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Advanced Drug Delivery Reviews 60 (2008) 757 – 767

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Advances in lipid nanodispersions for parenteral drug

delivery and targeting ☆
Panayiotis P. Constantinides a,⁎, Mahesh V. Chaubal b , Robert Shorr c
a
Biopharmaceutical and Drug Delivery Consulting, LLC, Gurnee, IL, USA
b
Baxter Healthcare, Round Lake, IL, USA
c
Cornerstone Pharmaceuticals, Cranbury, NJ, USA
Received 1 September 2007; accepted 20 October 2007
Available online 6 November 2007

Abstract

Parenteral formulations, particularly intravascular ones, offer a unique opportunity for direct access to the bloodstream and rapid onset of drug
action as well as targeting to specific organ and tissue sites. Triglyceride emulsions, liposomes and micellar solutions have been traditionally used
to accomplish these tasks and there are several products on the market using these lipid formulations. The broader application of these lipid
systems in parenteral drug delivery, however, particularly with new chemical entities, has been limited due primarily to the following reasons: a)
only a small number of parenteral lipid excipients are approved, b) there is increasing number of drugs that are partially or not soluble in
conventional oils and other lipid solvents, and c) the ongoing requirement for site-specific targeting and controlled drug release. Thus, there is
growing need to expand the array of targetable lipid-based systems to deliver a wide variety of drugs and produce stable formulations which can
be easily manufactured in a sterile form, are cost-effective and at least as safe and efficacious as the earlier developed systems. These advanced
parenteral lipid-based systems are at various stages of preclinical and clinical development which include nanoemulsions, nanosuspensions and
polymeric phospholipid micelles. This review article will showcase these parenteral lipid nanosystems and discuss advances in relation to
formulation development, processing and manufacturing, and stability assessment. Factors controlling drug encapsulation and release and in vivo
biodistribution will be emphasized along with in vitro/in vivo toxicity and efficacy case studies. Emerging lipid excipients and increasing
applications of injectable lipid nanocarriers in cancer chemotherapy and other disease indications will be highlighted and in vitro/in vivo case
studies will be presented. As these new parenteral lipid systems advance through the clinic and product launch, their therapeutic utility and value
will certainly expand.
© 2007 Elsevier B.V. All rights reserved.

Keywords: Parenteral delivery; Lipid-based systems; Nanoemulsions; Nanosuspensions; Polymeric phospholipid micelles; Manufacturing; Product stability; Cell and
tissue targeting; Biodistribution; Pharmacokinetics

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 758
2. Formulation and manufacturing aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 758
2.1. Nanoemulsions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 758
2.2. Nanosuspensions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 759

Abbreviations: PEG, polyethylene glycol; CMC, critical micelle concentration; PIT, phase-inversion temperature; PE, phosphatidylethanolamine; MTD, maximum
tolerated dose; Cmax, maximum plasma concentration; AUC, area under the plasma concentration–time curve; EPR, enhanced permeability retention; LCM, lipid-
coated microbubbles; LDL, low-density lipoprotein; DSPE, 1,2-Distearoyl-sn-Glycero-3-Phosphoethanolamine or distearoylphosphatidylethanolamine.
☆
This review is part of the Advanced Drug Delivery Reviews theme issue on “Lipid-Based Systems for the Enhanced Delivery of Poorly Water Soluble Drugs”.
⁎ Corresponding author. Biopharmaceutical and Drug Delivery Consulting, LLC, P. O. Box 7565, Gurnee, IL 60031, USA. Tel./fax: +1 847 599 9496; +1 224 430
0383 (Mobile).
E-mail address: constantinpp@aol.com (P.P. Constantinides).

0169-409X/$ - see front matter © 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.addr.2007.10.013
758 P.P. Constantinides et al. / Advanced Drug Delivery Reviews 60 (2008) 757–767

2.3. Polymeric phospholipid micelles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 759

2.4. Formulation considerations for nanoparticulate systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 760
3. In vitro/in vivo case studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 760
3.1. Nanoemulsions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 760
3.2. Nanosuspensions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 763
3.3. Polymeric phospholipid micelles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 764
4. Conclusions and future perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 764
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 765
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 765

