A toxic organic solvent-free technology for the preparation of PEGylated paclitaxel nanosuspension based on human serum albumin for effective cancer therapy

Abstract

Clinically, paclitaxel (PTX) is one of most commonly prescribed therapies against a wide range of solid neoplasms. Despite its success, the clinical applicability of PTX (Taxol®) is severely hampered by systemic toxicities induced by Cremophor EL. While attempts to bypass the need for Cremophor EL have been developed through platforms such as Abraxane™, nab™ relies heavily on the use of organic solvents, namely, chloroform. The toxicity introduced by residual chloroform poses a potential risk to patient health. To mitigate the toxicities of toxic organic solvent-based manufacture methods, we have designed a method for the formulation of PTX nanosuspensions (PTX-PEG [polyethylene glycol]-HSA [human serum albumin]) that eliminates the dependence on toxic organic solvents. Coined the solid-dispersion technology, this technique permits the dispersion of PTX into PEG skeleton without the use of organic solvents or Cremophor EL as a solubilizer. Once the PTX-PEG dispersion is complete, the dispersion can be formulated with HSA into nanosuspensions suitable for intravenous administration. Additionally, the incorporation of PEG permits the prolonged circulation through the steric stabilization effect. Finally, HSA-mediated targeting permits active receptor-mediated endocytosis for enhanced tumor uptake and reduced side effects. By eliminating the need for both Cremophor EL and organic solvents while simultaneously increasing antitumor efficacy, this method provides a superior alternative to currently accepted methods for PTX delivery.

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Open Access Full Text Article
http://dx.doi.org/10.2147/IJN.S92697
A toxic organic solvent-free technology for the
preparation of PEGylated paclitaxel nanosuspension
based on human serum albumin for effective cancer
therapy
Tingjie Yin*
Lihui Dong*
Bei Cui
Lei Wang
Lifang Yin
Jianping Zhou
Meirong Huo
Sta te Key L abo rat ory of Natural
Medicines, Department of
Pharmaceutics, China Pharmaceutical
University, Nanjing, People’s Republic
of China
*These authors contributed equally
to this work
Abstract: Clinically, paclitaxel (PTX) is one of most commonly prescribed therapies against a
wide range of solid neoplasms. Despite its success, the clinical applicability of PTX (Taxol®) is
severely hampered by systemic toxicities induced by Cremophor EL. While attempts to bypass
the need for Cremophor EL have been developed through platforms such as Abraxane™, nab™
relies heavily on the use of organic solvents, namely, chloroform. The toxicity introduced by
residual chloroform poses a potential risk to patient health. To mitigate the toxicities of toxic
organic solvent-based manufacture methods, we have designed a method for the formulation
of PTX nanosuspensions (PTX-PEG [polyethylene glycol]-HSA [human serum albumin]) that
eliminates the dependence on toxic organic solvents. Coined the solid-dispersion technology,
this technique permits the dispersion of PTX into PEG skeleton without the use of organic
solvents or Cremophor EL as a solubilizer. Once the PTX-PEG dispersion is complete, the
dispersion can be formulated with HSA into nanosuspensions suitable for intravenous admin-
istration. Additionally, the incorporation of PEG permits the prolonged circulation through the
steric stabilization effect. Finally, HSA-mediated targeting permits active receptor-mediated
endocytosis for enhanced tumor uptake and reduced side effects. By eliminating the need for
both Cremophor EL and organic solvents while simultaneously increasing antitumor efficacy,
this method provides a superior alternative to currently accepted methods for PTX delivery.
Keywords: human serum albumin, nanosuspension, paclitaxel, polyethylene glycol, solid-
dispersion technology
Introduction
Paclitaxel (PTX) is a clinically accepted front-line therapy against a wide range of
solid neoplasms since 1990. It works by promoting microtubule polymerization from
tubulin heterodimers, but inhibiting microtubule destruction. This leads to the induction
of cell apoptosis at the late G2/M phase.1 Despite its wide use, the practical applica-
tion of PTX is still limited by its poor aqueous solubility and low therapeutic index.2
In its commercial formulations (Taxol®, Bristol-Myers Squibb, New York, NY, USA),
PTX is formulated in a 50:50 mixture of Cremophor EL and dehydrated ethanol.3,4
Unfortunately, Cremophor EL has been attributed to significant side effects, such as
hypersensitivity, neurotoxicity, nephrotoxicity, and cardiotoxicity when intravenously
(IV) administrated at a large dose.5,6
Because of the inherent problems associated with Cremophor EL, a number of
alternative formulations including liposomes,7 lipid emulsions,8 polymeric micelles,9
Correspondence: Jianping Zhou;
Meirong Huo
State Key Laboratory of Natural
Medicines, Department of Pharmaceutics,
China Pharmaceutical University,
24 Tongjiaxiang, Nanjing 210009,
People’s Republic of China
Tel +86 25 8327 1102
Fax +86 25 8330 1606
Email cpu_zhoujp@163.com;
huomeirongcpu@163.com
Journal name: International Journal of Nanomedicine
Article Designation: Original Research
Year: 2015
Volume: 10
Running head verso: Yin et al
Running head recto: A toxic organic solvent-free technology for preparation of PTX-PEG-HSA
DOI: http://dx.doi.org/10.2147/IJN.S92697
Number of times this article has been viewed
This article was published in the following Dove Press journal:
International Journal of Nanomedicine
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cyclodextrin complexes,10 polymer–PTX conjugates,11
nanosuspensions,12 and nanoparticles (NPs)13 have been inves-
tigated to circumvent the need for Cremophor EL. Among
these alternatives, nanosuspensions in particular offer unique
advantages. Some examples include increased solubility for
hydrophobic drugs, greater bioavailability in vivo, improved
particle accumulation through the enhanced permeability and
retention (EPR) effect, excellent applicability to most hydro-
phobic drugs, and suitability for industrial scale-up.14 Conse-
quently, nanosuspension has been widely used as an effective
drug delivery system for hydrophobic anticancer drugs.
While nanosuspensions offer a solution for the chemical
formulation of hydrophobic drugs, targeting remains an issue
that still must be addressed. Human serum albumin (HSA)-
mediated targeting uses one of the major soluble proteins
in the blood circulation, HSA, and has been reported as a
promising biocompatible method for drug delivery.15 Biologi-
cally, HSA functions to maintain colloidal osmotic pressure
in the blood, alongside facilitating the transport of several
endogenous and exogenous substances.16,17 However, HSA
also naturally possesses specific targeting abilities to inflamed
and malignant tissues. Once targeted to the tumor site, HSA
is efficiently transported into tumor cells through gp60
(a 60 KDa glycoprotein) or SPARC (one secreted protein,
acidic and rich in cysteine) receptor-mediated endocytosis.18,19
As a result, HSA has been exploited in a multitude of drug
delivery systems due to its natural tumor targeting ability
and absence of toxicity or immunogenicity.19
Using nanosuspensions to mediate the chemical for-
mulation of PTX and HSA for tumor targeting, Ameri-
can Bioscience Inc. (Blauvelt, NY, USA) produced the
first biodegradable HSA-based PTX nanosuspension,
Abraxane™, by nab™. Abraxane™ is the first US Food and
Drug Administration (FDA)-approved chemotherapeutic
formulation based on nanotechnology.20 To date, the main
preparation methods of albumin-based formations can be
concluded into four techniques: emulsification,21 thermal
gelation,22 desolvation (coacervation),23 and nab™.24 While
the first three conventional techniques involve chemical
cross-linking processes or heat denaturation, nab™ oxidizes
sulfhydryl residues in albumin through homogenization to
form new cross-linking disulfide bonds, without denaturing
albumin. nab™ provides a unique window of opportunity for
albumin-based formulation, as it circumvents the destruction
of albumin’s biological characteristic while not requiring the
use of chemical crosslinkers. The lack of crosslinkers ensures
the absence of aldehyde byproducts, a prominent defect in
the conventional preparation methods.25,26
Albeit promising, the use of chloroform in nab™
manufacturing leads to chronic toxicities. In response to
the issue of chronic toxicities, we have developed a method
for the successful formulation of PTX nanosuspensions
that eliminates the dependence on toxic organic solvents
during manufacturing. Furthermore, NPs formed using this
method still retain their suitability for IV administration.
Polyethylene glycol (PEG) is a highly hydrophilic polymer
with superb biocompatibility and biodegradability, which has
been widely used as a hydrophilic solubilizing regent and
pharmacokinetic tailor.27,28 Therefore, PEG was employed to
effectively disperse PTX.29,30 In detail, PTX is first co-melted
with PEG to form a solid dispersion, which was then mixed
with the HSA solution and passed under a high pressure jet
to form the PTX-PEG-HSA nanosuspension with a narrow
size distribution. Because of the “steric stabilization” effect
of PEG, PTX-PEG-HSA nanosuspensions remain invisible
to the reticuloendothelial system (RES). As a result, PTX-
PEG-HSA nanosuspensions exhibit significantly prolonged
circulation and enhanced tumor accumulation while mitigat-
ing collateral damage to noncancerous tissues. To confirm
these advantages, the properties of the PTX-PEG-HSA nano-
suspension such as drug-loading (DL) capacity, pH, osmotic
pressure, compatible stability, drug release, and cell growth
inhibition against MDA-MB-231 cells were characterized
in vitro. Furthermore, the in vivo antitumor efficacy and
systemic toxicity of PTX-PEG-HSA were further evaluated
in MDA-MB-231 tumor-bearing nude mice in comparison
to PTX-HSA and Taxol®.
Materials and methods
Materials
HSA with a molecular weight (MW) of 65,000 Da was
purchased from Yumin Biotech Co., Ltd. (Shanghai,
People’s Republic of China). PTX and PEG (MW 6 kDa)
were purchased from Sunve Pharmaceutical Co., Ltd.
(Shanghai, People’s Republic of China) and Taihua Natural
Plant Pharmaceutical Co., Ltd. (Xi’an, Shanxi Province,
People’s Republic of China), respectively. Diazepam was
obtained from Nanjing Xiandao Chemical Co., Ltd. (Jiangsu,
People’s Republic of China). 3-(4,5-Dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide (MTT) was purchased
from Sigma-Aldrich (St Louis, MO, USA). The Cy5.5
N-hydroxysuccinimide ester (Cy5.5-NHS) and fluorescein
isothiocyanate (FITC) were obtained from Beijing Fanbo
Science and Technology Co., Ltd. (Beijing, People’s Republic
of China). HPLC-grade reagents were used as the mobile
phase in HPLC (high-performance liquid chromatography)

