Hyaluronic acid ion-pairing nanoparticles for targeted tumor therapy

Article abstract of DOI:10.1016/j.jconrel.2016.01.049

Hyaluronic acid (HA)-based doxorubicin (DOX) nanoparticles (HA-NPs) were fabricated via ion-pairing between positively charged DOX and negatively charged HA, which displayed near-spherical shapes with an average size distribution of 180.2 nm (PDI = 0.184). Next, HA-NPs were encapsulated in liposomal carriers to afford HA-based DOX liposomes (HA-LPs), which also showed near-spherical morphology with an average size of 130.5 nm (PDI = 0.201). HA-NPs and HA-LPs displayed desirable sustained-release profiles compared to free DOX, and moreover, HA-LPs were proven to prevent premature release of DOX from HA-NPs. Cell based studies demonstrated HA-NPs and HA-LPs were selectively taken up by CD44⁺ tumor cells, and DOX was released intracellularly to target the cell nuclei. Both HA-NPs and HA-LPs showed comparable levels of penetration efficiency in tumor spheroids. In vivo studies revealed that HA-NPs and HA-LPs significantly prolonged the blood circulation time of DOX, decreased accumulation in the normal tissues and enriched drugs into the tumors. Furthermore, HA-NPs and HA-LPs greatly enhanced therapeutic efficacy of DOX in tumor-bearing mice and minimized systemic toxicity against vital organs. In sum, HA-NPs and HA-LPs represent promising nanocarriers for CD44⁺ tumor-targeted delivery.

Full text of DOI:10.1016/j.jconrel.2016.01.049

Hyaluronic acid ion pairing nanoparticles for targeted tumor therapy
Wenhao Li, Xiaoli Yi, Xing Liu, Zhirong Zhang, Yao Fu, Tao Gong
PII:
DOI:
Reference:
S0168-3659(16)30044-X
doi: 10.1016/j.jconrel.2016.01.049
COREL 8105
To appear in:
Journal of Controlled Release
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Revised date:
Accepted date:
2 November 2015
19 January 2016
26 January 2016
Please cite this article as: Wenhao Li, Xiaoli Yi, Xing Liu, Zhirong Zhang, Yao Fu, Tao
Gong, Hyaluronic acid ion pairing nanoparticles for targeted tumor therapy, Journal of
Controlled Release (2016), doi: 10.1016/j.jconrel.2016.01.049
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ACCEPTED MANUSCRIPT
Hyaluronic acid ion pairing nanoparticles for targeted tumor therapy
*
*
Wenhao Li, Xiaoli Yi, Xing Liu, Zhirong Zhang, Yao Fu , Tao Gong
Key Laboratory of Drug Targeting and Drug Delivery Systems, Ministry of Education, West China
School of Pharmacy, Sichuan University, Chengdu 610041,China
*
Corresponding author.
Yao Fu
Key Laboratory of Drug Targeting and Drug Delivery Systems,
West China School of Pharmacy, Sichuan University,
No. 17, Section 3, Southern Renmin Road, Chengdu, 610041, China
Tel./fax: + 86 028 85503798. E-mail: yfu4@scu.edu.cn
Tao Gong
Key Laboratory of Drug Targeting and Drug Delivery Systems,
West China School of Pharmacy, Sichuan University,
No. 17, Section 3, Southern Renmin Road, Chengdu, 610041, China
Tel./fax: + 86 028 85501615. E-mail: gongtaoy@126.com
Graphical abstract
+
HA-NPs and HA-LPs have been developed to be selectively taken up by CD44 tumor cells,
which greatly improved antitumor efficacy and reduced systemic adverse effects in treating CD44
+
cancer.
1

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Abstract:
Hyaluronic acid (HA)-based doxorubicin (DOX) nanoparticles (HA-NPs) were fabricated via
ion-pairing between positively charged DOX and negatively charged HA, which displayed
near-spherical shapes with an average size distribution of 180.2 nm (PDI = 0.184). Next,
HA-NPs were encapsulated in liposomal carriers to afford HA-based DOX liposomes (HA-LPs),
which also showed near-spherical morphology with an average size of 130.5 nm (PDI = 0.201).
