamethasone from non-aqueous emulsions in the rat, Int. J.
Pharm. 331 (2007) 204–210, http://dx.doi.org/10.1016/j.ijpharm.2006.11.062.
[4] K. Hippalgaonkar, S. Majumdar, V. Kansara, Injectable lipid emulsions—advancements,
opportunities and challenges, AAPS PharmSciTech 11 (2010) 1526–1540, http://dx.
doi.org/10.1208/s12249-010-9526-5.
[5] K.B. Sutradhar, M.L. Amin, Nanoemulsions: increasing possibilities in drug delivery,
Eur. J. Nanomedicine. 5 (2013) 97–110, http://dx.doi.org/10.1515/ejnm-2013-0001.
 K. Hörmann, A. Zimmer / Journal of Controlled Release 223 (2016) 85–98
[6] A. Maali, M.T.H. Mosavian, Preparation and application of nanoemulsions in the last
decade (2000–2010), J. Dispers. Sci. Technol. 34 (2013) 92–105, http://dx.doi.org/
10.1080/01932691.2011.648498.
[7] S. Tamilvanan, S.R. Senthilkumar, R. Baskar, T.R. Sekharan, Manufacturing tech-
niques and excipients used during the formulation of oil-in-water type nanosized
emulsions for medical applications, J. Excip. Food Chem. 1 (2010) 11–29.
[8] S. Tamilvanan, Formulation of multifunctional oil-in-water nanosized emulsions for
active and passive targeting of drugs to otherwise inaccessible internal organs of the
human body, Int. J. Pharm. 381 (2009) 62–76, http://dx.doi.org/10.1016/j.ijpharm.
2009.08.001.
[9] A. Wretlind, Development of fat emulsions, JPEN J. Parenter. Enteral Nutr. 5 (1981)
230–235.
[10] A.G. Floyd, Top ten considerations in the development of parenteral emulsions,
Pharm. Sci. Technol. Today 4 (1999) 134–143.
[11] F. Leal-Calderon, M. Cansell, The design of emulsions and their fate in the body fol-
lowing enteral and parenteral routes, Soft Matter 8 (2012) 10213, http://dx.doi.org/
10.1039/c2sm26215k.
[12] V. Klang, C. Valenta, Lecithin-based nanoemulsions, J. Drug Deliv. Sci. Technol. 21
(2011) 55–76.
[13] S.M. Đorđević, T.S. Radulović, N.D. Cekić, D.V. Ranđelović, M.M. Savić, D.R. Krajišnik,
et al., Experimental design in formulation of diazepam nanoemulsions:
physicochemical and pharmacokinetic performances, J. Pharm. Sci. 102 (2013)
4159–4172, http://dx.doi.org/10.1002/jps.23734.
[14] J. Rossi, J.-C. Leroux, Principles in the development of intravenous lipid emulsions,
in: K.M. Wasan (Ed.), Role Lipid Excipients Modifying Oral Parenter, Drug Deliv,
John Wiley & Sons, New York 2007, pp. 88–123.
[15] Chylomicrons, By Xvazquez (Own Work. [GFDL (http://www.gnu.org/copyleft/
fdl.html), CC-BY-SA-3.0 or FAL], via Wikimedia Commons, https://commons.
wikimedia.org/wiki/File%3AChylomicron.svg2008 (accessed July 23, 2015).
[16] S. Tamilvanan, Oil-in-water lipid emulsions: implications for parenteral and ocular
delivering systems, Prog. Lipid Res. 43 (2004) 489–533, http://dx.doi.org/10.1016/
j.plipres.2004.09.001.
[17] M.N. Ton, C. Chang, Y.A. Carpentier, R.J. Deckelbaum, In vivo and in vitro properties
of an intravenous lipid emulsion containing only medium chain and fish oil triglyc-
erides, Clin. Nutr. 24 (2005) 492–501, http://dx.doi.org/10.1016/j.clnu.2005.03.001.
[18] L. Liu, T.K. Hitchens, Q. Ye, Y. Wu, B. Barbe, D.E. Prior, et al., Decreased reticuloendo-
thelial system clearance and increased blood half-life and immune cell labeling for
nano- and micron-sized superparamagnetic iron-oxide particles upon pre-
treatment with intralipid, Biochim. Biophys. Acta 1830 (2013) 3447–3453, http://
dx.doi.org/10.1016/j.bbagen.2013.01.021.