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A toxic organic solvent-free technology for preparation of PTX-PEG-HSA
analysis, and all other reagents were of analytical grade and
used without further purification. Distilled and deionized
water was used in all experiments.
Cells
MDA-MB-231 cells were provided by Origin Biosciences
Inc. (Nanjing, People’s Republic of China). Cells were
cultured in Dulbecco’s Modified Eagle’s Medium supple-
mented with 10% fetal bovine serum at 37°C in a 5% CO2
atmosphere.
Animals
BALB/c nude mice were obtained from Shanghai Institute of
Materia Medica, Chinese Academy of Sciences, Shanghai,
People’s Republic of China. All the animals were pathogen-
free and allowed to access food and water freely. All animals
were treated in accordance with the Guide for Care and Use
of Laboratory Animals, approved by China Pharmaceutical
University.
Preparation of PTX-PEG-HSA and PTX-
HSA nanosuspension
Preparation of PTX-PEG-HSA nanosuspension
First, the solid dispersion technique was used for the prepara-
tion of PTX-PEG suspension. PTX (5 mg) was cofused with
PEG (10, 15, 20 mg, MW 6,000) in 100 μL of anhydrous
ethanol for 1 hour at 60°C under stirring. The anhydrous
ethanol was rapidly evaporated and the PTX-PEG disper-
sion was formed. These cofused samples were named as
PEG/PTX (w/w) group at 2/1, 3/1, and 4/1, respectively.
As a result, the PTX-PEG solid dispersion was formed with
PTX incorporated inside the skeleton of the hydrophilic
modifiers.
Subsequently, 45 mg of HSA was dissolved in 10 mL of
phosphate-buffered saline (PBS, 0.1 M, pH 7.4). The solution
was then mixed with the PTX-PEG solid dispersion under
magnetic stirring for 15 minutes. Then, the mixture was pre-
mixed for 10 minutes by using a high-speed disperser. Finally,
the coarse suspension was homogenized at 20,000 psi for
6 cycles. The resulting solution was filtered through a 0.22 μm
pore-sized microfiltration membrane and then lyophilized.
Cy5.5-labeled HSA and FITC-labeled PEG were used
to prepare the PTX-HSA(Cy5.5)-PEG(FITC) nanosuspen-
sion following the same method described above. To obtain
Cy5.5-labeled HSA and FITC-labeled PEG, HSA or PEG
was agitated with Cy5.5-NHS or FITC for 8 hours away from
light in the corresponding buffer solution.31–33 The mixed
solution was then dialyzed and lyophilized.
Confocal laser scanning microscopy (CLSM, LeicaTCS
SP5, Leica, Heidelberg, Germany) was used to confirm the
successful insertion of PEG into the HSA nanosuspension.
The localization of Cy5.5-labeled HSA and FITC-labeled
PEG was observed under CLSM excited at 494 and 673 nm,
and emitted at 522 and 692 nm for FITC and Cy5.5, respec-
tively. The final images were analyzed using Leica Confocal
Software.
Preparation of PTX-HSA nanosuspension
PTX-HSA nanosuspension with a size range of 100–
150 nm was prepared as reported.19 Briefly, HSA was
dissolved in the water saturated with chloroform. Mean-
while, PTX was dissolved in the consolute solutions of
chloroform and ethanol (9:1, v:v). These two solutions
were then mixed and homogenized at 20,000 psi for 9
cycles. The resulting colloid was placed under rotary
evaporation at 25°C for 15 minutes under reduced pres-
sure and lyophilized.
Characterization of PTX-PEG-HSA
nanosuspension
Determination of DL and entrapment efciency
The DL (wt%) and entrapment efficiency (EE, %) of the
PTX-PEG-HSA nanosuspension was calculated by using
the following formula:
Drug
loading (%)
Weight of PTXinnanosuspension
Weight of PTXinnan
=oosuspension
Weight of HSAfed initially
100%
+×
Entrapment
efficiency
Weight of PTXinnanosuspension
Weight o
(%) =ffPTXfedinitially ×100%
PTX concentrations were measured by HPLC (LC-2010C,
Shimadzu Corporation, Kyoto, Japan) equipped with a
Lichrospher C18 column (4.6×250 mm, 5 μm). The mobile
phase was the mixed solution of methanol and water
(75:25, v/v), while the flow rate and detection wavelength
were set at 1 mL/min and 227 nm, respectively. The sample-
injected volume was 20 μL and the column temperature was
maintained at 30°C.
Particle size and zeta potential
The lyophilized PTX-PEG-HSA and PTX-HSA powders
were redissolved by 5% (w/v) glucose solution and diluted to
5 mg/mL. The particle size and zeta potential were measured
using Malvern Zetasizer Nano-ZS90 (Malvern Instruments,