HA-NPs and HA-LPs displayed desirable sustained-release profiles compared to free DOX, and
moreover, HA-LPs were proven to prevent premature release of DOX from HA-NPs. Cell based
+
studies demonstrated HA-NPs and HA-LPs were selectively taken up by CD44 tumor cells, and
DOX was released intracellularly to target the cell nuclei. Both HA-NPs and HA-LPs showed
comparable levels of penetration efficiency in tumor spheroids. In vivo studies revealed that HA-NPs
and HA-LPs significantly prolonged the blood circulation time of DOX, decreased accumulation in
the normal tissues and enriched drugs into the tumors. Furthermore, HA-NPs and HA-LPs greatly
enhanced therapeutic efficacy of DOX in tumor-bearing mice and minimized systemic toxicity against
+
vital organs. In sum, HA-NPs and HA-LPs represent promising nanocarriers for CD44
tumor-targeted delivery.
Keywords: Hyaluronic acid (HA); Doxorubicin (DOX); Nanoparticle; Ion-pairing; Liposome
1
. Introduction
Hyaluronic acid (HA) is an anionic polysaccharide consisting of repeating disaccharide units of
N-acetyl-D-glucosamine (1-β-4) and D-glucuronic acid (1-β-3) (Fig. 1a) [1], which is a critical
component of the extracellular matrix [2, 3]. As a naturally derived macromolecule, HA has been
extensively used in biomedical applications due to excellent biocompatibility, biodegradability,
low-toxicity and low-immunogenicity. HA has been used as a moisture protectant in cosmetics and
medical fields over the past few decades. Moreover, HA and modified HA have been extensively used
for ophthalmic surgery [4], arthritis treatment [5], tissue engineering [6] and drug delivery [7, 8].
Recently, cancer cells were found to overexpress CD44 receptor, which is a principal cell-surface
receptor for HA [9-12]. HA was proven to specifically bind to surface CD44 receptors on cancer cells,
and thus, HA has been explored as a targeting ligand in tumor-targeted drug delivery [13-17]. As a
tumor-targeting vector, the main application of HA was in the following aspects. The structure of HA
can be modified to generate a variety of HA derivatives, which retain biodegradability and
compatibility with improved stability [18-21]. HA has rich carboxyl and hydroxyl groups which can be
used as sites of chemical modification. Eun Ju Oh et al designed and synthesized a series of HA
derivatives successfully for the real-time imaging of HA derivatives using quantum dots (QDot),
which confirmed the presence of CD44 receptors in human organs (liver, kidney and spleen) [22]. HA
was also used in the surface modification of nanoparticles or liposomes by chemical coupling to
achieve tumor targeting or improve biocompatibility [16, 23-26]. Eliaz et al modified the surface of
cationic liposomes with HA by chemical coupling, which greatly improved the tumor target ability of
liposomes [24-26]. Regarding modified HA derivatives, they become novel materials after chemical
modification, and thus the biocompatibility, biodegradability and cytotoxicity of the materials remain
to be further elucidated.
In the current study, we aimed to fabricate a HA-based nanoparticle that not only carries the
advantages of HA but also maintains its structural integrity. Classical polymeric nanoparticles
2

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displayed the core-shell structure that usually consists of a hydrophilic shell and a hydrophobic core.
Due to the presence of negatively charged carboxyl groups, HAs were hypothesized to interact with
positively charged hydrophobic drugs by forming ion pair complexes thus resulting in the formation
of self-assembled nanocomplexes with positively charged hydrophobic drugs in the core and
negatively charged hydrophilic HAs in the shell. Owing to its high potency and broad spectrum of
anti-tumor activity, doxorubicin (DOX) is a leading therapeutic in various cancer chemotherapy [27].