[19] M.A.J. Assink, P.E. Spronk, H.J.M. van Kan, A. Braber, Intravenous lipid emulsion in
the treatment of verapamil intoxication, Neth. J. Crit. Care 17 (2013) 18–21.
[20] M.R. Fettiplace, K. Lis, R. Ripper, K. Kowal, A. Pichurko, D. Vitello, et al., Multi-modal
contributions to detoxification of acute pharmacotoxicity by a triglyceride micro-
emulsion, J. Control. Release 198 (2014) 62–70, http://dx.doi.org/10.1016/j.jconrel.
2014.11.018.
[21] K. Shi, Y. Xia, Q. Wang, Y. Wu, X. Dong, C. Chen, et al., The effect of lipid emulsion on
pharmacokinetics and tissue distribution of bupivacaine in rats, Anesth. Analg. 116
(2013) 804–809, http://dx.doi.org/10.1213/ANE.0b013e318284123e.
[22] S. Ganta, M. Talekar, A. Singh, T.P. Coleman, M.M. Amiji, Nanoemulsions in transla-
tional research—opportunities and challenges in targeted cancer therapy, AAPS
PharmSciTech (2014)http://dx.doi.org/10.1208/s12249-014-0088-9.
[23] T. Takino, K. Konishi, Y. Takakura, M. Hashida, Long circulating emulsion carrier
systems for highly lipophilic drugs, Biol. Pharm. Bull. 17 (1994) 121–125.
[24] S. Ganta, J.W. Paxton, B.C. Baguley, S. Garg, Pharmacokinetics and pharmacodynam-
ics of chlorambucil delivered in parenteral emulsion, Int. J. Pharm. 360 (2008)
115–121, http://dx.doi.org/10.1016/j.ijpharm.2008.04.027.
[25] X. Li, L. Du, C. Wang, Y. Liu, X. Mei, Y. Jin, Highly efficient and lowly toxic docetaxel
nanoemulsions for intravenous injection to animals, Pharmazie 66 (2011) 479–483,
http://dx.doi.org/10.1691/ph.2011.1015.
[26] D. Zhao, T. Gong, Y. Fu, Y. Nie, L.-L. He, J. Liu, et al., Lyophilized cheliensisin A submi-
cron emulsion for intravenous injection: characterization, in vitro and in vivo anti-
tumor effect, Int. J. Pharm. 357 (2008) 139–147, http://dx.doi.org/10.1016/j.
ijpharm.2008.01.055.
[27] J. Li, S. Nie, X. Yang, C. Wang, S. Cui, W. Pan, Optimization of tocol emulsions for the
intravenous delivery of clarithromycin, Int. J. Pharm. 356 (2008) 282–290, http://dx.
doi.org/10.1016/j.ijpharm.2007.12.046.
[28] S. Ramreddy, P. Kandadi, K. Veerabrahma, Formulation and pharmacokinetics of
diclofenac lipid nanoemulsions for parenteral application, PDA J. Pharm. Sci.
Technol. 66 (2012) 28–37, http://dx.doi.org/10.5731/pdajpst.2012.00735.
[29] J. Zheng, Y. Tang, M. Sun, Y. Zhao, Q. Li, J. Zhou, et al., Characterization, pharmacoki-
netics, tissue distribution and antitumor activity of honokiol submicron lipid emul-
sions in tumor-burdened mice, Pharmazie 68 (2013) 41–46, http://dx.doi.org/10.
1691/ph.2013.2062.
[30] A.M. Dierling, Z. Cui, Targeting primaquine into liver using chylomicron emulsions
for potential vivax malaria therapy, Int. J. Pharm. 303 (2005) 143–152, http://dx.
doi.org/10.1016/j.ijpharm.2005.07.015.
[31] J.-H. Jang, C.-K. Kim, H.-G. Choi, J.H. Sung, Preparation and evaluation of 2-
(allylthio)pyrazine-loaded lipid emulsion with enhanced stability and liver
targeting, Drug Dev. Ind. Pharm. 35 (2009) 363–368, http://dx.doi.org/10.1080/
03639040802363696.
[32] B. Nikonenko, V.M. Reddy, E. Bogatcheva, M. Protopopova, L. Einck, C.A. Nacy, Ther-
apeutic efficacy of SQ641-NE against Mycobacterium tuberculosis, Antimicrob.