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Malvern, UK). All of dynamic light scattering (DLS) mea-
surements were performed at 25°C and at a scattering angle
of 90°. The zeta potential values were calculated by the
Smoluchowski equation.
pH and osmotic pressure determination
The pH values of PTX-PEG-HSA (dissolved by 5% glucose
parenteral solution at 0.5 mg/mL of PTX) and PTX-HSA
(dissolved by 5% glucose parenteral solution at 0.5 mg/mL
of PTX) were measured using a microelectrode (Radiometer,
Copenhagen, Denmark). Determination of osmotic pressure
was conducted with an advanced freezing point osmometer
obtained from Advanced Instruments Inc (Norwood,
MA, USA).
Morphology observation
Transmission electron microscopy (TEM, H-7650, Hitachi,
Tokyo, Japan) and atomic force microscopy (AFM, Nano
Scope IIIa, Veeco, Santa Barbara, CA, USA) were employed
to observe the morphology and size distribution of PTX-PEG-
HSA nanosuspension. The samples for TEM imaging were
prepared with negative stain. Negative staining of samples
was performed as follows: a drop of sample solution was
placed onto a copper grid coated with carbon; the sample
drop was taped with a filter paper to remove surface water
and air-dried for 5 minutes, followed by the application of
0.01% phosphotungstic acid to deposit NPs on the grid. The
samples were air-dried before observation. The AFM obser-
vation was operated in tapping mode. A drop of properly
diluted micelles was placed on the surface of a clean silicon
wafer and dried under nitrogen flow at room temperature.
The AFM imaging was visualized in contact mode, using
high-resonance frequency (F0=129 kHz) pyramidal cantile-
vers with silicon probes having force constants of 20 Nm.
Scan speed was set at 2 Hz. Imaging was processed and the
widths of the particles were measured using Nanoscope
version 7.3 software.
DSC and WAXD analysis
The dispersion state of PTX in PTX-PEG and PTX-PEG-
HSA was evaluated by differential scanning calorimetry
(DSC) and wide angle X-ray diffraction (WAXD) analysis.
Analysis was conducted using an NETZSCH DSC 204
and an XD-3A powder diffraction meter (Bruker, AXS,
Karlsruhe, Germany) with CuKα-radiation, respectively.
In this study, the samples including blank HSA, PTX,
and the physical mixture of PTX and HSA were used as
controls.
Compatible stability of lyophilized PTX-
PEG-HSA
To investigate the stability of lyophilized PTX-PEG-HSA
upon resuspension, lyophilized powders of the PTX-PEG-
HSA nanosuspension were separately resuspended at
0.5 mg/mL in 0.9% sodium chloride parenteral solution and
5% glucose parenteral solution. The solutions were stored at
room temperature for 0, 8, 12, 18, or 24 hours, followed by
determination of PTX contents as described in the “Deter-
mination of DL and entrapment efficiency” section.
In vitro drug release from PTX-PEG-HSA
nanosuspension
The release profiles of PTX from the PTX-PEG-HSA nano-
suspension and the PTX-HSA nanosuspension were studied
using a simple dialysis method. The lyophilized PTX-PEG-
HSA or PTX-HSA powders containing 0.5 mg of PTX were
dispersed in 1 mL of 5% glucose solution with or without
supplement of 10% serum and placed inside a dialysis bag
(molecular weight cut off =12,000–14,000 Da). The entire
bag was immersed in a beaker containing 150 mL of PBS
containing 0.2% Tween 80 and shaken in a 37°C water bath
at 100 rpm. At predetermined time intervals, 1 mL of release
media was withdrawn and equivalent fresh release medium
was added. The amount of PTX released was determined by
HPLC analysis as described in the “Determination of DL and
entrapment efficiency” section.
In vitro cellular study
Cellular uptake
To evaluate the cellular uptake capacities of the PTX-PEG-
HSA nanosuspension, a Coumarin 6 (C6) fluorescence
probe was encapsulated into PTX-PEG-HSA and PTX-
HSA nanosuspensions accordant to our protocol for the
preparation of PTX-loaded nanosuspensions (preparation of
PTX-PEG-HSA nanosuspension). The DL of C6 in the both
suspensions was approximately 0.1%. Flow cytometry (BD
FACSCalibur, Becton Dickinson, San Jose, CA, USA) and
CLSM were used to determine the cell uptake capacities of
C6-PTX-PEG-HSA and C6-PTX-HSA. Cells were incubated
with C6-PTX-PEG-HSA or C6-PTX-HSA (C6 content:
60 μg/mL) for 1 and 4 hours at 37°C. In CLSM analysis, cells
were rinsed with PBS three times, followed by the addition
of Hoechst 33342 (10 μg/mL) to stain the cell nuclei. The
samples were then observed using an Olympus confocal
microscope (Olympus FV1000, Olympus Corporation,
Tokyo, Japan) at excitation and emission wavelengths of 488
and 530 nm, respectively. In the flow cytometry analysis, all