1. Introduction without the inherent creaming, flocculation, coalescence and

Triglyceride emulsions, liposomes and micellar solutions have
been traditionally employed to solubilize and parenterally deliver
poorly soluble drugs with several approved products on the market
[1–4]. These formulations, however, incorporate a small number
of approved parenteral excipients and have limitations in terms of
drug solubilization/loading and targeting, sustained release
characteristics, improved efficacy and pharmacokinetics, as well
as improved manufacturing and shelf-life [1–4]. Several of the
existing drugs and new chemical entities are not soluble in
conventional triglyceride oils/lipids and surfactants. The devel-
opment of advanced parenteral systems in recent years, such as
nanoemulsions [3–5], nanosuspensions [6,7] and polymeric
micelles [2,8–10] was the outgrowth of the aforementioned needs.
Both conventional and advanced parenteral lipid-based
systems represent a useful alternative to surfactant: alcohol
solutions, such as Cremophor EL:ethanol admixtures which
required to be diluted with saline or dextrose solutions to final
paclitaxel concentration of 0.3 to 1.2 mg/ml prior to adminis-
tration followed by long infusion times, typically over 3 h
[3,4,11,12]. Although such systems are good solvents for poorly
soluble drugs, they require special infusion apparatus at extra
expense and time. The ability of Cremophor to extract toxic
plasticizers from manufacturing and i.v. infusion tubing, is
largely responsible for the observed venous irritation and
anaphylactic reactions, such as respiratory distress [13,14].
Thus, a desired parenteral reformulation of a marketed drug
should have the following characteristics: a) is Cremophor-free,
b) exhibits high drug solubilization/loading capacity, c) is at least
as efficacious as the marketed formulation, d) it has a shelf-life of
at least 2 years, e) should be easily processed and manufactured
using standard equipment and f) is amenable to fast track
development status by addressing “unmet medical needs”.
Nanoemulsions or mini-emulsions are transparent or trans-
lucent oil-in-water (o/w) or water-in-oil droplets with a mean
droplet diameter in the range between 100 and 500 nm [1,3–
5,15–18]. They are also known as submicron emulsions and
unlike the thermodynamically stable microemulsions, nanoe-
mulsions are kinetically stable with great stability in suspension
due to their small droplet size [15]. Ostwald ripening is the
primary instability process, which can be reduced by the
addition of a second less soluble oil phase and/or addition of a
strongly adsorbed and water-insoluble polymeric surfactant
[15,19]. Advantages of nanoemulsions over macroemulsions or
coarse emulsions include, higher surface area and free energy
sedimentation associated with macroemulsions. They can be
formulated in a variety of formulations such as liquids, sprays,
foams, creams, ointments and gels.
Likewise, nanosuspensions of drugs are submicron colloidal
dispersions of drug particles which are stabilized by surfactants
[6,7]. They can be distinguished from nanoparticles which are
polymeric drug carriers [2,8–10,20] and solid lipid nanoparti-
cles, which are lipidic drug carriers [21]. One of the advantages
of nanosuspensions is their use to formulate drugs that are
insoluble in both water and oils and are usually employed when
the use of other lipid-based systems is limited. Particularly in
the case of high melting point compounds, solubilization in any
solvent is difficult, and nanosuspensions can be used to
maintain these drugs in a preferred crystalline state of
sufficiently small size for intravenous administration [7]. The
higher drug loading of nanosuspensions compared to nanoe-
mulsions for example, is afforded by the dense, solid state of
nanosuspensions. High drug loading is advantageous since the
administration volume can be significantly reduced [7].
Polymeric micelles are formed from amphiphilic copolymers
containing hydrophobic and hydrophilic blocks and are widely
used recently to solubilize a variety of poorly soluble drugs [2,8–
10,20]. There are three major types based on linear block
copolymers: a) common block copolymer micelle, b) drug-
conjugated block copolymer micelle and c) block ionomer
complex micelle [20]. While there are many options for the
hydrophobic block, polyethylene glycol (PEG) is the most often
used hydrophilic segment due to its flexibility, hydrophilicity and
biocompatibility [2,9,20]. The size of polymeric micelles is
typically less than 100 nm and are often more stable than
conventional surfactant micelles due to their lower critical micelle
concentration (CMC) values [2,8–10,20]. In addition to their high
drug solubilization capacity, extended drug release and circulation
times are other advantages over conventional micelles [20].
Within the scope of the present review article, the focus is on
polymeric micelles based on polymer-conjugated phospholipids
[2,9,10,22] and more comprehensive discussion on other types of
polymeric micelles can be found elsewhere [8–10,20].
2. Formulation and manufacturing aspects
2.1. Nanoemulsions
The methods used to produce nanoemulsions can be divided
into the high- and low-energy ones [15]. High-energy methods
 P.P. Constantinides et al. / Advanced Drug Delivery Reviews 60 (2008) 757–767
include high-pressure homogenization and microfluidization
which can be used at both laboratory and industrial scale as well
as ultrasonification which is primarily used at laboratory scale
[15,16,19]. These high-energy methods although very effective in
reducing droplet size, are undesirable for labile drugs and
macromolecules, such as proteins and nucleic acids. For these
labile molecules low-energy emulsification methods are preferred
which include, spontaneous emulsification, the solvent-diffusion
method and the phase-inversion temperature (PIT) method [15–
17,19,23,24]. A lipophilic drug can be added to the oil phase
whereas a hydrophilic one can be solubilized in the aqueous phase.
Co-solvents can be used, if needed, to aid drug solubilization.
During high-pressure homogenization, the coarse dispersion
of the oil and aqueous phase is passed through a small inlet
orifice at an operating pressure in the range of 500–5000 psi,
where the emulsion mixture is subjected to intense turbulence
and hydraulic shear which then produces a fine emulsion with an
extremely small droplet size [25]. Microfluidization uses a high-
pressure positive displacement pump operating at very high
pressures, up to 20,000 psi, which forces the emulsion product
through the interaction chamber which consists of a series of
microchannels. The emulsion flows through the microchannels
on to an impringement area resulting in very fine emulsion
droplets [26]. The operating pressure and the number of passes
of the coarse emulsion through the interaction chamber of the
microfluidizer determine the particle size of the fine emulsion.
The higher the operating pressure and the number of passes, the
smaller the droplet size of the final emulsion. The resulting
nanoemulsion can then be filtered through a 0.2 μm filter under
nitrogen to remove any large particles present resulting in a
uniform nanoemulsion. High-energy emulsification methods
can produce both o/w and w/o nanoemulsions.
Among the low-energy emulsification methods, solvent
diffusion and PIT generate o/w nanoemulsions, whereas
spontaneous emulsification produces w/o nanoemulsions [15–
17,19,23,24]. By rapid cooling of the microemulsion form in
the phase-inversion zone, or dilution with water whatever the
temperature, very stable o/w nanoemulsions can be produced
[19]. Dilution with water is more practical and adaptable and
preferred for industrial and pharmaceutical applications. How-
ever, it has recently been shown that under certain conditions,
the PIT method can also produce w/o nanoemulsions [19]. This
involves the inclusion of a lipophilic PEG-surfactant to generate
a microemulsion within the phase-inversion zone which upon
dilution with suitable oil produces highly monodispersed w/o
nanodroplets [19]. This type of nanoemulsions with an aqueous
core has increasing demand for hydrophilic drug delivery and
targeting, particularly for peptides/proteins [27] but is beyond
the scope of this review article.
2.2. Nanosuspensions
Nanosuspensions are useful as injectable dosage forms for
poorly soluble drugs. Stabilization of the nanoparticles in a
suspension form requires strong binding of surfactants onto the
particle surface. Furthermore high energy is often required in
production of such particles — both these aspects require the
759
Fig. 1. A typical plot showing the particle size mean and 99% for a model drug
nanosuspension as a function of homogenization cycle numbers.
drug to have a reasonably high melting point, so that the crystal
surface does not experience phase transition that can inhibit
binding of surfactants [6,7].
Nanosuspensions utilize similar high-energy processing
equipment as described for nanoemulsions. The amount of
energy required to break a drug crystal is significantly higher
than that required to reduce the particle size of a globule. Hence
high pressures and multiple passes are required in producing
nanosuspensions. Besides high-pressure homogenization and
microfluidization, media milling has also been utilized to
produce nanosuspensions. In each case, the particle size is
reduced by successive passes through the high-energy impact
zones. A typical particle size reduction profile is shown in Fig. 1.
It is seen that the particle size decreases rapidly in the early
phase, followed by a more gradual reduction. Besides particle
size reduction, homogenization processes also allow rapid
stabilization of the particles by facilitating contact and binding
of the surfactants to the newly formed crystal surfaces [6,7].
Aside from the high-energy processes, nanosuspensions can
also be prepared directly by crystallization or precipitation. If
the crystallization conditions are carefully chosen to maximize
the number of nuclei in a rapid supersaturation environment, the
particles produced would be very small — even in the
submicron range. Crystal growth inhibitors can be utilized to
restrict growth of the newly formed particles and surfactants can
be used to prevent their aggregation. Efficient removal of the
solvent is important in such a process, since presence of the
solvent can lead to Ostwald ripening [7].
Finally, the microprecipitation and high-energy approaches
can also be combined to exploit the advantages of both
processes. Such a combination process can be more efficient as
compared to homogenization alone. The particle size achieved
with the combination process was lower than that seen for direct
homogenization. Furthermore, the combination process allows
for the possibility of aseptic processing for thermally sensitive
molecules [7].
2.3. Polymeric phospholipid micelles
There are several methods to incorporate drugs into micelles.
These include, dialysis of organic solvents or micellar solutions of
760 P.P. Constantinides et al. / Advanced Drug Delivery Reviews 60 (2008) 757–767