As a result, we selected DOX as the model drug (Fig. 1b), and fabricated HA-based DOX
nanoparticles (HA-NPs) via electrostatic interactions (Fig. 1c). In our preliminary study, HA-NPs
displayed poor stability during storage at room temperature and significant burst effect in the plasma.
Next, HA-NPs were further encapsulated in phospholipid bilayers to afford HA-based DOX liposomes
(HA-LPs) (Fig. 1d), which were designed to reduce premature release of DOX from HA-NPs. The
method for HA-NP fabrication was proven simple and convenient, which successfully maintained the
structural integrity of HA without any chemical modification.
Fig. 1. Chemical structure of (a) HA and (b) DOX. Schemes of (c) HA-NPs and (d) HA-LPs
fabrication.
2
2
. Materials and methods
.1. Materials
Sodium hyaluronate was obtained from Freda Biopharm Co., Ltd. (Shandong, China). DOX
hydrochloride was obtained from Huafeng Technology Co., Ltd. (Beijing, China). Cholesterol was
obtained from Kelong Company (Chengdu, China). Lipoid E80 was obtained from Lipoid Co., Ltd.
(
(
Ludwigshafen, Germany). Solutol HS 15 was obtained from BASF Aktiengesellschaft
Ludwigshafen, Germany). Soybean oil for injection was purchased from Tieling Medical Oil Co., Ltd.
Other reagents and chemicals were analytical grade and purchased commercially.
Wistar rats and Kunming mice were obtained from experiment animal center of Sichuan University
(Chengdu, China). All animal studies presented were performed per the institutional guidelines and
approved by the Ethics Committee of Sichuan University.
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2
.2. Preparation
DOX hydrochloride was neutralized by the following procedure. Briefly, 10 mg of DOX
hydrochloride was dissolved in 2 ml of deionized water. Then the pH value was adjusted to about 10
to remove the hydrochloride salt. The solution was extracted with dichloromethane. Then
dichloromethane was removed by rotary evaporator to yield DOX.
2
.2.1. Preparation of HA-NPs
As shown in Fig. 1c, 3.6 mg of HA and 50 mg of Solutol HS 15 were dissolved in 10 ml of
deionized water. 100 μl of soybean oil for injection was added to HA mixture and the reaction was
stirred for 10 min at room temperature. Then, 2 ml of ethanol solution containing 5 mg of DOX was
added dropwise to the above solution. This reaction was stirred for 30 min at room temperature. Next,
the mixture was maintained for 30 min at 45 ºC to evaporate the ethanol by rotary evaporator. The
obtained suspension was sonicated for 3 min at 265 W (5 s pulses and 5 s rest, ultrasonic cell crasher)
and homogenized 10 times at 7500 psi (AH 100D; ATS Engineering Inc, Canada). Finally, HA-NPs
suspension was freeze dried for 48 h at -45 ºC in the presence of 5% (w/v) of mannitol as the
freeze-drying protective agent.
2
.2.2. Encapsulation of HA-NPs in liposomes
HA-NPs were loaded into liposomes passively by using thin film dispersion method to obtain
HA-LPs (Fig. 1d) [28]. Briefly, lipoid E80 (80 mg) and cholesterol (40 mg) were dissolved in
dichloromethane at the mass ratio of 2:1, and dried to a thin phospholipid film under rotary
evaporation. Next, dichloromethane was removed by rotary evaporation under vacuum for 1 h. The
phospholipid films were hydrated in 10 mL of above prepared HA-NPs suspension. The hydrated
vesicle suspension was homogenized 10 times at 7500 psi and freeze-dried for 48 h at -45 ºC in the
presence of 5% (w/v) of mannitol as the freeze-drying protective agent.
