Self-assembled nanoparticles from hyaluronic acid-paclitaxel prodrugs for direct cytosolic delivery and enhanced antitumor activity

Article abstract of DOI:10.1016/j.ijpharm.2015.07.069

A prodrug-based nanosystem obtained by formulating prodrug and nanotechnology into a system is one of the most promising strategies to enhance drug delivery for disease treatment. Herein, we report a new nanosystem based on HA-PTX conjugates (HA-PTX Ns),

Full text of DOI:10.1016/j.ijpharm.2015.07.069

Accepted Manuscript
Title: Self-assembled nanoparticles from hyaluronic
acid-paclitaxel prodrugs for direct cytosolic delivery and
enhanced antitumor activity
Author: Chaoran Xu Wei He Yaqi Lv Chao Qin Lingjia Shen
Lifang Yin
PII:
S0378-5173(15)30087-9
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.ijpharm.2015.07.069
IJP 15073
To appear in:
International Journal of Pharmaceutics
Received date:
Revised date:
Accepted date:
24-5-2015
18-7-2015
27-7-2015
Please cite this article as: Xu, Chaoran, He, Wei, Lv, Yaqi, Qin, Chao, Shen, Lingjia,
Yin, Lifang, Self-assembled nanoparticles from hyaluronic acid-paclitaxel prodrugs
for direct cytosolic delivery and enhanced antitumor activity.International Journal of
Pharmaceutics http://dx.doi.org/10.1016/j.ijpharm.2015.07.069
This is a PDF file of an unedited manuscript that has been accepted for publication.
As a service to our customers we are providing this early version of the manuscript.
The manuscript will undergo copyediting, typesetting, and review of the resulting proof
before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that
apply to the journal pertain.

Self-assembled nanoparticles from hyaluronic acid-paclitaxel prodrugs for direct
cytosolic delivery and enhanced antitumor activity
Chaoran Xu a,1, Wei He a, *, Yaqi, Lv a, Chao Qin a, Lingjia Shen b, Lifang Yin a,*
a
Department of Pharmaceutics, School of Pharmacy, China Pharmaceutical University, Nanjing
210009, PR China
b National Engineering and Research Center for Target Drugs, Lianyungang, 222047, PR China
*To whom correspondence should be addressed: He, W and Yin, LF.
Department of Pharmaceutics, School of Pharmacy, China Pharmaceutical University, Nanjing,
210009, PR China. Tel/Fax: +862583271018; E-mail: weihe@cpu.edu.cn (He, W) and
lifangyin@hotmail.com (Yin, LF).
Graphical abstract
1

Abstract
A prodrug-based nanosystem obtained by formulating prodrug and nanotechnology into a system is one
of the most promising strategies to enhance drug delivery for disease treatment. Herein, we report a
new nanosystem based on HA-PTX conjugates (HA-PTX Ns), which penetrated across cell membranes
into cytosol, thus enhancing paclitaxel (PTX) delivery. HA-PTX Ns were successfully obtained based
on HA-PTX, and their average particle size was approximately 200 nm. Importantly, unlike other
prodrug-based nanosystems, HA-PTX Ns obtained cellular entry without entrapment within the
lysosomal-endosomal system by using pathways including clathrin-mediated endocytosis,
microtubule-associated internalization, macropinocytosis and cholesterol-dependence. Due to
significant accumulation in tumors, HA-PTX Ns had more than a 4-fold decrease in tumor volume on
day 14 in contrast with PTX alone. In conclusion, HA-PTX Ns could enter cells, bypass the
lysosomal-endosomal system and improve PTX delivery.
Chemical compounds studied in this article
Paclitaxel (PubChem CID: 44155032); Fluorescein isothiocyanate (PubChem CID: 18730); MTT
(PubChem CID: 16218671); IR783 (PubChem CID: 46873808); Diphenylphosphinic chloride
(PubChem CID: 73910); Adipic acid dihydrazide (PubChem CID: 66117); Succinic anhydride
(PubChem CID: 7922); Carbodiimide hydrochloride (PubChem CID: 15908); triethylamine (PubChem
CID: 8471); Pyridine (PubChem CID: 1049).
Keywords: self-assembly, nanoparticles, prodrug, paclitaxel, cytosolic delivery, internalization
pathway, antitumor activity
2

1. Introduction
Nanosystems for drug delivery (NsDD) possess great potential to improve the therapeutic effect of
active compounds due to advantages such as enhanced solubility and drug stability, small nanoparticle
size, the ability to target drugs to the disease site, changes in pharmacokinetics and tissue
biodistribution and reduced side effects (Ashley et al., 2011; Cheng et al., 2012; Davis et al., 2008).
However, conventional nanocarriers suffer from drawbacks such as poor stability in physiological
conditions, a high tendency to recrystallize during storage, low drug-loading efficiency, premature
burst release and poor target site accumulation, therefore resulting in limitations of clinical applications
(Duhem et al., 2014; Mura et al., 2015; Zhang et al., 2013). Thus, novel NsDD that can overcome these
drawbacks are highly desirable.
Prodrug-based NsDD that integrate prodrug strategies and nanotechnology into a system are some of
the most promising ways to enhance the delivery of active compounds owing to their advantages
including enhanced drug availability in target sites, high drug-loading capacity, noncrystallization upon
encapsulation and controlled and prolonged drug release (Fang and Al-Suwayeh, 2012; Luo et al.,
2014). Generally, the currently available prodrug-based NsDD can be classified into three categories: (i)
prodrug-nanoparticles prepared by formulating the prodrug into the nanocarriers via noncovalent
interactions, (ii) small molecular weight prodrug-nanoparticles obtained by self-assembly of active
compounds and (iii) NsDD based on amphiphilic polymer-drug conjugates, which are prepared by
self-assembling the conjugates into nanoscaled particles. Due to the exceptionally high drug loading
and excellent stability under physiological environments, NsDD based on polymer-drug conjugates are
more efficient for drug delivery (Cai et al., 2015; Shen et al., 2010).
3