detergents [2,9,10,22]. In the former method, the drug in a volatile Table 1

organic solvent is added to a preformed aqueous dispersion of Choice of excipients for parenteral nanosuspensions
polymeric micelles and subsequently the organic solvent is Excipient Function IV SC/ Ocular Pulmonary
removed by evaporation. Alternatively, the drug is dissolved in a IM
detergent solution and upon mixing with a polymeric micelles Surfactant(s) Particle stabilization + + + +
dispersion, excess detergent is removed by dialysis. For the Buffer pH adjustment and control + + + +
Polysaccharides Viscosity enhancement − + + −
preparation of drug-loaded PEG-PE micelles which are discussed
Sugars Tonicity adjuster and/or + + + +
in this review article, the procedure involves [22]: a) a dry film of lyoprotectant
the drug and PEG-PE is formed by evaporation of an organic Additional Bioadhesives, matrices for − + + −
solvent solution of the mixture, and b) hydration of the dry firm polymers sustained release
with an aqueous solution/buffer by intensive shaking to form Preservatives Preservatives for multidose − + + +
products
drug-loaded micelles. Any insoluble drug is precipitated in a
Chelating Scavenging of metal ions + + + +
crystalline form and removed by filtration. In order to improve agents (depends on drug stability)
drug solubilization, if needed, additional micelle-forming com-
pounds, such as egg phosphatidylcholine, are added to PEG-PE
micelles [22]. Several poorly soluble drugs were successfully surfactants used to stabilize the nanoparticles are critical in
solubilized in PEG-PE micelles with loading efficiencies in the defining the stability of the product. Some of the commonly used
range of 1.5 to 50% w/w and tight association of the investigated surfactants for stabilization include poloxamers [28,29], phos-
drugs with the PEG-PE micelles was demonstrated by prolonged pholipids [30], and Tweens [31]. Buffers and tonicity agents play
dialysis against aqueous buffer under sink conditions [22]. an important role in maintaining stability of a suspension —
ionizable species may shield the electrostatic repulsion between
2.4. Formulation considerations for nanoparticulate systems particles, thereby reducing the overall stability.
Given the complex physical forces involved in potential
For each of the type of system discussed above, particle size, destabilization of nanoparticles, formulation of such systems
nature and density of coatings, and rate of dissolution of the requires specific accelerated stress tests. Higher temperature and
submicron particles are the primary factors that impact their fate humidity driven accelerated stability studies typically used for
and efficacy. Hence formulation considerations for all these other dosage forms, can be used to demonstrate long-term
systems can be discussed collectively, and learnings from one stability. However formulation screening requires additional
system can be directly applied to another. stress tests to explore potential long-term destabilizing factors
Physical stability is an essential requirement for a nanoparti- such as sedimentation, Ostwald ripening, and aggregation.
culate system. Poorly formulated particles can aggregate over
time leading to higher number of large particles, thereby creating 3. In vitro/in vivo case studies
a potential safety concern. Nanoparticles may also sediment over
time in storage. If not appropriately stabilized, the sediment may In this section, case studies are presented with various poorly
be difficult to resuspend. Finally, if the drug has finite solubility soluble drugs beginning with nanoemulsions, followed by
in the suspending buffer, the drug particles can dissolve and nanosuspensions and polymeric phospholipid-based micelles.
recrystallize on large particles, a phenomenon commonly known
as Ostwald ripening. Any of these issues can lead to a change in 3.1. Nanoemulsions
particle size and consequently, the behavior of the finished
product. Particle size of the final formulation is particularly Paclitaxel is a member of the class of drugs known as taxanes
important for parenteral dosage forms, since that may also and has been isolated from the bark or leaves (needles) of the
impact safety of the product. Hence development of a stable Pacific Yew tree, Taxus brevifolia and related species. Taxanes
nanosuspension has to take into account all these modes of have been found to be useful in the treatment of various cancers
physical instability. Processes such as spray drying, freezing or such as ovarian, breast, non-small cell lung and head and neck
lyophilization can be implemented to facilitate stabilization of carcinomas although allergic reactions were observed to
the particles. However these processes can cause additional formulation excipients [11,12,32,33]. A difficulty in adminis-
stress to the particles, thereby destabilizing them. For example trating taxanes is that the drug is typically insoluble in water.
drying can cause dehydration of the adsorbed stabilizers, causing Paclitaxel has been formulated in a 1:1 mixture of Cremophor
particle aggregation. Hence excipients and stabilizers have to be EL® (a polyethoxylated castor oil) and ethanol to create Taxol®
carefully chosen to stabilize the particles and prevent particle (Bristol-Myers Squibb, Inc) which per ml contains 6 mg
size change during processing and storage. In addition, the route paclitaxel, 527 mg Cremophor EL and 49.7% (v/v) ethanol.
of administration, desired pharmacokinetic profile, and addi- Reconstitution in a suitable carrier is required (stable for ∼ 12 h)
tional features (such as targeting) have to be considered. Some of along with in-line filtration to remove any drug, which may
the excipients used in nanosuspensions for different routes of crystallize out. Taxol® has been shown to be neurotoxic,
administration are listed in Table 1. Most of the excipients on the causing sensory and sometimes accompanying motor neuro-
list (such as tonicity adjusters, chelating agents, preservatives) pathy in patients. Specific neurological manifestations of this
are commonly used in parenteral solutions. However the condition, are numbness/paraesthesia, tingling/burning in the
 P.P. Constantinides et al. / Advanced Drug Delivery Reviews 60 (2008) 757–767 761

charged tumor vessel endothelia. These characteristics were

Fig. 2. Comparative tumor growth of mice implanted with B16 murine
melanoma. Mice were administered saline, drug-free vehicle control, tocol
emulsion (listed as TOCOSOL paclitaxel) and the reference formulation Taxol®
on a schedule of q3dx5 (values shown are mean ± SEM). The data points for the
40 and 60 mg/kg groups are overlaid. Reprinted from Ref. [3].
determined in early preclinical studies as shown in Figs. 2 and 3.
Fig. 2 presents antitumor activity data in mice implanted with
B16 melanoma on a q3dx5 schedule using saline, drug-free
nanoemulsion and Taxol® as controls. Tumor growth over
time was monitored using TOCOSOL paclitaxel at 20, 40 and
60 mg/kg and Taxol® at its MTD of 20 mg/kg. As can be seen
from the data in Fig. 2, Taxol® at 20 mg/kg showed minimal
antitumor activity with a log cell-kill value of 0.5 and 50%
mortality [3,38]. In contrast, TOCOSOL paclitaxel was much
better tolerated with no mortality at 20 and 40 mg/kg and log cell-
kill values of 1.8 and 3.0, respectively [3,38] as evident from the
complete tumor remission at 40 and 60 mg/kg (Fig. 2). The
improved antitumor activity of TOCOSOL paclitaxel compared
to Taxol® in B16-bearing mice was found to be correlated to its
enhanced tumor uptake as illustrated in Fig. 3. While the total
paclitaxel concentration in the blood was similar following
administration of these two formulations, paclitaxel concentration