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A toxic organic solvent-free technology for preparation of PTX-PEG-HSA
samples were rinsed with PBS three times to remove excess
culture media, harvested by trypsinization, and collected in
PBS to measure the fluorescence intensity.
Cytotoxicity evaluation
Approximately 100 μL of MDA-MB-231 cells at a con-
centration of 5×103 cells/well were seeded in each well
of a 96-well plate. The plate was incubated at 37°C in a
humidified atmosphere with 5% CO2. Subsequently, specific
populations of cells were treated with PTX-PEG-HSA, PTX-
HSA and Taxol® with increasing doses: 0.001–100 μg/mL
for 72 hours. After 72 hours of incubation, the MTT assay
was performed using previously reported procedures using
a microplate reader (SOFT max® PRO, Molecular Devices
Corporation, Sunnyvale, CA, USA).34 The toxicity of samples
was expressed as the inhibitory concentration at which 50%
of cell growth inhibition was obtained (IC50).
Apoptosis assay
Apoptosis of MDA-MB-231 cells induced by PTX-PEG-HSA
nanosuspensions were analyzed by flow cytometry using
AnnexinV-FITC/propidium iodide (PI) double staining
assay. MDA-MB-231 cells were cultured in 6-well plates at
a concentration of 5×104 cells/well and incubated at 37°C
in the presence of 5% CO2 for 24 h. Subsequently, the cells
were treated with 0.1 μg/mL of PTX-PEG-HSA, PTX-HSA,
or Taxol® for 24 hours, followed by three washes with cold
PBS. Cells were then harvested, washed, and resuspended
in 500 μL of PBS. Afterward, 5 μL of Annexin V-FITC and
5 μL of PI were added and incubated for 15 minutes at room
temperature in the dark. Finally, the cell suspension was
evaluated by a flow cytometry (BD FACSCalibur, Becton
Dickinson).
In vivo antitumor activity
Approximately 1×106 of MDA-MB-231 cells were inocu-
lated subcutaneously in the armpit region of athymic nude
mice. Once tumors grew to approximately 50 mm3, mice
were randomly divided into four groups and received dif-
ferent injections as follows: 1) saline (the control group,
n=5); 2) PTX-PEG-HSA (redissolved in 5% glucose, n=5);
3) PTX-HSA (redissolved in 5% glucose, n=5); 4) Taxol®
(n=5). All formulations were administered IV via the tail vein
at a PTX dose of 10 mg/kg. Treatments occurred once every
5 days for 15 days. The body weights of all mice were also
recorded, and tumor volumes were calculated as (a2×b)/2,
where a represents the smallest diameter and b the largest.
Tumors were excised from sacrificed mice after 20 days of
observation and weighed. The tumor weight inhibition rate
(IR) (%) was calculated using the following formula:
IR (%) TW TW
TW
controltest
control
=−×100%
where TWcontrol and TWtest represented the mean tumor
weight of the control group and treated groups, respectively.
Thereafter, the tumor tissues were fixed in 10% of formalin
and embedded in paraffin blocks to conduct hematoxylin
and eosin (H&E) staining and terminal deoxynucleotidyl
transferase-mediated nicked labeling (TUNEL) assays.35,36
Then the stained tissue sections were observed under a light
microscope (Olympus) for histologic examination.
Statistics
Statistical evaluation was performed through a two-tailed
Student’s t-test, and one-way analysis of variance. All data
are expressed as the mean ± standard deviation (SD), unless
otherwise noted. A value of P,0.05 was considered statisti-
cally significant.
Results and discussion
Preparation and characterization of PTX-
PEG-HSA nanosuspension
Preparation of PTX-PEG-HSA solid suspension
The need to solubilize hydrophobic drugs using organic
solvents is an undesirable option from a practical standpoint
due to the potential toxicity of the solvents. In this work,
we have replaced the need for toxic organic solvents by the
solid-dispersion technology in terms of solubilizing PTX. This
method was created to further optimize the preparation of
PTX-loaded HSA nanosuspensions based on nab™. The PTX
powder was co-melted with PEG to form a solid dispersion,
which effectively improved the dissolution rate and apparent
solubility of PTX. This solid was then mixed with an HSA
solution and passed under high pressure, analogous to the
operative description used in nab™. Thus, the PTX-PEG-HSA
nanosuspension obtained from solid-dispersion technology is
free of toxic chloroform residues that limit the clinical appli-
cation of the original nab™ formulation. In other words, the
chronic toxicities of residual chloroform in Abraxane™ can be
successfully mitigated using solid-dispersion technology.
We first investigated the optimal weight ratio of PEG
to PTX for the preparation of the PTX-PEG solid suspen-
sion through co-melting. The solid dispersion technique
incorporates PTX inside the hydrophilic skeleton structure