Paclitaxel (PTX), a highly hydrophobic drug with water-solubility less than 0.24 mg/L, is one of the
most commonly used active compounds in clinics for cancer therapy. PTX suppresses microtubule
dynamic instability, which is required for cellular division, and induces apoptosis (Li et al., 2015b;
Schiff et al., 1979). However, like many other anticancer drugs, its clinical use is greatly limited by its
inherent drawbacks including poor solubility, adverse side effects and poor tumor penetration (He et al.,
2015b). In order to solubilize PTX, Cremophor EL and dehydrated ethanol have to be added into the
product formulation, Taxol. However, the presence of Cremophor EL in Taxol would in turn generate
severe side effects, such as allergy, hypersensitivity, and anaphylactic reactions, thus affecting 20−40%
of patients and compromising the therapeutic index for PTX. Therefore, the development of a
Cremophor EL-free PTX formulation is urgent.
Hyaluronic acid (HA), a naturally occurring biocompatible polyanionic polysaccharide, is an anionic
biopolymer made up of alternating disaccharide units of D-glucuronic acid and N-acetyl-d-glucosamine
with a β (1 → 4) interglycosidic linkage (Yang et al., 2015). Due to its well-tolerated and
biocompatible nature, HA has been widely studied for biomedical applications including tissue
engineering, drug delivery and molecular imaging (Li et al., 2015a; Martens et al., 2015). Importantly,
HA is also utilized as a targeting ligand in drug carriers because it can effectively bind to the CD44
receptor, which is overexpressed in cancer cells (Liu et al., 2011; Zhong et al., 2015), thus achieving
active targeting for anticancer drug delivery.
Herein, we reported a new NsDD based on HA-PTX conjugates. Indeed, other groups reported that
HA-PTX is active for tumor therapy (Luo et al., 2000); however, few reports indicated that HA-PTX
conjugates could form nanoparticles. Here, we demonstrate that HA-PTX conjugates can self-assemble
4

into nanoparticles in aqueous conditions. Importantly, we demonstrated that HA-PTX Ns bypassed the
lysosomal-endosomal system, were well taken up by cancer cells and were located in cytosol and
nucleus. To our best knowledge, NsDDS that can obtain cellular entry without entrapment within the
lysosomal-endosomal system have not been reported until now. Such a discovery would be beneficial
to enlarge the application of based-polymer-drug conjugate nanosystems, severing as carriers for
targeting delivery of protein or gene drugs into the cytosol. Such NsDDS also enhanced antitumor
activities compared with free PTX, thus achieving active tumor therapeutic targeting without additional
modification of other ligands. Using HA-PTX self-assembled nanopartiles is a promising strategy for
enhancing PTX delivery for tumor treatment.
2. Methods and materials
2.1. Materials
PTX (99% purity) was obtained from Yew Biotechnology Co. Ltd. (Jiangsu, China). Taxol (marked
product of PTX) was from Bristol-Myers Squibb (China) Investment Co., Ltd. (Shanghai, China). IR
783 probe (90% purity), fluorescein isothiocyanate (FITC, 98% purity), hyaluronidase and MTT (98%
purity) were purchased from Sigma-Aldrich Co., Ltd. (St. Louis, MO, USA). Hyaluronic acid,
n-hydroxy two imide, diphenyl phosphoryl chloride, adipic acid dihydrazide and succinic anhydride
were purchased from Aladdin Industrial Inc. (Shanghai, China). The H22 cell line was purchased from
Nanjin Key GEN Biotech Co., Ltd. (Nanjing, China). Anhydrous pyridine, carbodiimide hydrochloride
(EDC) and triethylamine were from Lingfeng Chemical Reagent Co., Ltd. (Shanghai, China). Fetal
bovine serum, HBS, RPMI-1640, Dulbecco’s modified Eagle medium and trypsin were obtained from
Thermo Fisher Scientific Inc. (Waltham, MA, USA). DAPI and LysoTracker Red were obtained from
5