limbs, general diffuse weakness, and a loss of reflexes,

including those maintaining posture and gait [12,13,33].
Cremophor EL® is in part responsible for the neuropathy, as
it is itself not free of significant side effects [13,14].
Attempts to formulate paclitaxel in a long-term stable lipid
emulsion have been challenging [34–37]. The drug is reported to
be insoluble in lipid emulsions such as Intralipid®, which
contains primarily soybean oil, or Liposyn®, which contains a
mixture of soybean and safflower oils [34]. Heating paclitaxel in
either soybean oil or safflower oil, or even upon sonication, does
not result in the dissolution of appreciable amounts of drug, and
addition of paclitaxel to a lipid emulsion during a homogeniza-
tion step meets with equally disappointing results. Emulsions
incorporating up to 15 mg/mL of paclitaxel have been
formulated with triacetin, L-α-lecithin, Polysorbate 80, Pluronic
F-68, ethyloleate and glycerol. However, these emulsions are
toxic and unstable upon storage [35].
There is a clear need for a stable, easily prepared, bio-
compatible, efficacious formulation of taxanes, such as paclitaxel,
exhibiting minimal side effects that would promote uptake into
tumor cells with greater potency against drug-resistant cells. Oil-
in-water nanoemulsions incorporating paclitaxel meet these
criteria as it will be illustrated below.
A vitamin E-based nanoemulsion of paclitaxel (TOCOSOL
paclitaxel) was developed by SONUS Pharmaceuticals and it is
currently in Phase III clinical development [3,4,38–42]. It is a
Cremophor-free formulation of paclitaxel, a ready-to-use
injectable product that incorporates high drug loading (8–
10 mg/ml) in smaller dose administration volumes and shorter
infusion times. Based on the data to date, TOCOSOL paclitaxel
shows greater tumor accumulation than Taxol® and antitumor
efficacy allowing for the delivery of a greater “dose density” to
the tumor [43,44]. TOCOSOL paclitaxel nanodroplets manu-
factured by proprietary high shearing homogenization have a
Fig. 3. Comparative paclitaxel concentration in blood (top panel) and tumor
mean droplet diameter in the range of 40–80 nm, are stable and tissue (bottom panel) upon a single administration of 10 mg/kg dose of Taxol®
neutrally charged, which allows for diffusion into the tumor and TOCOSOL paclitaxel to mice implanted with B16 murine melanoma tumor
interstitium without impediment by the highly negatively cells. Reprinted from Ref. [3].
762 P.P. Constantinides et al. / Advanced Drug Delivery Reviews 60 (2008) 757–767
in the tumor was higher following administration of TOCOSOL
paclitaxel, resulting in 1.5-times higher Cmax and 2.2-times higher
AUC compared to the parameters produced by Taxol®. Higher
accumulation of TOCOSOL paclitaxel is due to the enhance
permeability retention (EPR) effect [44] as a result of the
extremely small size of the tocol nanoemulsion.
D'Arrigo et al. [45–64] published a number of studies in the
early 1990s describing a composition and method to produce
lipid-coated microbubbles (LCM) as an effective sonographic
contrast agent for localization, of experimental tumors in rats
[45–50]. LCM consists of glycerides, cholesterol and choles-
terol esters. Most of the tumors examined, were located in the
brain, liver and subcutaneous tissues. The observed sonographic
enhancement of tumor images [46–48] suggested LCM
accumulated rapidly and selectively within the cells of a
tumor mass. This is an important distinction from other tumor
mass accumulation promoting technologies and has been found
to be composition specific. The result was confirmed by
counter-staining histological samples, from each of the above
tumor locations, with either of two lipid-specific stains. Results
provided micrographic documentation of lipid-stained disc-like
structures accumulated in the tumor cells but not in the tissues in
which the tumors are embedded [46,50].
D'Arrigo et al. [45] evaluated LCM drug delivery using
glioblastoma C6 cells and gliosarcoma 9L cells, in tissue culture
and orthotopic rat models. After labeling LCM with the
fluorescent lipophilic dye “diO”, Barbarese et al. [51] reported
that analysis of LCM interactions by confocal laser scanning
microscopy with C6 and 9L cells in culture, showed that LCM
was first adsorb at the surface of the cells, and with time became
localized inside the cells. Binding and internalization proceeded
faster at 37 °C than at room temperature. Similar analysis of
such tumors in vivo, from rats injected with diO-labeled LCM,
again revealed that labeled LCM was actually inside the tumor
cells. Barbarese et al. [51] further found that staining of live
cells in tissue culture with DAMP, a dye that recognizes acidic
compartments, showed that the majority of internalized LCM
was associated with compartments containing DAMP. If the
same uptake mechanism were operative in vivo, it would
indicate that a portion of LCM bypasses the reticuloendothelial
system and become endocytosed directly by tumor cells.
LCM/Cremophor–ethanol drug delivery was also explored
using Taxol® (Bristol-Myers Squibb Co., NY, NY) in rat
orthotopic brain tumor models. Ho et al. [52] reported that the
LCM/Cremophor–ethanol Taxol® reduced tumor progression
in Fischer 344 rats inoculated with 9L glioma more effectively
than Taxol® alone.
Uptake into diverse tumor human types in cell culture or
xenographic models was not reported. Based upon the structure
of LCM and their known physicochemical properties [53–64],
D'Arrigo has suggested that LCM may bind apolipoprotein B-
100 (apo B-100), apolipoprotein E (apo E), and/or lipoprotein
lipase (LPL) in the bloodstream [65]. Apo B-100 is known to
mediate low-density lipoprotein (LDL) binding to cellular LDL
receptors, and it is widely reported that many tumor cells show
increased LDL receptor expression and activity [66–70].
Consequently, the proposed binding of plasma apo B-100 by
LCM could influence increased LDL receptor-mediated
endocytosis occurring within the tumor tissue. Enhanced LDL
receptor-mediated uptake of LCM by tumor tissue may explain
the rapidity and selectivity of LCM accumulation in tumors, as
compared to findings with liposomes [71,72].
Other LDL or alternative lipid receptor gene-family
members [73,74] may well participate in this receptor-mediated
uptake of LCM by tumor tissue [65]. For example, apo E and
LPL binding to various lipid particles (as similarly proposed
above for LCM) is widely reported to facilitate the uptake of the
lipid particles [e.g., apo E-enriched β -VLDL or LPL-enriched
VLDL, β -VLDL, and chylomicrons] by a large “multi-ligand”
endocytic receptor known as “LDL receptor-related protein” or
LRP [73].
In addition, LCM could likely bind (as do LDL with high
affinity) to macrophage scavenger receptors or be linked to lipid
raft phenomena. LCMs range in diameter from about 0.5 to
4 μm, with the vast majority having a diameter of approximately
0.5 μm or less [59,64].
Further development of the D'Arrigo LCM technology and
modification of chemical composition specifically for drug
delivery to tumors as a non-gas containing nanoemulsion with
increased drug payload and solubility of lipids over LCM have
been achieved by Cornerstone Pharmaceuticals (www.corner-
stonepharma.com, Cranbury, NJ) without the use of Cremo-
phor–ethanol. This improved technology called Emulsiphan,
like LCM has been shown to be rapidly taken up into cancer
cells selectively directly in culture and with increased efficacy
in human tumor xenograft mouse models. Antibodies directed
against the lipid transport domain of the LDL receptor however
did not block or decrease uptake and in some studies increased
Emulsiphan uptake. That material that was taken up into cells
was demonstrated by confocal microscopy using fluorescent
labeled preparations. Membrane fusion-lipid raft or other lipid
transport receptors may be involved in the selective uptake.
Further study to elucidate this mechanism is required. An
Emulsiphan lipid–oil–water nanoemulsion has been formulated
with paclitaxel resulting in the product candidate EmPAC. This
is a ready to infuse drug with increased tumor cell uptake and
greater safety and efficacy than Taxol® or liposomal formula-
tions of drug in comparative human tumor cell or xenograft
models. Increased uptake and binding of drug to microtubules
over the same concentration of paclitaxel formulated as Taxol®
or other approved formulations has been demonstrated in cells
in culture using anti-paclitaxel antibodies.
In surveying a variety of tumor types it was found that while
most solid tumors would take up the nanoemulsion they did not
do so to the same degree (Table 2). Level of uptake in parenteral
3 T3 L1 cells was not detectable. As shown in Fig. 4 comparison
of EmPAC and paclitaxel in Cremophor–ethanol indicated
EmPAC to be substantially more effective in tumor growth
delay with a 4–10-fold-higher dose of Taxol® required to
achieve the same result, depending on the model being studied.
The aggressively growing H460 NSLC line was used. That
increased cellular uptake that translates into increased efficacy
at lower doses is supported by the observation that microtubule
sites for taxane binding are saturable. Doses of drug above the
 P.P. Constantinides et al. / Advanced Drug Delivery Reviews 60 (2008) 757–767 763