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of hydrophilic carriers, which allows detection by DSC.37,38
As shown in Figure 1A, all diagrams exhibited the endother-
mic peak of PEG around 50°C. Furthermore, only the physi-
cal mixture of PEG + PTX and the group at 2/1 (PEG/PTX,
w/w) obtained the characteristic peaks of PTX in crystalline
state around 240°C. The disappearance of characteristic
peaks of PTX in groups at 3/1 and 4/1 indicated the suc-
cessful formation of PTX-PEG solid dispersion with PTX
existing in an amorphous state. During the preparation of
the solid dispersion, the optimal pharmaceutical parameter
was defined as the minimum amount of hydrophilic carri-
ers required for the hydrophobic drugs to be sufficiently
dispersed.39 Therefore, the PTX-PEG solid dispersion
was first prepared at a weight ratio of 1:3 with respect to
PTX:PEG. This mixture was then combined with the HSA
solution and homogenized to obtain the PTX-PEG-HSA
nanosuspension.
Furthermore, a double-fluorescent label method was used
to confirm the successful attachment of PEG within the HSA-
based nanosuspension. In this method, HSA and PEG were
first tagged with Cy5.5 and FITC, respectively. Thereafter,
the PTX-HSA(Cy5.5)-PEG(FITC) nanosuspension was
prepared following the procedures of PTX-PEG-HSA manu-
facture. The formation of PTX-HSA(Cy5.5)-PEG(FITC)
nanosuspension was evaluated visually under a CLSM. As
shown in Figure 1B, the merged green/red fluorescence
(indicated by white arrows) of PTX-HSA(Cy5.5)-PEG(FITC)
nanosuspensions was obtained via confocal microscopy
imaging. This result directly demonstrated that PTX-
PEG-HSA nanosuspensions could be formed through our
toxic organic solvent-free technology (with successful PEG
insertion). H-bonding network between PEG and vicinal
amino acids of HSA surrounding may play a major role in
the PEG/HSA complexation.40
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Figure 1 (A) DSC thermograms of (a) physical mixture of PEG + PTX; (b) the PTX-PEG solid dispersion prepared at weight ratio 2/1 (PEG/PTX); (c) the PTX-PEG solid
dispersion prepared at weight ratio 3/1 (PEG/PTX); (d) the PTX-PEG solid dispersion prepared at weight ratio 4/1 (PEG/PTX). (B) Confocal microscopy of PTX-HSA(Cy5.5)-
PEG(FITC).
Notes: For each panel, images from left to right show the uorescence of PEG-FITC (green), HSA-Cy5.5 (red), and the overlays of PTX-HSA(Cy5.5)-PEG(FITC) with
uorescence of PEG-FITC and HSA-Cy5.5 indicated by white arrows.
Abbreviations: DSC, differential scanning calorimetry; PEG, polyethylene glycol; PTX, paclitaxel; FITC, uorescein isothiocyanate; HSA, human serum albumin.

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A toxic organic solvent-free technology for preparation of PTX-PEG-HSA
Determination of DL and EE
All of the physicochemical characteristics of PTX-loaded
nanosuspensions are listed in Table 1. First, DL capability
was determined using HPLC. The DL (wt%) and EE (%)
of PTX-PEG-HSA was 9.1±0.2 wt% and 90.5%±0.6%,
respectively. PTX-HSA obtained a DL and an EE of
9.2±0.1 wt% and 91.4%±0.5%, respectively. The similar
DL and EE observed in these two HSA-based nanosuspen-
sions indicated that the unique drug dispersion by PEG did
not affect the specific binding interactions between PTX
and HSA.
Particle size and zeta potential evaluation
The particle size and zeta potential of PTX-PEG-HSA and
PTX-HSA were measured by DLS. As listed in Table 1,
the effective diameters of PTX-PEG-HSA and PTX-HSA
were 123.0 and 137.2 nm with polydispersity indices (PDI)
of 0.11 and 0.14, respectively, implying a narrow size
distribution. In addition, the particle surface of PTX nano-
suspensions were negatively charged with zeta potential of
approximately -43.6 mV for PTX-PEG-HSA and -39.9 mV
for PTX-HSA. The mean particle size distribution usually
affects the formulation stability, pharmacokinetics, and in
vivo tissue distribution of nanosuspension systems.41 As
compared with PTX-HSA, PTX-PEG-HSA nanosuspen-
sions shared a similar zeta potential but had a smaller mean
diameter and are less likely to be nonselectively recognized
by the RES. This decrease in RES uptake, therefore, allows
the particles to achieve long-circulation times in blood by
avoiding RES clearance.42 Additionally, the EPR effect was
reported to be a significant factor when the size of NPs was
approximately 100–200 nm.43 Furthermore, the relatively
high zeta potential provides a repelling force between the
particles. This electrostatic repulsion acts to the formulation’s
advantage to prevent undesired aggregation or precipitation
and thus, improves the formulation’s stability.44 Therefore, it
is reasonable to conclude that PTX-loaded nanosuspensions
with small diameters (123.0 nm) and negatively charged
zeta potentials (-43.6 mV) are suitable for targeted tumor
therapy through IV injection due to the reasons that have
just been listed.
pH and osmotic pressure determination
Both the PTX-PEG-HSA and PTX-HSA nanosuspensions
exhibited pH values around 7.4 and osmotic pressures in the
range of 300–340 mOsm/kg, which agrees with the optimal
measurements for IV injections.
Morphology observation
TEM and AFM were used to visualize the size and morphology
of PTX-PEG-HSA particles. As shown in Figure 2, the mor-
phology of both the nanosuspensions was spherical, and the
particles were highly dispersed with diameters approximately
100 nm. It was also noted that the particle size observed by
TEM and AFM is smaller than that determined by DLS.
In the DLS measurement, the hydrated dynamic diameter
of NPs was measured, ie, the colloidal hydrate layer is
calculated as one part of “diameter”. In the microscopy
determination, the colloidal hydrate layer is dried and the
observed true diameter decreased. Therefore, it makes sense
that the measurements observed with dehydrated samples in
TEM and AFM would be smaller than those determined with
hydrated samples during DLS.
DSC and WAXD analysis
To investigate the physical state of PTX in the nanosus-
pensions, DSC and WAXD analysis were conducted for
PTX-PEG-HSA with PEG, HSA, PTX, a physical mixture
of HSA + PTX, and a physical mixture of HSA + PTX-PEG
as controls. DSC diagrams of the six samples are shown in
Figure 3A. As illustrated in Figure 3A(a), the endothermic
peak at 217.3°C and exothermic peak at 244.5°C were
characteristic peaks of PTX in crystalline state, while the
exothermic peak at 208.9°C in Figure 3A(b) represented
the peak of decomposed HSA. Figure 3A(c) exhibited the
endothermic peak of PEG at around 50°C. Additionally, an
exothermic peak at 245.8°C was seen for the physical mix-
ture of HSA + PTX (Figure 3A(d)), but was nearly absent
for HSA + PTX-PEG (Figure 3A(e)) and PTX-PEG-HSA
nanosuspensions (Figure 3A(f)). All results conclude that
the PTX were physically encapsulated in these nanosus-
pensions with reduced crystallinity. The results of WAXD
analysis in Figure 3B further confirmed the existing state
Table 1 Physicochemical characteristics of PTX-PEG-HSA and PTX-HSA nanosuspension
Groups DL (wt%)aEE (%)aSize (nm) PDIbZeta potential (mV) pH OP (mOsm/kg)c
PTX-PEG-HSA 9.1±0.2 90.5±0.6 123.0±2.1 0.11 -43.6±1.8 7.3±0.1 322±4.2
PTX-HSA 9.2±0.1 91.4±0.5 137.2±1.9 0.14 -39.9±3.2 7.3±0.2 325±6.2
Notes: aMean diameters of the nanosuspensions, detected by DLS. bPolydispersity index (PDI) of the nanosuspensions, detected by DLS. cOsmotic pressure (OP) of the
nanosuspensions.
Abbreviations: PTX, paclitaxel; PEG, polyethylene glycol; HSA, human serum albumin; DLS, dynamic light scattering; DL, drug loading; EE, encapsulation efciency.