Agents Chemother. 58 (2014) 587–589, http://dx.doi.org/10.1128/AAC.01254-13.
[33] A. Alayoubi, S. Kanthala, S.D. Satyanarayanajois, J.F. Anderson, P.W. Sylvester, S.
Nazzal, Stability and in vitro antiproliferative activity of bioactive “Vitamin E”
97
fortified parenteral lipid emulsions, Colloids Surf. B: Biointerfaces 103 (2013)
23–30, http://dx.doi.org/10.1016/j.colsurfb.2012.10.003.
[34] A.Y. Alayoubi, J.F. Anderson, S.D. Satyanarayanajois, P.W. Sylvester, S. Nazzal, Con-
current delivery of tocotrienols and simvastatin by lipid nanoemulsions potentiates
their antitumor activity against human mammary adenocarcenoma cells, Eur. J.
Pharm. Sci. 48 (2012) 385–392, http://dx.doi.org/10.1016/j.ejps.2012.12.011.
[35] C.L. Krahn, R.P. Raffin, G.S. Santos, L.B. Queiroga, R.L. Cavalcanti, P. Serpa, et al.,
Isoflurane-loaded nanoemulsion prepared by high-pressure homogenization: in-
vestigation of stability and dose reduction in general anesthesia, J. Biomed.
Nanotechnol. 8 (2012) 849–858, http://dx.doi.org/10.1166/jbn.2012.1449.
[36] X. Chen, L. Zhang, X. Hu, X. Lin, Z. Yu, X. Tang, Formulation and preparation of a sta-
ble intravenous Disulfiram-loaded lipid emulsion, Eur. J. Lipid Sci. Technol.
(2014)http://dx.doi.org/10.1002/ejlt.201400278 (n/a–n/a).
[37] M. Shawer, P. Greenspan, S. ØIe, D.R. Lu, VLDL-resembling phospholipid-submicron
emulsion for cholesterol-based drug targeting, J. Pharm. Sci. 91 (2002) 1405–1413,
http://dx.doi.org/10.1002/jps.10117.
[38] T.C. Contente, I.F. Kretzer, F.B. Filippin-Monteiro, D.A. Maria, R.C. Maranhão, Associ-
ation of daunorubicin to a lipid nanoemulsion that binds to low-density lipoprotein
receptors enhances the antitumour action and decreases the toxicity of the drug in
melanoma-bearing mice, J. Pharm. Pharmacol. 66 (2014) 1698–1709, http://dx.doi.
org/10.1111/jphp.12296.
[39] R.C. Maranhão, E.R. Tavares, A.F. Padoveze, C.J. Valduga, D.G. Rodrigues, M.D. Pereira,
Paclitaxel associated with cholesterol-rich nanoemulsions promotes atherosclerosis
regression in the rabbit, Atherosclerosis 197 (2008) 959–966, http://dx.doi.org/10.
1016/j.atherosclerosis.2007.12.051.
[40] M.L.N. Dias, J.P. Carvalho, D.G. Rodrigues, S.R. Graziani, R.C. Maranhão, Pharma-
cokinetics and tumor uptake of a derivatized form of paclitaxel associated to a
cholesterol-rich nanoemulsion (LDE) in patients with gynecologic cancers, Can-
cer Chemother. Pharmacol. 59 (2007) 105–111, http://dx.doi.org/10.1007/
s00280-006-0252-3.
[41] B. Madhusudhan, D. Rambhau, S.S. Apte, D. Gopinath, 1-O-alkylglycerol stabilized
carbamazepine intravenous o/w nanoemulsions for drug targeting in mice, J. Drug
Target. 15 (2007) 154–161, http://dx.doi.org/10.1080/10611860601141150.
[42] C.-J. Wen, T.-C. Yen, S.A. Al-Suwayeh, H.-W. Chang, J.-Y. Fang, In vivo real-time fluo-
rescence visualization and brain-targeting mechanisms of lipid nanocarriers with
different fatty ester:oil ratios, Nanomedicine (London) 6 (2011) 1545–1559,
http://dx.doi.org/10.2217/nnm.11.46.
[43] L. Wei, G. Li, Y.-D. Yan, R. Pradhan, J.O. Kim, Q. Quan, Lipid emulsion as a drug deliv-
ery system for breviscapine: formulation development and optimization, Arch.