Table 2
Uptake of Emulsiphan nanoparticles by human tumor cell lines
Tumor cell lineage Cell line Fluorescence intensity via N
FACS a (mean ± SEM)
Breast MDA-MB-231 120.02 ± 26.37 4
HS578T 100.65 ± 12.48 4
MX-1 86.51 ± 13.28 3
Lung H460 93.60 ± 20.34 5
A549 48.82 ± 8.07 4
H23 189.03 ± 37.86 4
H522 184.67 ± 40.58 4
Central nervous system SF-539 219.63 ± 23.33 10
SF-268 100.41 ± 7.17 3
SF-295 232.74 ± 45.79 2
U-87 234.07 ± 25.54 2
Colon HT-29 46.44 ± 5.68 12
COLO-205 35.17 ± 6.89 4
SW-620 32.11 ± 7.79 4
HCT-15 31.59 ± 16.66 3 Fig. 5. EMPAC versus Taxol® uptake by brain and lung cells.
Caco-2 52.87 ± 5.52 4
Liver Hep-3B 537.69 ± 27.71 7
Uterine and MES-SA 204.92 ± 39.38 4
drug-resistant uterine MES-SA-DX5 180.57 ± 27.88 4 lower more frequent dosing can be use more safely without
MES-SA-MX2 220.81 ± 32.85 6 risking decreased efficacy. Typically 2–10-fold increases in
Ovarian SKOV-3 65.81 ± 11.42 4
EmPAC uptake can be observed on short-term (1 h) incubations
Pancreatic PAN 02 211.99 ± 35.76 4
PAN 03 73.07 ± 22.30 4 depending on cell line and conditions as for brain SF539 and
a lung A549 cells (Fig. 5).
Average fluorescence intensity per cell was obtained for each fluorescent
Emulsiphan-treated sample, by comparing and subtracting fluorescence from the EmPAC is being prepared for investigation in human clinical
same respective untreated cell line. trials. The Emulsiphan technology has been shown to be
applicable to additional taxanes, camptothecins and other
known chemotherapeutic drugs.
ability to saturate these sites are major contributors to side
effects. 3.2. Nanosuspensions
Tumor growth inhibition studies where cells were exposed
briefly to alternative taxane preparations showed an increased In vivo behavior of nanosuspensions strongly depends on three
“apparent” potency for EmPAC. The data supports the notion factors: (a) particle size distribution; (b) dissolution rates; and
that higher dosing can be made even more effective or that (c) nature and density of coatings [7]. Particles that dissolve
rapidly (in order of seconds to minutes) will show pharmacoki-
netic behavior that is similar to solutions. For example, Clement et
al. demonstrated that a phospholipid-based nanosuspension of
flurbiprofen had the same pharmacokinetic behavior and in vivo
distribution as a high-pH solution formulation of the drug after
intravenous injection in rats [74]. This similarity between the
crystalline suspension and the solution formulations was due to
the rapid dissolution of the particles in the blood. Slow dissolving
particles on the other hand are either taken up by the
reticuloendothelial system, or can be targeted to specific tissues
by making them small and adding specific coatings. For example,
small (b 200 nm), PEGylated nanoparticles are known to have
stealth properties, allowing for passive targeting to tumor cells,
via the process of enhanced permeation retention (EPR) effect
[72,75]. Shenoy et al. [75] tested polyethylene oxide-modified
poly(beta-amino ester) nanoparticles in mice containing human
ovarian carcinoma xenograft (SKOV-3). The PEO-based particles
showed higher tumor selectivity via the EPR effect [44].
Macrophage targeting of nanoparticles has been demonstrated
by Rabinow et al. for a poorly soluble drug, Itraconazole [76]. In
Fig. 4. Comparative tumor growth of H460-tumor bearing mice treated with
EmPAC versus Taxol®. Test agents were administered i.p. 3× weekly at the this study, particles greater than 200 nm were seen to be taken up
indicated doses and percent changes in tumor size values represent mean ± SEM by circulating macrophages. The drug then released from the cells
(n = 10). gradually, allowing for sustained release over 3 days. These
764 P.P. Constantinides et al. / Advanced Drug Delivery Reviews 60 (2008) 757–767
Fig. 6. Selective accumulation of PE-PEG micelles in EL4 T lymphoma tumor in mice: (A) pharmacokinetics, (B) AUC. Reprinted from Ref. [22].
examples demonstrate the importance of in vitro characterization
of particle size, surface charge, and dissolution rates in
qualitatively predicting the fate of the nanosuspension in vivo.
Nanosuspensions have been investigated extensively as a
delivery system for poorly soluble drugs. Liversidge et al.
demonstrated the efficacy of nanoparticulate formulations of
various anticancer drugs (piposulfan, camptothecin, etoposide,
and paclitaxel) in a CH3 mouse model [77]. Formulation of the
drugs as a nanosuspension led to an improved acute toxicity
profile. Peters et al. developed a nanosuspension of an anti-
infective drug, clofazamine and compared its in vivo biodis-
tribution with a liposomal formulation [78]. It was seen that the
nanosuspension with a particle size mean of around 380 nm,
primarily targeted the reticuloendothelial system, with a
distribution quite similar to liposomes. The advantage that
nanosuspensions provided in this situation was better stability,
compared to liposomes. Scholer et al. utilized nanosuspensions
for the intravenous delivery of an antimalarial drug, atova-
quone, in a murine model [79]. Intravenously injected
atovaquone nanosuspension was distributed in various sites of
infection, such as the liver, brain, and lung. Administration of
atovaquone nanosuspension at a dose of 10 mg/kg led to serum
drug concentrations similar to those attained by oral adminis-
tration of a 10-fold-higher dose of atovaquone suspension.
Rabinow et al. developed a nanosuspension of Itraconazole
for intravenous administration [76]. This nanosuspension
showed a unique pharmacokinetic profile, with significant pro-
longation of the circulation half-life of the drug, as compared to
a cyclodextrins-based solution formulation. This prolonged
half-life may be a result of sequestration of the particles into the
macrophages, and subsequent gradual release of the dissolved
drug.
Dou et al. have developed a unique approach to use
macrophages as a targeting vehicle for delivery of antiretroviral
drugs [30]. A nanosuspension of indinavir was loaded into bone
marrow derived macrophages, and injected into HIV-1-challenged
humanized mice. The targeted delivery system significantly
reduced numbers of virus-infected cells in plasma, lymph nodes,
spleen, liver, and lung, and led to CD4 (+) T-cell protection.
A nanosuspension of Itraconazole stabilized by Poloxamer
388 was tested in human clinical trials [29]. It was seen that the
nanosuspension allowed for higher tolerable dose. Also, the
pharmacokinetic profile of the nanosuspension was seen to be
similar to a cyclodextrins-based solution formulation. This