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Figure 2 (A) TEM images of free PTX (a), PTX-PEG-HSA (b), and PTX-HSA (c), and (B) the AFM images of PTX-PEG-HSA.
Abbreviations: TEM, transmission electron microscopy; PTX, paclitaxel; PEG, polyethylene glycol; HSA, human serum albumin; AFM, atomic force microscopy.
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Figure 3 (A) DSC thermograms of (a) PTX; (b) lyophilized HSA; (c) PEG; (d) a physical mixture of HSA + PTX; (e) a physical mixture of HSA + PTX-PEG; (f) PTX-PEG-HSA.
(B) Powder X-ray diffraction patterns for (a) PTX; (b) lyophilized HSA; (c) PEG; (d) a physical mixture of HSA + PTX; (e) a physical mixture of HSA + PTX-PEG;
(f) PTX-PEG-HSA.
Abbreviations: DSC, differential scanning calorimetry; PTX, paclitaxel; PEG, polyethylene glycol; HSA, human serum albumin.

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A toxic organic solvent-free technology for preparation of PTX-PEG-HSA
of PTX loaded in PTX-PEG-HSA nanosuspensions. As
shown in Figure 3B(a), PTX had three intense peaks at 5°,
9°, and 12° and small peaks at 15°–30°. The HSA gave a
broad and weak peak at 15°–30°. The typical crystal peaks
of PTX and HSA were all present in the WAXD diagram of
a physical mixture of PTX + HSA. However, the PTX-PEG-
HSA nanosuspensions possessed negligible characteristic
peaks of PTX and exhibited one obvious peak, similar to
that of free HSA, which also demonstrated that the PTX
were encapsulated in nanosuspensions with a significantly
reduced crystalline state.45
Compatible stability for clinical
application
The lyophilized powders of this formulation are usually
redissolved by 0.9% sodium chloride or 5% glucose injection
for clinical application. Therefore, the stability and compat-
ibility of resuspensions are a crucial criterion for formulation
design. In this study, PTX-PEG-HSA redissolved in 0.9%
sodium chloride or 5% glucose injection was analyzed for the
amount of unincorporated PTX and particle size. As listed in
Table 2, there was no significant change of the two indexes
within 24 hours, suggesting that both the 0.9% sodium chlo-
ride and 5% glucose injections can be used for resuspending
the PTX-PEG-HSA from lyophilized powders.
In vitro drug release
Drug release rate was determined to examine whether a
burst release pattern was present, or whether the drug could
be slowly dissipated from the polymer matrix for exerting
potency. Therefore, PTX-PEG-HSA and PTX-HSA nano-
suspensions were incubated in PBS at pH 7.4, and the PTX
release profiles were determined. As shown in Figure 4A,
both PTX-PEG-HSA and PTX-HSA nanosuspensions
exhibited steady and continual release profiles with no initial
burst release. The results suggested that PTX molecules were
indeed encapsulated into the hydrophobic micropockets of
HSA and not lodged on the particle surface. This finding
agreed with the results of DSC and WAXD analysis. To
simulate the in vivo drug release, the release evaluation of
PTX-PEG-HSA and PTX-HSA in 10% serum supplemented
medium was also conducted. The results shown in Figure 4B
exhibited a similar slow drug release behavior of PTX-PEG-
HSA within 24 hours to that obtained in PBS solution and
an increased drug release from PTX-HSA as compared with
that in PBS solution. These results indicated that PEG in
PTX-PEG-HSA could protect NPs from protein interaction
and the PTX-PEG-HSA could achieve a less drug preleakage
during blood circulation, therefore improving the circulative
stability and also mitigating side effects. Another point worth
noting is that the amount of PTX released from PTX-HSA
was lower than that from PTX-PEG-HSA over the same time
intervals in PBS without serum. Within the first 4 days, only
approximately 35% of PTX was released from PTX-HSA,
while the release amount in PTX-PEG-HSA group reached
up to 53%. This result may be attributed to the insertion of
PEG within PTX-PEG-HSA nanosuspensions that furthers
drug diffusion. Finally, the cumulative PTX release per-
centage of both preparations reached 80% after 10 days in
serum-free media, indicating successful PTX release from
designed nanosuspensions.
In vitro cellular studies
Cellular uptake and intracellular distribution
With confirmed physicochemical properties of PTX-PEG-
HSA nanosuspension, investigation into the cellular uptake
behavior of the nanosuspension using a C6 as fluorescent
marker was conducted. The results from flow cytometry
analysis are shown in Figure 5A. MDA-MB-231 cells incu-
bated with the C6-PTX-PEG-HSA and C6-PTX-HSA both
exhibited fluorescence of C6 at 1 and 4 hours, indicating
a time-dependent cellular internalization mechanism.
Meanwhile, the fluorescence intensity in the PTX-PEG-
HSA group was approximately equal to that in PTX-HSA
group at 1 and 4 h, respectively. This result demonstrates
that the PEGylation did not adversely affect the cell uptake
of PTX-PEG-HSA, which could be mediated through the
60 KD glycoprotein (gp60) or SPARC receptor on the cell
membrane.46 The highly effective cellular internalization of
PTX-loaded nanosuspensions was also confirmed via CLSM
analysis. The time dependence of endocytosis and strength
of intracellular fluorescence is shown in Figure 5B and is in
agreement with the results from flow cytometry analysis. In
addition, the internalized fluorescence of C6 was distributed
Table 2 The compatible stability of PTX-PEG-HSA with 5%
glucose and 0.9% sodium chloride injections (n=3)
Time (h) 5% glucose 0.9% sodium chloride
Size (nm) PTX
content (%)
Size (nm) PTX
content (%)
0122.5±2.9 100.0±3.2 124.5±2.2 100.0±2.1
8124.7±7.9 100.1±5.1 125.2±4.6 100.8±3.0
12 125.8±6.1 101.1±1.2 128.4±5.9 99.7±7.7
18 128.4±9.2 100.8±2.5 126.7±8.3 101.3±6.1
24 126.9±3.2 99.1±1.9 127.4±6.5 101.2±3.8
Abbreviations: PTX, paclitaxel; PEG, polyethylene glycol; HSA, human serum
albumin.