Pharm. Res. 35 (2012) 1037–1043, http://dx.doi.org/10.1007/s12272-012-0611-z.
[44] F. Xiong, H. Wang, Y.-J. Chen, K.-K. Geng, N. Gu, J.-B. Zhu, Characterization,
biodistribution and targeting evaluation of breviscapine lipid emulsions following
intravenous injection in mice, Drug Deliv. 18 (2011) 159–165, http://dx.doi.org/
10.3109/10717544.2010.528068.
[45] X. Zhang, X. Sun, J. Li, X. Zhang, T. Gong, Z. Zhang, Lipid nanoemulsions loaded with
doxorubicin-oleic acid ionic complex: characterization, in vitro and in vivo studies,
Pharmazie 66 (2011) 496–505, http://dx.doi.org/10.1691/ph.2011.0379.
[46] K. Ogawara, Y. Yoshizawa, K. Un, T. Araki, T. Kimura, Challenges of Drug Delivery
Systems That Contribute to Cancer Chemotherapy Nanoparticle-based Passive
Drug Targeting to Tumors : Considerations and Implications for Optimization, 36
(2013) 698–702.
[47] J. Seki, S. Sonoke, A. Saheki, H. Fukui, H. Sasaki, T. Mayumi, A nanometer lipid
emulsion, lipid nano-sphere (LNS), as a parenteral drug carrier for passive
drug targeting, Int. J. Pharm. 273 (2004) 75–83, http://dx.doi.org/10.1016/j.
ijpharm.2003.12.022.
[48] K. Qi, M. Al-Haideri, T. Seo, Y.A. Carpentier, R.J. Deckelbaum, Effects of particle size
on blood clearance and tissue uptake of lipid emulsions with different triglyceride
compositions, J. Parenter. Enter. Nutr. 27 (2003) 58–64.
[49] T. Onodera, I. Kuriyama, T. Andoh, H. Ichikawa, Y. Sakamoto, E. Lee-Hiraiwa, et al.,
Influence of particle size on the in vitro and in vivo anti-inflammatory and anti-
allergic activities of a curcumin lipid nanoemulsion, Int. J. Mol. Med. 35 (2015)
1720–1728, http://dx.doi.org/10.3892/ijmm.2015.2186.
[50] F. Bruxel, S. Cojean, A. Bochot, H. Teixeira, C. Bories, P.-M. Loiseau, et al., Cationic
nanoemulsion as a delivery system for oligonucleotides targeting malarial topo-
isomerase II, Int. J. Pharm. 416 (2011) 402–409, http://dx.doi.org/10.1016/j.
ijpharm.2011.01.048.
[51] C.M. Keck, M. Jansch, R.H. Müller, Protein adsorption patterns and analysis on IV
nanoemulsions—the key factor determining the organ distribution, Pharmaceutics
5 (2012) 36–68, http://dx.doi.org/10.3390/pharmaceutics5010036.
[52] A. Torcello-Gómez, M. Wulff-Pérez, M.J. Gálvez-Ruiz, A. Martín-Rodríguez, M.
Cabrerizo-Vílchez, J. Maldonado-Valderrama, Block copolymers at interfaces: inter-
actions with physiological media, Adv. Colloid Interf. Sci. 206 (2014) 414–427,
http://dx.doi.org/10.1016/j.cis.2013.10.027.
[53] F.M. Veronese, A. Mero, The impact of PEGylation on biological therapies, BioDrugs
22 (2008) 315–329, http://dx.doi.org/10.2165/00063030-200822050-00004.
[54] D.E. Owens, N.A. Peppas, Opsonization, biodistribution, and pharmacokinetics of
polymeric nanoparticles, Int. J. Pharm. 307 (2006) 93–102, http://dx.doi.org/10.
1016/j.ijpharm.2005.10.010.
[55] F. Liu, D. Liu, Long-circulating emulsions (oil-in-water) as carriers for lipophilic drugs,
Pharm. Res. 12 (1995) 1060–1064, http://dx.doi.org/10.1023/A:1016274801930.
[56] A. Alayoubi, S. Alqahtani, A. Kaddoumi, S. Nazzal, Effect of PEG surface conformation
on anticancer activity and blood circulation of nanoemulsions loaded with
tocotrienol-rich fraction of palm oil, AAPS J. 15 (2013) 1168–1179, http://dx.doi.
org/10.1208/s12248-013-9525-z.