suggests that the particles dissolved upon dilution in blood
allowing for a solution like distribution. Another intravenous
nanoparticle formulation tested in clinical trials was Spartaject
Busulfan — a formulation stabilized by dimyritoylphosphatidyl-
choline, and dilauroylphosphatidyl choline in a buffer containing
mannitol [80]. Twelve patients with chronic myeloid leukemia
were treated in a Phase I/II clinical trial. The nanoparticle
formulation demonstrated low intra-patient variability compared
to the oral formulation. Furthermore, the nanoparticle formulation
indicated reduced toxicity compared to other formulations.
Finally, a paclitaxel nanosuspension in albumin matrix has been
approved for the treatment of metastatic breast cancer [81]. Data
from various clinical studies for this product have been published
[81–83]. Most significantly, a pivotal Phase III trial involving 454
breast cancer patients, showed that a high dose of paclitaxel could
be administered without any premedication with steroid and
antihistamines (typically required to alleviate Cremophor related
hypersensitivity). The infusion time could also be reduced, given
the absence of Cremophor EL. The nanosuspension formulation
also showed a higher response rate, higher time to tumor
progression, and lower incidence of grade 4 neutropenia.
3.3. Polymeric phospholipid micelles
Long circulation times of PEG-PE micelles were demonstrated
in mice by measuring blood clearance as a function of the mole-
cular size of the PEG block [22]. As the size of the PEG block
increases, the circulation half-lives in the blood increased
from about 1 to 2 h as a result of steric hindrance against opsonin
penetration to the hydrophobic core of the micelle [22].
Preferential accumulation of PEG-PE micelles to tumors is
shown in Fig. 6 using EL4 T-cell lymphoma murine tumor model
[22]. Micelles from distearoylphosphatidylethanolamine (DSPE)
incorporating either PEG750 or PEG2000, showed selective
tumor accumulation (Fig. 6). The slightly better accumulation
of PEG750-DSPE compared to PEG2000-DSPE may be related to
the small cutoff size of the EL4 vasculature [22].
4. Conclusions and future perspectives
Nanoemulsions, nanosuspensions and polymeric phospho-
lipid micelles constitute novel parenteral formulations of
 P.P. Constantinides et al. / Advanced Drug Delivery Reviews 60 (2008) 757–767
approved drugs or new chemical entities. They are at different
stages of preclinical and clinical development. It is evident from
the preceding discussion that there have been major develop-
ments and advances with these novel parenteral nanocarriers
both at the preclinical and clinical phases. These nanosized
particles with a very large interfacial area can influence the
transport and delivery properties of the incorporated drugs and
provide for site-specific targeting. Their enhanced delivery
characteristics (improved efficacy and better tolerance), along
with improved biodistribution and pharmacokinetics have been
well documented with cancer chemotherapeutic agents and
other drugs. Furthermore, improvements in manufacturing
methods and product stability have been achieved. As these
new parenteral lipid formulations advance through the clinic
and product launch, their therapeutic utility and value will
certainly expand.
Acknowledgements
We would like to thank Dr. Vladimir P. Torchilin of
Northeastern University for the discussion on polymeric
phospholipid micelles and permission to include some of his
published data in this review article. We would also like to thank
Rajunder Bhasin, Claudia Moore, Sameer Kulkarni and Lan
Hnuygen of Cornerstone Pharmaceuticals for their skillful
contributions to the Emulsiphan work.
References
[1] J. Rossi, J.-C. Leroux, Principles in the development of intravenous lipid
emulsions, in: K. Wasan (Ed.), Role of Lipid Excipients in Modifying Oral
and Parenteral Drug Delivery, Wiley-Interscience, Hoboken, New Jersey,
2007, pp. 88–123.
[2] V.P. Torchilin, Lipid-based parenteral drug delivery systems: biological
implications, in: K. Wasan (Ed.), Role of Lipid Excipients in Modifying
Oral and Parenteral Drug Delivery, Wiley-Interscience, Hoboken, New
Jersey, 2007, pp. 48–87.
[3] P.P. Constantinides, A. Tustian, D.R. Kessler, Tocol emulsions for drug
solubilization and parenteral delivery, Adv. Drug Del. Rev. 56 (2004)
1243–1255.
[4] P.P. Constantinides, J. Han, S.S. Davis, Advances in the use of tocols as
drug delivery vehicles, Pharm. Res. 23 (2006) 243–255.
[5] D.K. Sarker, Engineering of nanoemulsions for drug delivery, Cur. Drug
Deliv. 2 (2005) 297–310.
[6] R.H. Muller, K. Peters, Nanosuspensions for the formulation of poorly
soluble drugs I. Preparation by a size reduction technique, Int. J. Pharm.
160 (1998) 229–237.
[7] B.E. Rabinow, Nanosuspensions in drug delivery, Nature Rev. 3 (2004)
785–796.
[8] D. Sutton, N. Nasongkla, E. Blanco, J. Gao, Functionalized micellar
systems for cancer targeted drug delivery, Pharm. Res. 24 (2007)
1029–1046.
[9] V.P. Torchilin, Micellar nanocarriers: pharmaceutical perspectives, Pharm.
Res. 24 (2007) 1–16.
[10] G.S. Kwon, Polymeric micelles for delivery of poorly water-soluble
compounds, Crit. Rev. Ther. Drug Carr. Syst. 20 (2003) 357–403.
[11] A. Sparreboom, L. van Zuylen, E. Brouwer, W.J. Loos, P. de Bruijn,
H. Gelderblom, M. Pillay, K. Nooter, G. Stoter, J. Verweij, Cremophor
EL-mediated alteration of paclitaxel distribution in human blood: clinical
pharmacokinetic implications, Cancer Res. 59 (1999) 1454–1457.
[12] L. van Zuylen, J. Verweij, A. Sparreboom, Role of formulation vehicles in
taxane pharmacology, Invest. New Drugs 19 (2001) 125–141.
765
[13] D. Dye, J. Watkins, Suspected anaphylactic reaction to Cremophor EL, Br.
Med. J. 280 (1980) 1353.
[14] H. Gelderblom, J. Verweij, K. Nooter, A. Sparreboom, Cremophor EL: the
drawbacks and advantages of vehicle selection for drug formulation, Eur. J.
Cancer 37 (2001) 1590–1598.
[15] T. Tadros, P. Izquierdo, J. Esquena, C. Solans, Formation and stability of
nanoemulsions, Adv. Colloid Interface Sci. 109 (2004) 303–318.
[16] S. Shafiq-un-Nabi, F. Shakeel, S. Talegaonkar, J. Ali, S. Baboota, A. Ahuja,
R.K. Khar, M. Ali, Formulation development and optimization using
nanoemulsion technique: a technical note, AAPS Pharm. Sci. Tech. 8 (2)
(2007) 28.
[17] P.P. Constantinides, Seang H. Yiv, Particle size of phase inverted water-in-
oil microemulsions under different dilution and storage conditions, Int. J.
Pharm. 115 (1995) 225–234.
[18] E.C. Unger, T. Porter, W. Culp, R. Labell, T. Matsunaga, R. Zutshi,