Beyotime Institute of Biotechnology (Haimen, China). All of the other chemicals were of analytical
reagent grade and were obtained from Sinopharm Chemical Reagent (Shanghai, China).
Male ICR mice (18–22 g) were purchased from the College of Veterinary Medicine at Yangzhou
University (License No: SCXK (Su) 2012-0004, Yangzhou, China). The animals used in the
experiments received care in compliance with the Principles of Laboratory Animal Care and the Guide
for the Care and Use of Laboratory Animals. The experiments followed the protocol approved by the
China Pharmaceutical University Institutional Animal Care and Use Committee.
2.2. Preparation of and characterization of HA-PTX Ns
2.2.1. Synthesis of PTX-NHS
The synthesis of n-hydroxy two imide two phenyl phosphate (SDPP) was performed as described
below. In brief, 230 mg n-hydroxy two imide, 34 μL diphenyl phosphoryl chloride and 280 μL
triethylamine were added into dichloromethane in order, the mixture was stirred for 12 h under room
temperature, and the reaction mixture was placed into a mortar and ground with ethyl ether.
Subsequently, the deposition was dissolved with acetic ether and washed with water and
saturated sodium chloride solution. Finally, the organic phase was mixed with anhydrous sodium and
dried under reduced pressure.
The synthesis of PTX-semi succinyl salt (PTX-semi) was performed as described below. Briefly, 540
mg PTX and 76 mg succinic anhydride were dissolved in dichloromethane. Upon addition of 513 μL
anhydrous pyridine, the reaction mixture was stirred for three days at room temperature. After
concentration under vacuum conditions, the sample was purified with column chromatography using
6

acetic ether and n-hexane (1:1, v/v) as eluents.
The synthesis of PTX-NHS was next. After dissolving 300 mg PTX-semi and 164 mg SDPP in
acetonitrile, the reaction mixture was mixed with 176 μL triethylamine and stirred at room temperature
for 24 h. Upon condensation under vacuum conditions, the mixture was separated with a mixture of
acetic ether and n-hexane (1:1, v/v) as eluents (Greenwald et al., 1996).
2.2.2. Synthesis of HA-adipic acid dihydrazide (HA-ADH)
Low molecular weight (MW) HA was prepared as follows: 2 g high MW HA (1.5 MDa) was first
dissolved in PBS (pH 6.5, 4 mg/mL), and then hyaluronidase (10 UI/mg) was added into the enzymatic
reaction solution, which was kept at 37 ˚C for 1 h and stopped at 90 ˚C. After that, the sample was
purified by dialyzing with a dialysis bag (MWCO = 3500 Da) and filtering through a 0.22-μm cellulose
acetate membrane. Finally, the purified sample was freeze-dried for use.
HA-ADH synthesis is listed below. ADH (52 mg) was placed into 15 mL low MW HA solution (3
mg/mL), and the reaction mixture was adjusted to pH 4.75 with 0.1 M HCl. It was then blended with
12 mg EDC and reacted for 24 h at pH 4.75. The reaction was stopped by pH regulation to 7.0 with 0.1
M sodium hydroxide. The sample was dialyzed successively against 0.1 M sodium hydroxide, 25%
ethanol/water and water (MW cut off 3.5 kDa). The purified sample was finally filtered through a
0.22-μm cellulose acetate membrane and freeze-dried.
2.2.3. Synthesis of HA-PTX conjugation
Thirty milliliters of 1 mg/mL HA-ADH in PBS was mixed with 2:1 (v/v) DMF/water containing 8 mg
PTX-NHS, and then the mixture was stirred for 24 h at room temperature. Subsequently, the reaction
7

mixture was paced into a dialysis bag (MWCO = 3500 Da) and dialyzed against 25% ethanol/water and
water. After filtration through a 0.22-μm membrane, the sample was freeze-dried (Luo et al., 2000).
The final activated conjugation and all of the intermediates were characterized by UV, IR and NMR
spectroscopy.
2.2.4. Preparation and characterization of HA-PTX Ns
Five milligrams HA-PTX conjugation was dispersed in 5 mL water, and the solution was sonicated for
10 min with probe ultrasonication (20–25 kHz, Ningbo Scientz Biotechnology Co. Ltd., China) at 100
W. The ultrasound pulse was turned off for 3 s with a period of 3 s. During the process, the temperature
was controlled by placing the sample in an ice-water bath. The final HA-PTX Ns was obtained by
filtering the sample through a 0.22-μm membrane.
Particle size and polydispersity index (PI) were determined with a 90Plus Particle Size Analyzer
(Brookhaven Instruments, Holtsville, NY) at room temperature based on the DLS principle. Raw data
were obtained over 5 min at an angle of 90°. The diameter of HA-PTX Ns was expressed with
intensity-weighted Gaussian distribution (with Chi-squared value <3).
HA-PTX Ns morphology was examined with TEM (JEM-1230, Tokyo, Japan) at an acceleration
voltage of 200 kV. In total, one drop of sample was placed on a copper mesh and dried at 25 °C after
removing the excessive sample with filter paper, and one drop of 2% (w/w) phosphotungstic acid was
then deposited on the copper and left for 1 min. After removing the extra phosphotungstic acid, the
copper was dried at room temperature for 10 min.
8