2
.2.3. Preparation of DOX liposomes
Due to the presence of phospholipid bilayers on the surface of HA-LPs, the tumor targeting of
HA-LPs may be affected. To study the influence of phospholipid bilayers for tumor cell uptake and
cytotoxicity, we prepared DOX liposomes (DOX-LPs) as controls. DOX-LPs were prepared using
ammonium sulfate gradient method [29, 30]. Briefly, lipoid E80 (80 mg) and cholesterol (40 mg)
were dissolved in dichloromethane at the mass ratio of 2:1, and dried to a thin phospholipid film under
rotary evaporation. The residual dichloromethane was removed similarly as previously described. The
phospholipid films were hydrated in 10 mL of ammonium sulfate solution (300 mM). The hydrated
vesicle suspension was homogenized 10 times at 7500 psi. The excess ammonium sulfate was
separated using a Sephadex G-75 minicolumn and then gained blank liposomes suspension. Next, 2
ml of DOX solution (4 mg/mL) was drop wise added to the blank liposomes suspension. This reaction
was stirred at 55 ºC for 20 min and finally gained DOX-LPs suspension.
2
.3. Characterization
The particle size distribution and zeta potentials of HA-NPs and HA-LPs were determined by
dynamic light scattering (DLS) (Zetasizer Nano ZS90, Malvern, UK). The morphologies of HA-NPs
and HA-LPs were examined by transmission electron microscopy (H-600, Hitachi, Japan) with 2%
phosphotungstic acid staining.
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The drug loading capacity (DL) and entrapment efficiency (EE) of DOX was determined by
fluorescence spectroscopy (Thermo Scientific Varioskan Flash, Thermo Fisher Scientific, USA). DL
and EE were calculated according to the following equations [31]:
DL (wt.%) = (weight of loaded drug/weight of nanoparticles) × 100%
EE (wt.%) = (weight of loaded drug/weight of feeding drug) × 100%
To investigate the stability of HA-NP and HA-LP suspensions, HA-NP and HA-LP suspensions
were placed at room temperature. Change in particle size was recorded over time [17, 32].
2
.4. In vitro release
The in vitro release of DOX from HA-NPs and HA-LPs was measured using the dialysis setup [33,
4]. Initially, 0.5 mL of HA-NP, HA-LP suspensions and free DOX solution were respectively placed
3
into dialysis bags (MWCO 8000 Da). Then the dialysis bags were immersed into 100 mL of phosphate
buffer solution (PBS, pH 7.4) and incubated at 37 °C under constant shaking at 100 rpm. At
predetermined time intervals, 1.0 mL of release medium was collected and replaced with 1.0 mL of
fresh medium. The amount of DOX released was measured using fluorescence spectrophotometer (Ex
=
498 nm, Em = 584 nm).
2
.5. Cell cultures
The mouse melanoma cells (B16F10, high expression of CD44), human lung carcinoma cells
(A549, high expression of CD44), murine hepatoma cells (H22, high expression of CD44) [17,35,36]
and renal tubular epithelial cells (HK2, no expression of CD44) were cultured in a 5% CO2
atmosphere at 37 ºC in RPMI-1640 supplemented with 10% fetal bovine serum (FBS), streptomycin
-1
-1
(
50 U mL ) and penicillin (50 U mL ).
2
.6. In vitro cellular uptake study
B16F10, A549, H22 and HK2 cells were cultured in 12-well plates at a seeding density of 1 × 10
5
cells per well and incubated for 24 h. Then replaced the original medium with free DOX, HA-NPs,
HA-LPs and DOX-LPs (final DOX concentration was 5 μg/mL) containing RPMI-1640. The cells
were incubated for 2 h and 4 h at 37 ºC and washed three times with PBS. Next, cells were suspended
and centrifuged at 3000 rpm for 3 min. The supernatants were removed and the cells were washed two
times with PBS. After centrifugation, cells were resuspended in 0.5 mL PBS. The fluorescent intensity
TM
of cells was measured by flow cytometer (Cytomics FC 500, Beckman Coulter, USA). Moreover,
the cellular uptake mechanism was researched in the presence of free HA (10 mg/mL). Also, HK2
cells served as a negative control cell line with no expression of CD44 receptor.