Therapeutic applications of lipid-coated microbubbles, Adv. Drug Del.
Rev. 56 (2004) 1291–1314.
[19] N. Anton, P. Saulnier, A. Beduneau, J.P. Benoit, Salting-out effect induced
by temperature cycling of a water/nonionic surfactant/oil system, J. Phys.
Chem. B 111 (2007) 3651–3657.
[20] L.Y. Qiu, Y.H. Bae, Polymer architecture and drug delivery, Pharm. Res.
23 (2006) 1–30.
[21] S.A. Wissing, O. Kayser, R.H. Muller, Solid lipid nanoparticles for
parenteral delivery, Adv. Drug Del. Rev. 56 (2004) 1257–1272.
[22] A.N. Lukyanov, V.P. Torchilin, Micelles from lipid derivatives of water-
soluble polymers as delivery systems for poorly soluble drugs, Adv. Drug
Del. Rev. 56 (2004) 1273–1289.
[23] T. Foster, W. Von Rybinski, A. Walde, Influence of microemulsion phases
on the preparation of fine-disperse emulsions, Adv. Colloid Interface Sci.
58 (1995) 119–149.
[24] R. Rons, I. Carrera, J. Caelles, J. Rouch, P. Panizza, Formation and
properties of miniemulsions formed by microemulsions dilution, Adv.
Colloid Interface Sci. 106 (2003) 129–143.
[25] C. Washington, S.S. Davis, The production of parenteral feeding
emulsions by microfluidizer, Int. J. Pharm. 44 (1988) 169–176.
[26] S. Pinnamaneni, N.G. Das, S.K. Das, Comparison of oil-in-water
emulsions manufactured by microfluidization and homogenization,
Pharmazie 58 (2003) 554–558.
[27] I. Jorgensen, E.H. Moeller, M. van de Weert, H.M. Nielsen, S. Frokjaer,
Preparing and evaluating delivery systems for proteins, Eur. J. Pharm. Sci.
29 (2006) 174–182.
[28] J. Moschwitzer, R.H. Muller, New method for the effective production of
ultrafine drug nanocrystals, J. Nanosci. Nanotechnol. 6 (2006) 3145–3153.
[29] J.W. Mouton, A. van Peer, K. de Beule, A. Van Vliet, J.P. Donnelly, P.A.
Soons, Pharmacokinetics of itraconazole and hydroxyitraconazole in
healthy subjects after single and multiple doses of a novel formulation,
Antimicrob. Agents Chemother. 50 (2006) 4096–4102.
[30] H. Dou, C.J. Destache, J.R. Morehead, R.L. Mosley, M.D. Boska, J. Kingsley,
S. Gorantla, L. Poluektova, J.A. Nelson, M. Chaubal, J. Werling, J. Kipp, B.E.
Rabinow, H.E. Gendelman, Development of a macrophage-based nanoparticle
platform for antiretroviral drug delivery, Blood 108 (8) (2006 Oct 15)
2827–2835.
[31] B. Van Eerdenbrugh, L. Froyen, J.A. Martens, N. Blaton, P. Augustijns,
M. Brewster, G. Van den Mooter, Characterization of physico-chemical
properties and pharmaceutical performance of sucrose co-freeze-dried
solid nanoparticulate powders of the anti-HIV agent loviride prepared by
media milling, Int. J. Pharm. 338 (2007) 198–206.
[32] E.K. Rowinsky, R.C. Donehower, Paclitaxel (Taxol®), New Engl. J. Med.
332 (1995) 1004–1014.
[33] A.K. Singla, A. Garg, D. Aggarwal, Paclitaxel and its formulations, Int. J.
Pharm. 235 (2002) 179–192.
[34] L.C. Collins-Gold, R.T. Lyons, L.C. Bartholow, Parenteral emulsions for
drug delivery, Adv. Drug Del. Rev. 5 (1990) 189–208.
[35] B.D. Tarr, T.G. Sambandan, S.H. Yalkowsky, A new parenteral emulsion
for the administration of Taxol®, Pharm. Res. 4 (1987) 162–165.
[36] B. Lundberg, A submicron lipid emulsion coated with amphipathic
polyethylene glycol for parenteral administration of paclitaxel (Taxol®l),
J. Pharm. Pharmacol. 49 (1997) 16–21.
766 P.P. Constantinides et al. / Advanced Drug Delivery Reviews 60 (2008) 757–767
[37] P. Simamora, R-M. Dannenfelser, S.E. Tabibi, S.H. Yalkowsky, Emulsion
formulations for intravenous administration of paclitaxel, J. Pharm. Sci.
Technol. 52 (1998) 170–172.
[38] P.P. Constantinides, K.J. Lambert, A.K. Tustian, B. Schneider, S. Lalji,
W. Ma, B. Wentzel, D. Kessler, D. Worah, S.C. Quay, Formulation
development and antitumor activity of a filter-sterilizable emulsion of
paclitaxel, Pharm. Res. 17 (2000) 175–182.
[39] S.C. Spigel, S.F. Jones, F.A. Greco, J.D. Hainsworth, N.R. Dickson,
N.T. Willcutt, S.W. Calvert, I.K. Rothman, D.A. Welker, J.L. Pratt, G.C.
Brandt, H.A. Burris III, TOCOSOL Paclitaxel (S-8184 Vitamin E
Paclitaxel Emulsion): Preclinical and Phase I Data, ASCO, 2002.
[40] A. Gorelov, S. Gorelov, P. Kavlov, O. Golubeva, M. Stewart, Paclitaxel
Injectable Emulsion: Phase 2a Study of Weekly Administration in Patients
with Metastatic or Locally Advanced Unresectable or Recurrent Urothelial
Transitional Cell Cancer (TCC), June 2004 ASCO, Abstract no. 4586.
[41] A. Lissianskaya, M. Gershanovich, N. Ognerubov, O. Golubeva, J. Pratt,
Paclitaxel Injectable Emulsion: Phase 2a Study of Weekly Administration
in Patients with Platinum-Resistant Ovarian Cancer, June 2004 ASCO,
Abstract no. 5047.
[42] N. Bogdanova, S. Tjulandin, N. Ognerubov, V. Semlgazov, B. Afanasjev,
A. Mahson, D. Krasnozhon, G. Manlkhas, O. Vtoraya, I. Mitashok,
V. Astakhov, M. Byakhov, J. Smith, J. Pratt, M. Bolton, R. Daifuku, A
Phase 2b Multicenter Evaluation of the Safety and Efficacy of TOCOSOL
Paclitaxel as Initial Treatment of Patients with Metastatic Breast Cancer
(MBC), 2005 SABCS, Abstract No. 1070.
[43] M.R. Dreher, W. Liu, C.R. Michelich, M.W. Dewhirst, F. Yuan, A. Chilkoti,
Tumor vascular permeability, accumulation, and penetration of macro-
molecular drug carriers, J. Natl. Cancer Inst. 98 (2006) 335–344.
[44] A.K. Iyer, G. Khaled, J. Fang, H. Maeda, Exploiting the enhanced
permeability and retention effect for tumor targeting, Drug Discov. Today
11 (2006) 812–818.
[45] J.S. D'Arrigo, R.H. Simon, S.Y. Ho, Lipid-coated uniform microbubbles
for earlier sonographic detection of brain tumors, J. Neuroimaging 1
(1991) 134–139.
[46] R.H. Simon, S.Y. Ho, C.R. Perkins, J.S. D'Arrigo, A quantitative
assessment of tumor enhancement by ultrastable lipid-coated microbubbles
as a contrast agent, Invest. Radiol. 27 (1992) 29–34.
[47] J.S. D'Arrigo, S.Y. Ho, R.H. Simon, Detection of experimental rat liver tumors
by contrast-assisted ultrasonography, Invest. Radiol. 28 (1993) 218–222.
[48] R.H. Simon, S.Y. Ho, D.F. Uphoff, S.C. Lange, J.S. D'Arrigo,
Applications of lipid-coated microbubble ultrasonic contrast to tumor
therapy, Ultrasound Med. & Biol. 19 (1993) 123–125.
[49] R.H. Simon, S.Y. Ho, J.S. D'Arrigo, A. Wakefield, S.G. Hamilton, Lipid-
coated ultrastable microbubbles as a contrast agent in neurosonography,
Invest. Radiol. 25 (1990) 1300–1304.
[50] J.S. D'Arrigo, Contrast-assisted tumor detection, Drug News & Perspect.
4 (1991) 164–167.
[51] E. Barbarese, S.Y. Ho, J.S. D'Arrigo, R.H. Simon, Internalization of
microbubbles by tumor cells in vivo and in vitro, J. Neuro-Oncology 26
(1995) 25–34.
[52] S.Y. Ho, E. Barbarese, J.S. D'Arrigo, C. Smith-Slatas, R.H. Simon,