2.3. Cellular uptake and internalization pathway
H22 cells were seeded on 24-well plates at a density of 1×104 cells/well and cultured for 24 h at 37 °C.
Then, the cells were incubated with FITC labeled HA-PTX Ns (FITC-HA-PTX Ns) or PTX
(FITC-PTX) for 4 h at various FITC concentrations at 37 °C or 4 °C. At the end of the experiment, the
cells were washed three times with PBS, and the florescence intensity was measured with flow
cytometry (BD FACSCalibur, USA).
H22 cells were seeded on 6-well plates at a density of 1×105 cells/well and cultured for 24 h at 37 °C.
The culture medium was then replaced with fresh medium, which contained endocytotic pathway
inhibitors including cytochalasin-D (10 μg/mL), nystatin (10 μM), chlorpromazine (10 μg/mL),
nocodazole (20 μM), methyl-β-cyclodextrin (M-β-CD, 2.5 mM), monensin (200 nM) and NaN3 (10
mM) + deoxyglucose (DG, 50 mM). After treatment with the inhibitors for 30 min at 37 °C, the cells
were incubated with FITC-HA-PTX Ns for 4 h, in which the FITC concentration in each well was 2
μg/mL. Cellular fluorescence intensity was determined using flow cytometry after trypsinization.
2.4. Intracellular location
H22 cells were seeded on a 30-mm cell culture dish with a glass bottom at a density of 1×105 cells/dish
and cultured for 24 h. The cells were then treated with FITC-HA-PTX Ns or FITC-PTX for 4 h (2
μg/mL FITC in PBS). Subsequently, 1 μL Lyso-Tracker Red was added, and the cells were incubated
for 2 h, washed three times with PBS and stained with DAPI for 15 min. The cells were examined
using CLSM (LECIATCS SP5 II, Heidelberg, Germany).
9

2.5. In vitro cytotoxicity
To assess the cytotoxicity, the cell viabilities against HA and HA-ADH without PTX were studied. H22
cells were seeded on 96-well plates at a density of 5×103 cells/well and cultured for 24 h. The cells
were then incubated with HA and HA-ADH at various concentrations. Cell viability was evaluated
using an MTT assay.
2.6. Cell apoptosis assay
H22 cells were seeded on 12-well plates at a density of 1×105 cells/well and cultured for 24 h. The cells
were then incubated with HA-PTX Ns, Taxol or saline for 72 h at a PTX concentration of 1, 000 ng/mL.
The cell apoptosis was detected with an FCM analysis using an Annexin V-FITC/PI apoptosis
detection kit (Bender Medsystems, Vienna, Austria).
2.7. In vivo imaging
H22 tumor-bearing mice were prepared as follows: 0.2 mL cell suspension at a density of 1×107
cells/mL was injected subcutaneously into the scapula region of the mice. Treatment began as the
tumor size reached a volume of 10×10×5 mm3.
The mice were anesthetized with a 4% novochlorhydrate solution, and fluorescence images were taken
with an in vivo imaging system (In-Vivo FX PRO, Carestream, Canada) at 1, 2 and 4 h after
intravenous administration of 0.5 mg/kg IR783 via the tail vein.
10

2.8. Antitumor activity
H22 tumor-bearing mice were randomly divided into three groups (9 mice per group): saline, Taxol and
HA-PTX Ns. Mice were injected intravenously via the tail vein at a dose of 20 mg/kg of PTX based on
body weight. At predetermined time points, tumor volume was measured.
2.9. Statistical Analysis
The data are expressed as the mean ±standard deviation, and differences were considered to be
statistically significant when the P value was less than 0.05.
3. Results
3.1. Synthesis of HA-PTX
The HA-PTX preparation method is presented in Fig. 1. Two intermediates (PTX-NHS and HA-ADH)
were first obtained. PTX-NHS was synthesized by linking NHS to C2’-hydroxyl groups of PTX via
substitution and esterification reactions while HA-ADH was prepared through grafting free amino
groups of ADH to HA with EDC as a condensation agent. Subsequently, HA-PTX conjugates were
obtained via a nucleophilic reaction between free HA and PTX-NHS amino groups. The chemical
1
structures of intermediates and HA-PTX conjugates were confirmed using H NMR, UV and IR
spectroscopy (Figs. 2 and 3). As demonstrated in Fig. 2 (A and B), the dd split peaked at the methylene
proton at 2.33 and 2.12 ppm from succinimide and succinic acid, respectively, as indicated by
PTX-NHS formation, whereas the methylene proton signals at 1.3–1.4 ppm, suggesting grafting of
ADH to HA. However, in the 1H NMR spectra of HA-PTX (Fig. 2C), the characteristic peaks were not
observed, which might be because its MW was too high. Thus, the UV vis spectra were further used to
11

confirm HA-PTX structure. As depicted in Fig. 2(D), the characteristic peaks at 200 and 230 nm that
belonged to HA and PTX (Pouyani and Prestwich, 1994), respectively, were observed, therefore
demonstrating the presence of PTX and HA in the conjugations. HA-PTX, HA-ADH and PTX-NHS
formation was also verified by IR spectra. For PTX-NHS, there were two activated carbonyl groups,
which made two characteristic peaks appear at 1750–1850 cm-1. As indicated in Fig. 3(A), two
absorption peaks located at 1786.2 and 1818.1 cm-1 indicating SDPP were linked to PTX (Li et al.,
2011). In contrast, characteristic peaks for the carbonyl groups did not appear in HA-PTX with a
characteristic absorption related to HA at approximately 3500 cm-1, while other absorption peaks
exhibited as a simple superimposition of HA-ADH and PTX-NHS, therefore confirming the formation
of HA-PTX (Fig. 3 (B and C)). Calculating by the equation of PTX loading (%) = amount of PTX in
conjugation/amount of PTX addition × 100%, the drug loading in HA-PTX Ns was approximately
47.2%.
3.2. Preparation and Characterization of HA-PTX Ns
The procedure for HA-PTX Ns preparation is depicted in Fig. 1. HA-PTX Ns were prepared by
dispersing HA-PTX conjugations into aqueous solution followed by ultrasonication. The size in
HA-PTX Ns diameter as determined by the DLS principle was approximately 200 nm with a PI value
less than 0.3 (Fig. 4A), which thus suggested a narrow size distribution (He et al., 2013). To examine
the morphology and further confirm the particle size of HA-PTX Ns, TEM observation was performed.
As demonstrated in Fig. 4(B), spherical particles are displayed that have a diameter of 50–80 nm
without any aggregation. The smaller size, in contrast with the DLS data, might be because of the
procedure that was used for the TEM test sample preparation. The nanoparticle size during TEM
12