2
.7. Confocal laser scanning microscopy (CLSM)
The cell uptake and intracellular distribution profiles of HA-NPs and HA-LPs were evaluated in
5
B16F10 and HK2 cells by CLSM. Cells were seeded on coverslips at a density of 1 × 10 cells/well in
a 6-well plate containing 2 mL of RPMI-1640. After 24 h incubation, the original medium was
replaced with free DOX, HA-NPs and HA-LPs (final DOX concentration was 5 μg/mL) containing
RPMI-1640. After 2 h and 4 h, cells were washed with PBS and fixed with 4% paraformaldehyde for
1
5 min. The cell membranes were stained with FITC-labeled phalloidin for 30 min and the cell nuclei
were stained with DAPI for 5 min. Finally, cells were viewed with CLSM (FV1000, Olympus, USA).
5

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2
.8. Cytotoxicity assay
The cytotoxicities of free DOX, HA-NPs, HA-LPs and DOX-LPs were evaluated by MTT assay.
4
Cells were cultured in 96-well plates at a density of 1 × 10 cells/well in 100 μL of RPMI-1640
medium, and then incubated in a 5% CO atmosphere at 37 ºC for 24 h. The medium was replaced
2
with 150 μL of fresh medium containing free DOX, HA-NPs, HA-LPs and DOX-LPs (concentrations
were from 0.5 to 10 μg/mL), and then incubated for 24 h. Next, MTT solution (20 μL, 5 mg/mL in
PBS) was added into each well. Cells were further incubated for 4 h under 37 °C. Then the medium
was discarded and cells were dissolved by 150 μL of dimethyl sulfoxide (DMSO). The absorbance
was measured using a microplate reader (Thermo Scientific Varioskan Flash, USA) at 490 nm. Cell
viability (%) was calculated according to the equation (A /Acontrol) × 100%, where Atest and Acontrol
test
represent the absorbance of cells treated with different test samples and culture medium, respectively.
2
.9. Formation of B16F10 multicellular tumor spheroids (MCTs)
MCTs were prepared with B16F10 cells using the hanging drop methods [37, 38]. 2% (w/v)
agarose solution was dissolved in PBS by heating for 30 min at 100 ºC and sterilized by autoclave.
Each well was coated with a thin layer (0.4 mL) of the agarose solution, and then filled with 800 μL of
RPMI-1640 medium. Next, 20 μL of cell suspensions (containing 500 cells) was suspended on the lid
of 24-well plate. After 72 h, MCTs were moved to the bottom of the well containing 800 μL of
medium. Then, MCTs were allowed to grow for 3 days, and the MCTs with 200 μm in diameter were
selected for tumor penetration study.
2
.10. Penetration in MCTs
The penetration and distributions of free DOX, HA-NPs, HA-LPs and DOX-LPs within MCTs were
determined by CLSM. B16F10 MCTs with a diameter of about 200 μm were collected after about 6
days of growth. Several spheroids were hand-picked for each experiment. Free DOX, HA-NPs,
HA-LPs and DOX-LPs (DOX concentration was 5 μg/mL) were added to the suspension of MCTs,
respectively, and then cultured at 37 °C for 4 h. Next, the medium was discarded and MCTs were
washed two times with PBS before observing.
2
.11. Pharmacokinetics
Wistar rats (220 - 230 g) were divided into three groups randomly (n = 5). Free DOX, HA-NPs and
HA-LPs were administered intravenously (DOX concentration was 2 mg/kg). At scheduled time
points (5, 15, 30, 45, 60, 120, 180, 240, 360, 480, 600 and 720 min) after injection, 200 μL of blood
were collected intraorbitally [39]. The concentrations of DOX were determined by LC-MS. Briefly,
1
00 μL of plasma sample was deproteinized with 400 μL of acetonitrile, vortexed for 10 min, and then
centrifuged at 10,000 rpm for 5 min. Then the supernatant was determined by LC-MS (Agilent 6410,
Agilent Technologies, USA) [40].