Evaluation of lipid-coated microbubbles as a delivery vehicle for Taxol in
brain tumor therapy, Neurosurgery 40 (1997) 1260–1268.
[53] J.S. D'Arrigo, Improved method for studying the surface chemistry of
bubble formation, Aviat. Space Environ. Med. 49 (1978) 358–361.
[54] J.S. D'Arrigo, Y. Mano, Bubble production in agarose gels subjected to
different decompression schedules, Undersea Biomed. Res. 6 (1979)
93–98.
[55] J.S. D'Arrigo, Physical properties of the nonionic surfactants surrounding
gas cavitation nuclei, J. Chem. Phys. 71 (1979) 1809–1813.
[56] J.S. D'Arrigo, Structural features of the nonionic surfactants stabilizing
long-lived bubble nuclei, J. Chem.Phys. 72 (1980) 5133–5138.
[57] J.S. D'Arrigo, Aromatic proteinaceous surfactants stabilize long-lived
gas microbubbles from natural sources, J. Chem.Phys. 75 (1981)
962–968.
[58] J.S. D'Arrigo, Glycoprotein surfactants stabilize long-lived gas micro-
bubbles in the environment, in: J.E. Zajic (Ed.), Microbial-Enhanced Oil
Recovery, PennWell Press, Tulsa, 1983, pp. 124–140.
[59] J.S. D'Arrigo, Biological surfactants stabilizing natural microbubbles in
aqueous media, Adv. Colloid. Interface Sci. 19 (1983) 253–307.
[60] J.S. D'Arrigo, C. Saiz-Jimenez, N.S. Reimer, Geochemical properties and
biochemical composition of the surfactant mixture surrounding natural
microbubbles, J. Colloid & Interface Sci. 100 (1984) 96–105.
[61] J.S. D'Arrigo, Surface properties of microbubble-surfactant monolayers at
the air/water interface, J. Colloid & Interface Sci. 100 (1984) 106–111.
[62] J.S. D'Arrigo, Stable Gas-in-Liquid Emulsions: Production in Natural Waters
and Artificial Media, Elsevier Science Publishers, Amsterdam, 1986.
[63] J.S. D'Arrigo. Surfactant mixtures, stable gas-in-liquid emulsions, and
methods for the production of such emulsions from said mixtures. U.S.
Patent No. 4,684,479 (1987).
[64] J.S. D'Arrigo, T. Imae, Physical characteristics of ultrastable lipid-coated
microbubbles, J. Colloid & Interface Sci. 149 (1992) 592–595.
[65] J.S. D'Arrigo, Uptake of lipid-coated microbubbles by tumors: drug
delivery by receptor-mediated endocytosis, Bk. Abstr.: 216th Am. Chem.
Soc. Meeting, American Chemical Society, Boston, August 1998.
[66] S. Vitols, G. Gahrton, A. Ost, C. Peterson, Elevated low density lipoprotein
receptor activity in leukemic cells with monocytic differentiation, Blood
63 (1984) 1186–1193.
[67] D. Gal, M. Ohashi, P.C. MacDonald, H.J. Buchsbaum, E.R. Simpson,
Low-density lipoprotein as a potential vehicle for chemotherapeutic agents
and radionucleotides in the management of gynecologic neoplasms, Am. J.
Obstet. Gynecol. 139 (1981) 877–885.
[68] S.G. Vitols, M. Masquelier, C.O. Peterson, Selective uptake of a toxic
lipophilic anthracycline derivative by the low-density lipoprotein receptor
pathway in cultured fibroblasts, J. Med. Chem. 28 (1985) 451–454.
[69] S. Vitols, C. Peterson, O. Larsson, P. Holm, B. Aberg, Elevated uptake of
low density lipoproteins by human lung cancer tissue in vivo, Cancer Res.
52 (1992) 6244–6247.
[70] M.J. Rudling, V.P. Collins, C.O. Peterson, Delivery of aclacinomycin A to
human glioma cells in vitro by the low density lipoprotein pathway, Cancer
Res. 43 (1983) 4600–4605.
[71] S.K. Huang, K.D. Lee, K. Hong, D.S. Friend, D. Papahadjopoulos,
Microscopic localization of sterically stabilized liposomes in colon
carcinoma-bearing mice, Cancer Res. 52 (1992) 5135–5143.
[72] S.M. Moghimi, J. Szebeni, Stealth liposomes and long circulating
nanoparticles: critical issues in pharmacokinetics, opsonization and
protein-binding properties, Progress. Lipid Res. 42 (2003) 463–478.
[73] M. Krieger, J. Herz, Structures and functions of multiligand lipoprotein
receptors: Macrophage scavenger receptors, and LDL receptor-related
protein (LRP), Ann. Rev. Biochem. 63 (1994) 601–637.
[74] M. Clement, W. Pugh, I. Parikh, Tissue distribution and plasma clearance
of a novel microcrystalline-coated flurbiprofen formulation, Pharmacolo-
gist 34 (1992) 204.
[75] D. Shenoy, S. Little, R. Langer, M. Amiji, Poly(ethylene oxide)-modified
poly(beta-amino ester) nanoparticles as a pH-sensitive system for tumor-
targeted delivery of hydrophobic drugs: part 2. In vivo distribution and
tumor localization studies, Pharm. Res. 22 (2005) 2107–2114.
[76] B. Rabinow, J. Kipp, P. Papadopoulos, J. Wong, J. Glosson, J. Gass,
C. Sun, T. Wielgos, R. White, C. Cook, K. Barker, K. Wood, Itraconazole
IV nanosuspension enhances efficacy through altered pharmacokinetics
in the rat, Int. J. Pharm. 339 (2007) 251–260.
[77] E. Merisko-Liversidge, P. Sarpotdar, J. Bruno, S. Hajj, L. Wei, N. Peltier,
J. Rake, J.M. Shaw, S. Pugh, L. Polin, J. Jones, T. Corbett, E. Cooper,
G.G. Liversidge, Formulation and antitumor activity evaluation of
nanocrystalline suspensions of poorly soluble anticancer drugs, Pharm.
Res. 13 (1996) 272–278.
[78] K. Peters, S. Leitzke, J. Diederichs, K. Borner, H. Hahn, R. Muller,
S. Ehlers, Preparation of a clofazimine nanosuspension for intravenous
use and evaluation of its therapeutic efficacy in murine Mycobacterium
avium infection, J. Antimicrob. Chemother. 45 (2000) 77–83.
[79] N. Scholer, K. Krause, O. Kayser, R.H. Muller, K. Borner, H. Hahn,
O. Liesenfeld, Atovaquone nanosuspensions show excellent therapeutic
effect in a new murine model of reactivated toxoplasmosis, Antimicrob.
Agents Chemother. 45 (2001) 1771–1779.
[80] E. Olavarria, M. Hassan, A. Eades, C. Nilsson, A. Timms, J. Matthews,
C. Craddock, E. Kanfer, J. Apperley, J. Goldman. A phase I/II study of
 P.P. Constantinides et al. / Advanced Drug Delivery Reviews 60 (2008) 757–767
multiple-dose intravenous busulfan as myeloablation prior to stem cell
transplantation. Leukemia. 14 (200) 1954–1959.
[81] W. Gradishar, S. Tjulandin, N. Davidson, H. Shaw, N. Desai, P. Bhar,
M. Hawkins, J. O'Shaughnessy, Phase III trial of nanoparticle albumin-
bound paclitaxel compared with polyethylated castor oil-based
paclitaxel in women with breast cancer, J. Clin. Oncol. 23 (2005)
7794–7803.
[82] N.K. Ibrahim, N. Desai, S. Legha, P. Soon-Shiong, R.L. Theriault, E.
Rivera, B. Esmaeli, S.E. Ring, A. Bedikian, G.N. Hortobagyi, J.A.
767
Ellerhorst, Phase I and pharmacokinetic study of ABI-007, a Cremophor-
free, protein-stabilized, nanoparticle formulation of paclitaxel, Clin.
Cancer Res. 8 (2002) 1038–1044.
[83] N.K. Ibrahim, B. Samuels, R. Page, D. Doval, K.M. Patel, S.C. Rao, M.K.
Nair, P. Bhar, N. Desai, G.N. Hortobagyi, Multicenter phase II trial of ABI-
007, an albumin-bound paclitaxel, in women with metastatic breast cancer,
J. Clin. Oncol. 23 (2005) 6019–6026.