analysis would become smaller because of the high energy of the electron beam and thus production of
local heating in the particles (Blasi et al., 2011).
3.3. Cellular uptake and internalization pathway
The uptake of FITC labeled HA-PTX Ns or PTX in H22 cells at 37 °C or 4 °C is displayed in Fig. 5
(A). HA-PTX Ns or PTX uptake generally increased with an increase in the amount of the two PTX
formulations, indicating that uptake was concentration-dependent. Importantly, the fluorescence
intensity of FITC-HA-PTX Ns, despite of FITC concentrations and temperature, was significantly
higher than FITC-PTX, therefore indicating enhanced cellular uptake from the former. Interestingly,
irrespective of concentrations, considerable HA-PTX Ns uptake was also observed at 4 °C, which
indicated that HA-PTX Ns were taken up following a nonendocytic mechanism (He et al., 2015a;
Hillaireau and Couvreur, 2009).
To study internalization mechanisms，HA-PTX Ns uptake was assessed in H22 cells that had been
pretreated with inhibitors. Inhibitors such as CPZ, Cyto-D, nystatin, nocodazole, monensin, M-CD,
BFA and NaN3 with DG (NaN3+DG), which inhibited clathrin-mediated endocytosis, macropinocytosis,
caveolin-mediated
internalization,
microtubule-related
internalization,
lysosome-involved
internalization, cholesterol-dependence, Golgi apparatus-related internalization and energy-dependent
mechanisms, respectively, were involved in the experiment. As displayed in Fig. 5 (B), the fluorescence
intensity was reduced significantly by several inhibitors including CPZ, monensin, nystatin and M-CD.
These data thus indicated that cellular uptake was controlled by more than one pathway, which
included clathrin-mediated endocytosis, microtubule-associated internalization, macropinocytosis and
cholesterol-dependence.
13

3.4. In vitro cytotoxicity and cell apoptosis
To assess HA-PTX Ns cytotoxicity, cells were cultured with HA and HA-ADH without PTX for 24 h at
37 °C. As demonstrated in Fig. 6 (A), cell viability was greater than 90% irrespective of the
concentrations, thus indicating better compatibility.
An Annexin V-FITC/PI kit was used to stain the early and late apoptotic cells, and the percentage of
apoptotic cells was measured using FCM. The total percentage of apoptosis in H22 cells was 15.0%,
2.8% and 0.68% for HA-PTX Ns, Taxol and saline (Fig. 6B), respectively, thereby indicating that
HA-PTX Ns was most efficient in inducing apoptosis in cancer cells.
3.5. Intracellular location
To examine the intracellular localization of FITC-labeled HA-PTX Ns following their internalization,
the cellular distribution of FITC-HA-PTX Ns was determined by CLSM. As demonstrated in Fig. 7(A),
green fluorescence was observed throughout the cytosol and nucleus. In contrast, lysosomal staining of
the cells that had been treated with FITC-HA-PTX Ns did not display lysosomal localization of
HA-PTX Ns (Fig. 7B). These results thus confirmed that the cellular uptake of HA-PTX was not
involved in the lysosomal-endosomal system
3.6. In vivo imaging
To examine its tumor-targeting capacity, in vivo fluorescence imaging was performed in tumor-bearing
mice that had been injected with IR783-HA-PTX Ns at 1, 2 and 4 h post-administration. As
demonstrated in Fig. 8, strong fluorescence intensity in tumors was observed, with increased intensity
within 1 to 4 h after injection, therefore demonstrating significant HA-PTX Ns accumulation in tumors.
14

Indeed, besides the target tissue, HA-PTX Ns were also distributed into other tissues such as the liver
and brain.
3.7. Antitumor activity
PTX formulations, Taxol and HA-PTX Ns, were injected intravenously into H22 tumor-bearing mice
via the tail vein using saline as a control. Tumor volumes for each group were recorded, and the change
in tumor volume was calculated every three days to evaluate antitumor activity. The time versus tumor
volume is present in Fig. 9. Tumor volume in the saline group increased rapidly without any inhibition
of tumor growth. Tumor growth from the Taxol-injected group (a marked product) was significantly
suppressed after 14 days of administration. In contrast, tumor growth from the HA-PTX Ns-treated
group was slowed dramatically, and the tumor volume at each time point was markedly reduced,
leading to more than a 4- and 5-fold decrease on day 14 compared with Taxol and saline, respectively,
therefore indicating a significant improvement in antitumor activity.
4. Discussion
The HA-PTX conjugations could self-assemble into nanoparticles with a particle size of approximately
200 nm. HA-PTX conjugations were synthesized by coupling hydrophobic PTX-NHS molecules to the
hydrophilic HA with an esterification reaction. Due to the presence of a number of active groups
(–COOH) in the HA structural skeleton, a considerable amount of PTX molecules would be readily
grafted onto the polymer, forming a comb-like copolymer prodrug and resulting in a high drug payload
(Li et al., 2011; Luo et al., 2014). Importantly, the HA-PTX conjugations, which have hydrophobic
moieties and a hydrophilic skeleton and thus have an amphiphilic nature, can act as an amphiphilic
15