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supplemented media at pH 7.4.
Note: Data are presented as the mean ± SD (n=3).
Abbreviations: PTX, paclitaxel; PEG, polyethylene glycol; HSA, human serum albumin; PBS, phosphate-buffered saline; SD, standard deviation.
throughout the cytoplasm, the optimal location for PTX to
induce cytotoxicity. Therefore, all the results here indicated
that our designed nanosuspension could be quickly taken into
tumor cells and reside in the preferred intracellular location
for inducing optimal cytotoxicity.
Cytotoxicity and apoptosis assay
To further investigate whether the highly efficient intra-
cellular PTX delivery platform (PTX-PEG-HSA) could
achieve strong cytotoxicity against tumor cells, an MTT
assay of PTX-PEG-HSA against MDA-MB-231 cells was
performed using PEG-HSA and Taxol® as controls. After
24 hours of incubation, Taxol® had significantly higher IC50
values against MDA-MB-231 cells than PTX-HSA and PTX-
PEG-HSA (P,0.001, Figure 6A). This phenomenon could
be attributed to a number of reasons. First, Taxol® primarily
utilizes passive diffusion to enter the cell, and this is solely
dependent on the concentration gradient. On the other hand,
the two HSA-based nanosuspensions could be transported
into tumor cells through active binding of HSA to the gp60
or SPARC receptor on cell membrane. Furthermore, PTX in
the HSA-based nanosuspensions accumulated in the tumor
cells, while free PTX was rapidly secreted back outside the
cells.47 In addition, the IC50 of the PTX-PEG-HSA group was
1.76-fold lower than that of PTX-HSA group. As stated, the
higher cytotoxicity of PTX-PEG-HSA on tumor cells may be
attributed to its faster drug release behavior, which has been
confirmed by the in vitro drug release experiment.
The significant inhibitory effect of PTX-PEG-HSA
on MDA-MB-231 cells was also demonstrated by an
AnnexinV-FITC/PI double staining assay, and cell apoptosis
was measured via flow cytometry. The representative images
obtained from flow cytometry shown in Figure 6C mirrored
results from the MTT assay. In detail (Figure 6B), the early
apoptosis (Annexin V+/PI-) value of MDA-MB-231 cells
treated with PTX-PEG-HSA, PTX-HSA, and Taxol® was
12.9%, 10.6%, and 10.6%, respectively. The percentage of
late apoptosis/necrosis cells (AnnexinV+/PI+) was 10.3%,
6.69%, and 3.45%, respectively. These results indicated
that PTX-PEG-HSA could induce elevated apoptosis over
PTX-HSA and Taxol®.
In vivo antitumor activity
To investigate the in vivo antitumor potential of PTX-PEG-
HSA nanosuspensions, the antitumor efficacy was further
evaluated on BALB/c nude mice bearing MDA-MB-231
xenografts with PTX-HSA and Taxol® as controls. The
changes of tumor volume over 20 days of treatment and
the IR (%) values of these PTX formulations calculated on
the basis of tumor weight are depicted in Figure 7A and B,
respectively. As indicated, all PTX formulations exhibited
effective inhibition on tumor growth when compared with
saline group. However, the PTX-PEG-HSA nanosuspension
achieved the strongest tumor inhibition of all four groups,
with an IR value of 89.24%, followed by Taxol® (79.96%)
and PTX-HSA (75.08%).
Furthermore, the remarkable antitumor efficacy of the
PTX-PEG-HSA nanosuspensions is also well supported by
H&E histological evaluation and TUNEL assay. Unlike the
tumors in saline control group with histologic characteristics

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A toxic organic solvent-free technology for preparation of PTX-PEG-HSA
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Figure 5 Flow cytometry (A) and CLSM (B) measurements of the intracellular uptake of C6-PTX-PEG-HSA and C6-PTX-HSA at 1 and 4 hours in MDA-MB-231 cells.
Note: Results were expressed as the mean ± SD (n=5).
Abbreviations: CLSM, confocal laser scanning microscopy; PTX, paclitaxel; PEG, polyethylene glycol; HSA, human serum albumin; SD, standard deviation; C6, Coumarin 6.
including hyper chromatic nuclei, scant cytoplasm, and
closely arranged tumor cells, the tumors of mice treated
with PTX-PEG-HSA, PTX-HSA or Taxol® all exhibited
cell shrinkage and intercellular blank (Figure 8). Yet,
tumors treated with PTX-PEG-HSA exhibited the great-
est degree of apoptosis observed through TUNEL assay
and the lowest tumor cellularity. The enhanced efficacy of
these two HSA-based nanosuspensions over Taxol® might

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Figure 6 Cytotoxicity and apoptosis assay of MDA-MB-231 cells incubation with different formulations.
Notes: (A) Viability of MDA-MB-231 cells after incubation with different formulations at different concentrations after 24 hours. The values are represented as mean ± SD
(n=5). *P,0.01 versus Taxol®, **P,0.05 versus PTX-HSA. (B) Different counts of apoptosis and necrotic cells after treatment with different formulations. *P,0.01.
(C) Representative results of Annexin V-FITC/PI double labeling assay. Cells were treated with saline (a), PTX-PEG-HSA (b), PTX-HSA (c), or Taxol® (d) for 24 hours.
Early apoptosis (AnnexinV+/PI-), late apoptosis and necrotic cells (AnnexinV+/PI+) are shown.
Abbreviations: PTX, paclitaxel; PEG, polyethylene glycol; HSA, human serum albumin; SD, standard deviation; PI, propidium iodide; FITC, uorescein isothiocyanate.