[57] D. Deshpande, D.R. Janero, M. Amiji, Engineering of an ω-3 polyunsaturated fatty
acid-containing nanoemulsion system for combination C6-ceramide and
98 K. Hörmann, A. Zimmer / Journal of Controlled Release 223 (2016) 85–98
17β-estradiol delivery and bioactivity in human vascular endothelial and smooth
muscle cells, Nanomedicine 9 (2013) 885–894, http://dx.doi.org/10.1016/j.nano.
2013.02.007.
[58] S. Ganta, P. Sharma, J.W. Paxton, B.C. Baguley, S. Garg, Pharmacokinetics and phar-
macodynamics of chlorambucil delivered in long-circulating nanoemulsion, J. Drug
Target. 18 (2010) 125–133, http://dx.doi.org/10.3109/10611860903244199.
[59] B.B. Lundberg, V. Risovic, M. Ramaswamy, K.M. Wasan, A lipophilic paclitaxel
derivative incorporated in a lipid emulsion for parenteral administration, J. Control.
Release 86 (2003) 93–100, http://dx.doi.org/10.1016/S0168-3659(02)00323-1.
[60] Y. Yoshizawa, Y. Kono, K. Ogawara, T. Kimura, K. Higaki, PEG liposomalization of
paclitaxel improved its in vivo disposition and anti-tumor efficacy, Int. J. Pharm.
412 (2011) 132–141, http://dx.doi.org/10.1016/j.ijpharm.2011.04.008.
[61] X. Jing, L. Deng, B. Gao, L. Xiao, Y. Zhang, X. Ke, et al., A novel polyethylene glycol
mediated lipid nanoemulsion as drug delivery carrier for paclitaxel, Nanomedicine
10 (2014) 371–380, http://dx.doi.org/10.1016/j.nano.2013.07.018.
[62] H. Zhao, H. Lu, T. Gong, Z. Zhang, Nanoemulsion loaded with lycobetaine–oleic acid
ionic complex: physicochemical characteristics, in vitro, in vivo evaluation, and
antitumor activity, Int. J. Nanomedicine 8 (2013) 1959–1973, http://dx.doi.org/10.
2147/IJN.S43892.
[63] R.H. Müller, Colloidal Carriers for Controlled Drug Delivery and Targeting: Modifica-
tion, Characterization and In Vivo Distribution, Taylor & Francis, 1991.
[64] J.M. Morachis, E.A. Mahmoud, A. Almutairi, Physical and chemical strategies for
therapeutic delivery by using polymeric nanoparticles, Pharmacol. Rev. 64 (2012)
505–519, http://dx.doi.org/10.1124/pr.111.005363.
[65] H. Zhou, J. Zhang, Q. Jin, G. Liu, Y. Long, M. Duan, et al., Targeting of coenzyme Q10
via d-alpha-tocopheryl polyethylene glycol 1000 succinate-based nanoemulsion to
the heart, Mater. Lett. 109 (2013) 20–22, http://dx.doi.org/10.1016/j.matlet.2013.
07.057.
[66] S. Raha, T. Paunesku, G. Woloschak, Peptide-mediated cancer targeting of
nanoconjugates, Wiley Interdiscip. Rev. Nanomed. Nanobiotechnol. 3 (2011)
269–281, http://dx.doi.org/10.1002/wnan.121.
[67] P.A. Jarzyna, T. Skajaa, A. Gianella, D.P. Cormode, D.D. Samber, S.D. Dickson, et al.,
Iron oxide core oil-in-water emulsions as a multifunctional nanoparticle platform
for tumor targeting and imaging, Biomaterials 30 (2009) 6947–6954, http://dx.
doi.org/10.1016/j.biomaterials.2009.09.004.
[68] A. Gianella, P.A. Jarzyna, V. Mani, S. Ramachandran, C. Calcagno, J. Tang, et al.,
Multifunctional nanoemulsion platform for imaging guided therapy evaluated in
experimental cancer, ACS Nano 5 (2011) 4422–4433, http://dx.doi.org/10.1021/
nn103336a.