material and therefore self-assemble into supramolecular nanostructures in aqueous conditions via
noncovalent interactions. Such supramolecular nanostructures were based on HA-PTX conjugations,
which would facilitate PTX delivery because of advantages of the nanoparticle drug delivery system,
thereby allowing enhanced cancer therapy.
We have demonstrated that HA-PTX Ns were taken up well by cancer cells and were located in the
cytosol and nucleus without detainment by the lysosomal-endosomal system. It was evident that a large
amount of HA-PTX Ns obtained cellular uptake at 4 °C (Fig. 5A). Because of membrane fluidity
inhibition at low temperature, nanoformulation accumulation in cells can only rely on direct penetration
across the membrane into the cytosol (Lee et al., 2012). Moreover, the lysosomal staining of cells that
had been incubated with FITC-HA-PTX Ns did not exhibit the colocalization of FITC signals with
LysoTracker (Fig. 7B) therefore further confirming its cellular entry via penetration across the
membrane. It might be related to the hydrophobic area from the hydrophobic PTX with a relatively
high MW onto the surface of supramolecular nanostructures. Previous reports indicated that the
presence of specific hydrophobic content on the surface of nanomedicines such as protein-coated
nanoparticles and carbon nanotubes could help the nanomedicines obtain direct cytosol delivery via
cholesterol or lipid rafts from the interaction between the hydrophobic site and the cell membrane (He
et al., 2015a; Verma et al., 2008). As displayed in Fig. 5(B), the cholesterol-dependence pathway was
involved in the cellular uptake of HA-PTX Ns, which partly supports our speculation. Nevertheless,
more experiments should be performed to elucidate how the nanoparticles obtained cytosolic delivery.
HA-PTX Ns had a significant enhancement in antitumor activity compared with Taxol, which resulted
from a combination of passive and active targeting mechanisms. First, HA reportedly acted as a
16

targeting moiety that can specifically bind to the CD44 receptor, which is highly expressed in cancer
cells, thus allowing HA-conjugated nanoparticles or prodrugs to obtain enhanced cellular uptake and
cytotoxicity (Oh et al., 2010; Zhong et al., 2015). The uptake was confirmed by cellular uptake assays
and in vivo imaging. As depicted in Fig. 5(A), cellular accumulation of HA-PTX Ns was markedly
higher than PTX alone, irrespective of the drug concentrations. As well known, there are considerable
amounts of esterases in the cell cytosol and lysosomes (Zhang etal., 2013). Thus, Once HA-PTX Ns
enter the cells, the ester linkage in HA-PTX conjugations will be easily broken after exposure to
intracellular esterase, thereby leading to release of free PTX. A significant accumulation of HA-PTX
Ns in tumors was observed from the in vivo image performance (Fig. 8). The percentage of apoptosis
in H22 cells induced by HA-PTX Ns was 5-fold as great as that of Taxol (Fig. 6B). Second, nanosized
HA-PTX Ns facilitated the improved antitumor activity because they promoted penetration into the
tumor interstitium via leaky vasculature (Petros and DeSimone, 2010; Rodriguez et al., 2013). Indeed,
more experiments such as pharmacokinetics, biodistribution and biocompatibility should be performed
to study the in vivo performance of HA-PTX Ns and confirm their targeting mechanisms.
5. Conclusions
In this study, supramolecular nanostructures of approximately 200 nm in diameter based on the
self-assembly of HA-PTX conjugations in aqueous conditions were successfully obtained. Importantly,
HA-PTX Ns obtained cytosolic delivery via bypassing the lysosomal-endosomal system, which was
dependent on more than one internalization pathway. Few reports indicate that the nanosystems, when
self-assembled from polymer-drug conjugates, can penetrate across the cell membrane directly. Such a
discovery would help broaden the application of based-polymer-drug conjugate nanosystems, which
17

serve as carriers for biological macromolecular drugs that need to rapidly escape from the
lysosomal-endosomal system into the cytosol. As expected, HA-PTX Ns displayed a significant
enhancement in antitumor activity compared with Taxol owing to a combination of passive and active
targeting mechanisms. In conclusion, HA-PTX Ns can directly translocate across the membrane into
cytosol and is a promising strategy for enhancing PTX delivery for tumor treatment.
Acknowledgments
This study was supported by the National Natural Science Foundation of China (No. 81402869 and No.
81473152), the Natural Science Foundation of the Jiangsu Province (No. BK20140671), the Fostering
Plan of University Scientific and Technological Innovation Team of Jiangsu Qing LanProject (2014)
and the Creation of Major New Drugs National Major Projects (2014ZX09507004-001,
2014ZX09507004-002).
18