2
.12. Biodistribution
H22 tumor bearing mice were divided into three groups randomly (n = 15). Free DOX, HA-NPs
and HA-LPs were administered intravenously (DOX concentration was 5 mg/kg). The mice were
sacrificed at 0.25, 1 and 2 h after administration. The major organs (heart, spleen, liver, lung and
kidney) and tumor were excised, washed with physiological saline and dried with filter paper carefully
for ex vivo imaging of DOX fluorescence through an in vivo imaging system (Quick View 3000,
6

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Bio-Real, Austria).
After collecting ex vivo imaging of DOX fluorescence, these organs were weighed and
homogenized with two fold of PBS (g/ml). The concentrations of drug in different organs were
determined by LC-MS. Sample postprocessing method was consistent with pharmacokinetics.
2
.13. In vivo toxicity and tolerability
weeks old male Kunming mice were used to evaluate the maximum tolerated dose (MTD) of free
6
DOX, HA-NPs and HA-LPs. Mice in each group were given a single i.v. injection of free DOX,
HA-NPs and HA-LPs at an equivalent dose of 5, 10, 15, 20, and 25 mg/kg DOX (n = 10). The
physical status and body weight of mice were monitored for 14 d, and MTD was defined as previously
described [41].
2
.14. In vivo antitumor efficacy
Male Kunming mice (initial body weight was 16 - 18 g) were used for researching in vivo antitumor
6
efficacy. Approximately 1 × 10 H22 cells were resuspended in 200 μL of serum-free medium and
3
subcutaneously injected into the right flanks of mice. When tumors grew to 50 - 100 mm in volume,
all mice were divided into four groups (five for each) randomly. On day 8, 10, 12, 14, 16, and 18 after
administration, physiological saline, free DOX, HA-NPs and HA-LPs (DOX concentration was 5
mg/kg) were injected to mice intravenously, respectively. Then the mice were recorded for weight loss
(
as an index of systemic toxicity) and tumor volumes every day. Tumor volumes were calculated using
2
V = l·s /2, where l and s represent the longest and shortest diameter of tumor. After 21 days, tumors
were excised and weighed. Tumor inhibitory rate (TIR) was calculated using the following formula
[
17]:
TIR = (Wsaline – Wdrug)/Wsaline × 100%
2
.15. Safety evaluation
Bone marrow suppression and cardiac toxicity have been the most serious side effects related to
DOX. In order to evaluate the level of bone marrow suppression, physiological saline, free DOX,
HA-NPs and HA-LPs were injected to male Wistar rats (200 - 220 g). All rats were randomly divided
into 4 groups (five for each). Physiological saline, free DOX, HA-NPs and HA-LPs (DOX
concentration was 5 mg/kg) were injected to rats intravenously, respectively. At the 3rd day after
+
administration, 0.5 mL of blood was collected in Eppendorf tubes (containing EDTA-2K salt). Red
blood cells (RBC), white blood cells (WBC) and platelets (Plt) were counted by automated
hematology analyzer (MEK-6318K, Nijon-kohden, Shinjuku-ku, Japan) as indicators of bone marrow
suppression [40, 42].
Male Wistar rats (200 - 220 g) were used for evaluating the cardiac toxicity of free DOX, HA-NPs
and HA-LPs. Before administration, all rats were fasted for 12 h and then were randomly divided into
4
groups (five for each). Physiological saline, free DOX, HA-NPs and HA-LPs (DOX concentration
was 5 mg/kg) were intravenously injected to rats once every two days for 4 times, respectively. On
day 8, all rats were sacrificed and heart samples were excised and processed for histological analysis
(hematoxylin and eosin stain) [40].
2
.16. Statistical analysis
All data represent mean ± standard deviations (S.D.). Student’s t-test was performed for comparison
7

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between two groups. A P-value < 0.05 was considered statistically significant, and a P-value < 0.01
was considered highly significant.