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A toxic organic solvent-free technology for preparation of PTX-PEG-HSA
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37;3(*+6$
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6DOLQH
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6DOLQH 37;+6$ 37;3(*
+6$
7D[RO
Figure 7 Tumor growth inhibition (A), tumor weight (B), and the percentage of tumor weight IR observed in MDA-MB-231 tumor-bearing nude mice treated with saline,
Taxol®, PTX-HSA, and PTX-PEG-HSA (n=5). *P,0.01, **P,0.05.
Abbreviations: IR, inhibition rate; PTX, paclitaxel; HSA, human serum albumin; PEG, polyethylene glycol.
6DOLQH
+(781(/
37;+6$ 37;3(*+6$7D[RO
Figure 8 H&E staining and TUNEL analysis of tissue sections of tumor excised from the mice 20 days after treatment.
Abbreviations: PTX, paclitaxel; HSA, human serum albumin; PEG, polyethylene glycol; H&E, hematoxylin and eosin; TUNEL, terminal deoxynucleotidyl transferase-
mediated nicked labeling.
be explained by the combination of EPR effect and HSA
binding receptor-mediated cellular internalization, which
significantly improved PTX accumulation in tumors.48,49
Moreover, the additional PEGylation of PTX-PEG-HSA
prolonged the in vivo half-life of particles in circulation
through its unique “stealth function” to prevent RES rec-
ognition, thereby leading to a further increased target dis-
tribution of PTX into tumors. Finally, the accelerated drug
release behavior of PTX-PEG-HSA over PTX-HSA further
enhanced the induction of apoptosis. Consequently, a more
effective anticancer activity was achieved by PTX-PEG-
HSA compared with PTX-HSA.
It is known that in vivo toxicity evaluation is essential for
systemic drug delivery. Therefore, we investigated animal
behavior and body weight during formulation treatments.
First, all mice in PTX-PEG-HSA and PTX-HSA groups
showed normal behavior during the entire treatment, while
the mice treated with Taxol® frequently demonstrated
decreased activity and appetite. Additionally, mice in the
Taxol® group exhibited significant weight loss (Figure 9). In
contrast, mice receiving HSA formulation-based treatments
revealed negligible changes in body weight. The obvious side
effects of Taxol® treatment have already been well explained
by the dose-limiting toxicity of the nonionic surfactant

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Yin et al
Cremophor® EL alongside the nonspecific distribution of
PTX.50 The marked reduction of systemic toxicity achieved
by HSA nanosuspensions was mainly due to the effective
encapsulation of PTX into the HSA-based particles, which
avoided Cremophor EL-related side effects. Furthermore, the
well-defined selective accumulation of HSA nanosuspensions
in tumor sites through both the EPR and protein-receptor-
mediated endocytosis enhanced the tumor specificity and
reduced systemic cytotoxicity.51 Additionally, the HSA-based
particles demonstrated decreased leakage of PTX into the
bloodstream during circulation as compared with Taxol®,
which further mitigated side effects.52
Conclusion
The solid-dispersion technology has allowed the formulation
of PTX-PEG-HSA, a powerful nanosuspension-based deliv-
ery system for PTX that circumvents the need for organic
solvents and Cremophor EL. PTX molecules were success-
fully dispersed by PEG through co-melting, followed by
combination, and homogenized with HSA solution. Particles
were confirmed to be of a small size (123.0 nm) with smooth
spherical morphology. Furthermore, the PTX molecules in
PTX-PEG-HSA were dispersed in a molecular or amorphous
state. The stability of this formulation was evident through
the steady, continued drug release behavior without an initial
burst release effect, which suggested the promising potential
of PTX-PEG-HSA as a safe systemic PTX delivery platform.
The high cellular internalization of PTX-PEG-HSA as
demonstrated by flow cytometry and CLSM analysis shows
that stable NPs could still be internalized into the cell. In
addition, the strong cytotoxicity of PTX-PEG-HSA against
MDA-MB-231 cells indicates that encapsulated PTX can
be released in the intracellular environment for significant
antitumor efficacy. More importantly, in vivo antitumor
investigation on nude mice bearing MDA-MB-231 cancer
xenografts further confirmed that PTX-PEG-HSA achieved
improved anticancer activity and reduced systemic toxic-
ity over PTX-HSA and Taxol®. The use of PEG and HSA
receptor-mediated targeting has allowed for prolonged blood
circulation and significant increases in tumor uptake, all of
which have translated to enhanced antitumor efficacy with
decreased collateral damage to healthy tissues. Thus, PTX-
PEG-HSA without dependence on toxic organic solvents
proves a greater alternative to commonly accepted PTX
delivery systems. Future work in this direction should focus
on in vivo pharmacokinetics and biodistribution of PTX-
PEG-HSA on tumor-bearing mice, as well as evaluation
of the safety and activity of the delivery system in a more
clinically accurate model.
Acknowledgments
This work was supported by the project of the National
Natural Science Foundation of China (number 81102397),
the Natural Science Foundation of Jiangsu Province (number
BK2012761), Qing Lan Project of Jiangsu Province (number
02432009), Innovative Project for Graduate Cultivation
of Jiangsu Province (number CXZZ11-0807), the Project
Program of State Key Laboratory of Natural Medicines,
China Pharmaceutical University (numbers JKGQ201107,
JKPZ2013004), Key New Drug Innovation Project from
the Ministry of Science and Technology of China (number
2009ZX09310004), Fostering Plan of University Scientific
and Technological Innovation Team of Jiangsu Qing Lan
Project (2014), and National Basic Research Program of
China (973 Program, number 2009CB903300).
Disclosure
The authors report no conflicts of interest in this work.
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