References
Ashley, C.E., Carnes, E.C., Phillips, G.K., Padilla, D., Durfee, P.N., Brown, P.A., Hanna, T.N., Liu, J.,
Phillips, B., Carter, M.B., Carroll, N.J., Jiang, X., Dunphy, D.R., Willman, C.L., Petsev, D.N., Evans,
D.G., Parikh, A.N., Chackerian, B., Wharton, W., Peabody, D.S., Brinker, C.J., 2011. The targeted
delivery of multicomponent cargos to cancer cells by nanoporous particle-supported lipid bilayers.
Nature Materials 10, 389–397.
Blasi, P., Giovagnoli, S., Schoubben, A., Puglia, C., Bonina, F., Rossi, C., Ricci, M., 2011. Lipid
nanoparticles for brain targeting I. Formulation optimization. International Journal of Pharmaceutics
419, 287–295.
Cai, K., He, X., Song, Z., Yin, Q., Zhang, Y., Uckun, F.M., Jiang, C., Cheng, J., 2015. Dimeric drug
polymeric nanoparticles with exceptionally high drug loading and quantitative loading efficiency.
Journal of the American Chemical Society 137, 3458–3461.
Cheng, Z., Al Zaki, A., Hui, J.Z., Muzykantov, V.R., Tsourkas, A., 2012. Multifunctional nanoparticles:
cost versus benefit of adding targeting and imaging capabilities. Science 338, 903–910.
Davis, M.E., Chen, Z., Shin, D.M., 2008. Nanoparticle therapeutics: an emerging treatment modality
for cancer. Nature Review Drug Discovery 7, 771–782.
Duhem, N., Danhier, F., Pourcelle, V., Schumers, J.M., Bertrand, O., Leduff, C.S., Hoeppener, S.,
Schubert, U.S., Gohy, J.F., Marchand-Brynaert, J., Preat, V., 2014. Self-assembling
doxorubicin-tocopherol succinate prodrug as a new drug delivery system: synthesis, characterization,
and in vitro and in vivo anticancer activity. Bioconjugate Chemistry 25, 72–81.
Fang, J.-Y., Al-Suwayeh, S.A., 2012. Nanoparticles as delivery carriers for anticancer prodrugs. Expert
19

Opinion on Drug Delivery 9, 657–669.
Greenwald, R.B., Gilbert, C.W., Pendri, A., Conover, C.D., Xia, J., Martinez, A., 1996. Drug delivery
systems:ꢀ water soluble taxol 2‘-poly(ethylene glycol) ester prodrugsdesign and in vivo effectiveness.
Journal of Medicinal Chemistry 39, 424–431.
He, W., Jin, Z., Lv, Y., Cao, H., Yao, J., Zhou, J., Yin, L., 2015a. Shell-crosslinked hybrid nanoparticles
for direct cytosolic delivery for tumor therapy. International Journal of Pharmaceutics 478, 762–772.
He, W., Lu, Y., Qi, J., Chen, L., Hu, F., Wu, W., 2013. Nanoemulsion–templated shell-crosslinked
nanocapsules as drug delivery systems. International Journal of Pharmaceutics 445, 69–78.
He, W., Lv, Y., Zhao, Y., Xu, C., Jin, Z., Qin, C., Yin, L., 2015b. Core–shell structured gel-nanocarriers
for sustained drug release and enhanced antitumor effect. International Journal of Pharmaceutics 484,
163–171.
Hillaireau, H., Couvreur, P., 2009. Nanocarriers' entry into the cell: relevance to drug delivery. Cellular
and Molecular Life Sciences: CMLS 66, 2873–2896.
Lee, J., Sharei, A., Sim, W.Y., Adamo, A., Langer, R., Jensen, K.F., Bawendi, M.G., 2012.
Nonendocytic delivery of functional engineered nanoparticles into the cytoplasm of live cells using a
novel, high-throughput microfluidic device. Nano Letters 12, 6322–6327.
Li, G.L., Liu, J.Y., Pang, Y., Wang, R.B., Mao, L.M., Yan, D.Y., Zhu, X.Y., Sun, J., 2011. Polymeric
micelles with water-insoluble drug as hydrophobic moiety for drug delivery. Biomacromolecules 12,
2016–2026.
Li, J., Yin, T., Wang, L., Yin, L., Zhou, J., Huo, M., 2015a. Biological evaluation of redox-sensitive
micelles based on hyaluronic acid-deoxycholic acid conjugates for tumor-specific delivery of paclitaxel.
20