3
3
. Results and discussion
.1. Preparation, optimization, characterization of HA-NPs and HA-LPs
The nanoparticle formulation was optimized based upon excipients, HA to DOX feeding ratio, and
reaction time. Solutol HS 15 was used to improve the encapsulation efficiency of DOX. 1% (v/v) of
soybean oil was used to facilitate DOX to form a hydrophobic core. The formulation and reaction
condition has been optimized in terms of HA molecular weight, solvent, HA-to-DOX feeding ratio,
the concentration of soybean oil, the reaction time, freeze-drying protective agents and other
excipients. The effect of reaction condition on the HA-NPs was shown in supplementary data. By
using 1% (v/v) of soybean oil for injection, the average particle size of HA-NPs was found to be about
1
80 nm (Fig. 2a), which was consistent with the particle size observed in TEM (Fig. 2c). Due to the
presence of carboxylic groups in HA, HA-NPs showed an average negative surface charge of -20.3
mV in water (Table 1). When coated with a phospholipid bilayer, HA-LPs showed an average particle
size of about 130 nm (Fig. 2b) and a negative zeta potential of -8.2 mV. HA-LPs showed a smaller
average particle size compared with HA-NPs, which might be due to the presence of phospholipid
membrane. As is previously discussed, HA-NPs were fabricated via ion-pairing which resulted in a
relative loose structure thus displaying a larger particle size. Phospholipids are a type of
amphiphiles, which may help reduce the surface tension of the colloidal particle solution, thus
resulting in a denser structure and a smaller particle size [43, 44]. In addition, nanoparticles with slight
negative charges and particle size of 150 nm were suggested to accumulate in tumors more efficiently
[
45-47]. Therefore, HA-NPs and HA-LPs might have great potential in tumor-targeted drug delivery.
Fig. 2. Size distribution and morphology of nanoparticles: Dynamic light scattering (DLS) size
measurement of (a) HA-NPs and (b) HA-LPs; TEM image of (c) HA-NPs and (d) HA-LPs.
The average loading capacity of DOX in HA-NPs was determined as 8.5 ± 1.3% (w/w, n=3) (Table
1
). After the encapsulation of HA-NPs into liposomes, the average content of DOX in HA-LPs
decreased to 2.8 ± 0.3%, which may be attributed to the usage of phospholipids and cholesterol. The
encapsulation efficiency of HA-NPs was consistently above 80%, illustrating that the drug was well
incorporated into nanoparticles. The unconjugated DOX was removed from the HA-NPs by a
Sephadex G-75 minicolumn.
8

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Table 1
Characterization of HA-NPs and HA-LPs
Entry
Particle size (nm)
Zeta potential
mV)
DL (%)
EE (%)
(
HA-NPs
HA-LPs
180.2±12.3
130.5±10.1
-20.3±3.2
-8.2±1.7
8.5±1.3
2.8±0.3
81.0±2.4
85.3±3.1
3
.2. Stability of HA-NP and HA-LP suspensions
Because of the high molecular weight of HA, HA-NP suspensions flocculated easily after a period
of time in our previous study. The in vitro stability of HA-NPs and HA-LPs were evaluated in plasma
and PBS (pH 7.4). In PBS, both HA-NPs and HA-LPs remained stable and no significant size change
was observed (Fig. 3a). When incubated in plasma, the particle size of HA-LPs increased
approximately 10 nm compared with the original size, while the particle size of HA-NPs increased
approximately 50 nm after 48 h. These results proved the enhanced plasma stability of HA-LPs
compared to HA-NPs.
Fig. 3. (a) The stability of HA-NPs and HA-LPs in plasma and PBS (pH 7.4) for 48 h. Data represent
mean ± S.D. (n = 3). (b) DOX release profiles of free DOX, HA-NPs and HA-LPs in PBS at pH 7.4.
Data represent mean ± S.D. (n = 3).