[69] S. Hak, E. Helgesen, H.H. Hektoen, E.M. Huuse, P.A. Jarzyna, W.J.M. Mulder, et al., The
effect of nanoparticle polyethylene glycol surface density on ligand-directed tumor
targeting studied in vivo by dual modality imaging, ACS Nano 6 (2012) 5648–5658,
http://dx.doi.org/10.1021/nn301630n.
[70] X. Zhang, H. Eden, X. Chen, Peptides in cancer nanomedicine: drug carriers, targeting
ligands and protease substrates, J. Control. Release 159 (2012) 2–13, http://dx.doi.
org/10.1016/j.jconrel.2011.10.023.Peptides.
[71] D. Deshpande, S. Kethireddy, F. Gattacceca, M. Amiji, Comparative pharmacokinetics
and tissue distribution analysis of systemically administered 17-β-estradiol and its
metabolites in vivo delivered using a cationic nanoemulsion or a peptide-modified
nanoemulsion system for targeting atherosclerosis, J. Control. Release 180 (2014)
117–124, http://dx.doi.org/10.1016/j.jconrel.2014.02.009.
[72] B.B. Lundberg, G. Griffiths, H.J. Hansen, Conjugation of an anti-B-cell lymphoma
monoclonal antibody, LL2, to long-circulating drug-carrier lipid emulsions, J.
Pharm. Pharmacol. 51 (1999) 1099–1105, http://dx.doi.org/10.1211/
0022357991776787.
[73] D. Goldstein, T. Nassar, G. Lambert, J. Kadouche, S. Benita, The design and evaluation
of a novel targeted drug delivery system using cationic emulsion-antibody conju-
gates, J. Control. Release 108 (2005) 418–432, http://dx.doi.org/10.1016/j.jconrel.
2005.08.021.
[74] D. Goldstein, O. Gofrit, A. Nyska, S. Benita, Anti-HER2 cationic immunoemulsion as a
potential targeted drug delivery system for the treatment of prostate cancer, Cancer
Res. 67 (2007) 269–275, http://dx.doi.org/10.1158/0008-5472.CAN-06-2731.
[75] Y.K. Song, D. Liu, K. Maruyama, T. Takizawa, Antibody mediated lung targeting of
long-circulating emulsions, PDA J. Pharm. Sci. Technol. 50 (1996) 372–377.
[76] E. Ishida, C. Managit, S. Kawakami, M. Nishikawa, F. Yamashita, M. Hashida,
Biodistribution characteristics of galactosylated emulsions and incorporated
probucol for hepatocyte-selective targeting of lipophilic drugs in mice, Pharm.
Res. 21 (2004) 932–939, http://dx.doi.org/10.1023/B:PHAM.0000029280.39882.ff.
[77] W. Yeeprae, S. Kawakami, Y. Higuchi, F. Yamashita, M. Hashida, Biodistribution char-
acteristics of mannosylated and fucosylated O/W emulsions in mice, J. Drug Target.
13 (2005) 479–487, http://dx.doi.org/10.1080/10611860500293367.
[78] W. Yeeprae, S. Kawakami, F. Yamashita, M. Hashida, Effect of mannose density on
mannose receptor-mediated cellular uptake of mannosylated O/W emulsions by
macrophages, J. Control. Release 114 (2006) 193–201, http://dx.doi.org/10.1016/j.
jconrel.2006.04.010.
[79] P. Kandadi, M.A. Syed, S. Goparaboina, K. Veerabrahma, Albumin coupled lipid
nanoemulsions of diclofenac for targeted delivery to inflammation, Nanomedicine
8 (2012) 1162–1171, http://dx.doi.org/10.1016/j.nano.2011.12.006.
[80] J. D'Arrigo, Stable Nanoemulsions: Self-assembly in Nature and Nanomedicine,
Elsevier, 2011http://dx.doi.org/10.1016/B978-0-444-53798-0.00022-5.
[81] H.-C. Wu, D.-K. Chang, Peptide-mediated liposomal drug delivery system targeting
tumor blood vessels in anticancer therapy, J. Oncol. 2010 (2010) 723798, http://
dx.doi.org/10.1155/2010/723798.
[82] Y. Zhao, J. Gao, X. Sun, H. Chen, L. Wu, W. Liang, Enhanced nuclear delivery and
cytotoxic activity of hydro-xycamptothecin using o/w emulsions, J. Pharm. Pharm.
Sci. 10 (2007) 61–70.