International Journal of Pharmaceutics 483, 38–48.
Li, Y., Wu, Z., He, W., Qin, C., Yao, J., Zhou, J., Yin, L., 2015b. Globular protein-coated paclitaxel
nanosuspensions: interaction mechanism, direct cytosolic delivery, and significant improvement in
pharmacokinetics. Molecular Pharmaceutics 12, 1485–1500.
Liu, Y., Sun, J., Cao, W., Yang, J., Lian, H., Li, X., Sun, Y., Wang, Y., Wang, S., He, Z., 2011. Dual
targeting folate-conjugated hyaluronic acid polymeric micelles for paclitaxel delivery. International
Journal of Pharmaceutics 421, 160–169.
Luo, C., Sun, J., Sun, B., He, Z., 2014. Prodrug-based nanoparticulate drug delivery strategies for
cancer therapy. Trends in Pharmacological Sciences 35, 556–566.
Luo, Y., Ziebell, M.R., Prestwich, G.D., 2000. A hyaluronic acid−taxol antitumor bioconjugate targeted
to cancer cells. Biomacromolecules 1, 208–218.
Martens, T.F., Remaut, K., Deschout, H., Engbersen, J.F.J., Hennink, W.E., van Steenbergen, M.J.,
Demeester, J., De Smedt, S.C., Braeckmans, K., 2015. Coating nanocarriers with hyaluronic acid
facilitates intravitreal drug delivery for retinal gene therapy. Journal of Controlled Release 202, 83–92.
Mura, S., Bui, D.T., Couvreur, P., Nicolas, J., 2015. Lipid prodrug nanocarriers in cancer therapy.
Journal of Controlled Release. 208, 25–41.
Oh, E.J., Park, K., Kim, K.S., Kim, J., Yang, J.-A., Kong, J.-H., Lee, M.Y., Hoffman, A.S., Hahn, S.K.,
2010. Target specific and long-acting delivery of protein, peptide, and nucleotide therapeutics using
hyaluronic acid derivatives. Journal of Controlled Release 141, 2–12.
Petros, R.A., DeSimone, J.M., 2010. Strategies in the design of nanoparticles for therapeutic
applications. Nature Review Drug Discovery 9, 615–627.
21

Pouyani, T., Prestwich, G.D., 1994. Functionalized derivatives of hyaluronic-acid oligosaccharides -
drug carriers and novel biomaterials. Bioconjugate Chemistry 5, 339–347.
Rodriguez, P.L., Harada, T., Christian, D.A., Pantano, D.A., Tsai, R.K., Discher, D.E., 2013. Minimal
"Self" peptides that inhibit phagocytic clearance and enhance delivery of nanoparticles. Science 339,
971–975.
Schiff, P.B., Fant, J., Horwitz, S.B., 1979. Promotion of microtubule assembly in vitro by taxol. Nature
277, 665–667.
Shen, Y., Jin, E., Zhang, B., Murphy, C.J., Sui, M., Zhao, J., Wang, J., Tang, J., Fan, M., Van Kirk, E.,
Murdoch, W.J., 2010. Prodrugs forming high drug loading multifunctional nanocapsules for
intracellular cancer drug delivery. Journal of the American Chemical Society 132, 4259–4265.
Verma, A., Uzun, O., Hu, Y., Hu, Y., Han, H.S., Watson, N., Chen, S., Irvine, D.J., Stellacci, F., 2008.
Surface-structure-regulated cell-membrane penetration by monolayer-protected nanoparticles. Nature
Materials 7, 588–595.
Yang, C., Wang, X., Yao, X., Zhang, Y., Wu, W., Jiang, X., 2015. Hyaluronic acid nanogels with
enzyme-sensitive cross-linking group for drug delivery. Journal of Controlled Release 205, 206–217.
Zhang, H., Wang, J., Mao, W., Huang, J., Wu, X., Shen, Y., Sui, M., 2013. Novel SN38
conjugate-forming nanoparticles as anticancer prodrug: in vitro and in vivo studies. Journal of
Controlled Release 166, 147–158.
Zhong, Y., Zhang, J., Cheng, R., Deng, C., Meng, F., Xie, F., Zhong, Z., 2015. Reversibly crosslinked
hyaluronic acid nanoparticles for active targeting and intelligent delivery of doxorubicin to drug
resistant CD44+ human breast tumor xenografts. Journal of Controlled Release 205, 144–154.
22

Figure captions
Fig. 1. Flow chart of the procedure for HA-PTX conjugations and preparation of HA-PTX Ns.
Fig. 2. 1H-NMR spectra of (A) PTX-NHS, (B) HA-ADH and (C) HA-PTX; Ultraviolet spectrum of (A)
HA-PTX.
Fig. 3. Infrared spectra of (A) PTX-NHS, (B) HA-ADH and (C) HA-PTX.
Fig. 4. Characterization of HA-PTX Ns. (A) Particle size and distribution. (B) TEM picture. The scale
bar is 100 nm.
Fig. 5. (A) Uptake of PTX and FITC-HA-PTX Ns with different FITC concentrations in H22 cells at 4
or 37 ˚C. * represents p<0.01.
(B) Endocytic mechanism of HA-PTX Ns in H22 cells. The cells were incubated with FITC labeled
HA-PTX Ns (100 ng/mL) for 2 h in the presence of various inhibitors. * represents p<0.05. (n=5).
Fig. 6. (A) In vitro cytotoxicity of HA and HA-ADH (n=5). (B) H22 cell apoptosis induced by
HA-PTX Ns and Taxol, which saline was used as control.
Fig. 7. Location of FITC-labeled HA-PTX Ns in H22 cells that had been treated with PTX
formulations containing 2 μg/mL FITC for 4 h. (A) Nuclei (blue area) were stained with DAPI. (B)
Lysosomes (red area) were stained with LysoTracker Red.
Fig. 8. In vivo imaging of H22 tumor-bearing mice that had been injected with IR783 labeled HA-PTX
Ns at 1, 2 and 4 h. Each mouse was injected with IR783 labeled HA-PTX Ns at a dose of 200 ng
IR783.
Fig. 9. Tumor growth inhibition of free PTX and HA-PTX Ns using saline as a control, *, ** and ***
indicate p<0.05, p<0.01 and p<0.001, respectively.
23

24

25

26

27

28

29