3
.3. In vitro release
Fig. 3b shows the in vitro release profile of free DOX, HA-NPs and HA-LPs. After a 24 h
incubation period, approximately 95.3% cumulative release was observed for free DOX, while about
8.2% cumulative release percentage was achieved by HA-NPs and 48.2% by HA-LPs. DOX was
5
shown to release gradually from HA-NPs and HA-LPs for up to 5 days in the dialysis setup.
Cumulative release of DOX from HA-NPs was significantly lower than free DOX, which was
attributed to the formation of ion pairs between HA and DOX. Cumulative release of DOX from
HA-LPs was slightly lower than HA-NPs, because the liposomal bilayer delayed the release of the
drug. This indicated that the HA-NPs and HA-LPs had a good sustained-release effect in vitro.
3
.4. In vitro cell uptake study
To investigate the cellular internalization, HA-NPs and HA-LPs were incubated with B16F10 cells
+
+
+
(
CD44 ), A549 cells (CD44 ) and H22 cells (CD44 ) (Fig. 4, group A). To elucidate the cellular
-1
uptake mechanism of HA-NPs, cells were incubated with HA solution (10 mg/mL , 100 kDa)
simultaneously and various preparations (Fig. 4, group B). At the same time, HK2 cells (CD44 ) were
-
cultured as a negative control.
9

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Fig. 4. Cellular uptake of free DOX, HA-NPs, HA-LPs and DOX-LPs after incubation with B16F10
cells, A549 cells, H22 cells and HK2 cells for 2 h and 4 h, observed by flow cytometric analysis.
Group A: cells were incubated with preparations solely; Group B: cells were simultaneously incubated
with HA solution (10 mg/mL, 100 kDa) and preparations. Data represent mean ± S.D. (n = 3). *: p <
0
.05; **: p < 0.01.
As shown in Fig. 4 (group A), the fluorescence intensity of B16F10 treated with HA-NPs was about
.5 times higher than these treated with DOX at 2 h and was about 1.5 times higher at 4 h. The
2
fluorescence intensity of HA-LPs was slightly lower than that of HA-NPs while significantly higher
than free DOX (p < 0.01). The fluorescence intensity of DOX-LPs was significantly lower (p < 0.01)
compared with free DOX, which proved that the presence of a phospholipid bilayer might impact the
cellular uptake. However, the negative impact was not significant for HA-LPs, possibly due to
HA-NPs encapsulated inside. When B16F10 cells were incubated with HA solution simultaneously
(group B), the cell uptake efficiencies of HA-NPs and HA-LPs were both reduced substantially
compared to that of HA-NPs and HA-LPs in the HA-free medium. For DOX-LPs, no significant
differences in fluorescence intensity were observed due to the absence of HA. Results were similar in
A549 cells and H22 cells. The differences between HA-containing delivery systems and HA-absent
systems might be explained by the fact that free HA macromolecules could compete for the binding
sites on the CD44 receptors thus resulting in the decreased cellular uptake of HA-NPs. In comparison,
DOX, HA-NPs and HA-LPs showed no significant differences in cell uptake efficiency in HK2 cells.
These results further proved that HA-NPs can bind to CD44 receptor specifically and internalize into
tumor cells via CD44 receptor-mediated endocytosis.
3
.5. Intracellular distribution under CLSM
To investigate the intracellular release of DOX, HA-NPs and HA-LPs were incubated with B16F10
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cells. At the same time, HK2 cells were used as a negative control. As shown in Fig. 5A, red
fluorescence representing DOX was observed in the cell nuclei of B16F10 cells after 2 h incubation
with HA-NPs and HA-LPs, which became the clear visual evidence of the cell internalization of
HA-NPs and HA-LPs and the release of the loaded DOX. Cells treated with HA-NPs and HA-LPs
showed the clearly increased perinuclear and nuclear fluorescence accumulation compared with free
DOX. After 4 h of incubation, more obvious differences were observed. The DOX fluorescence
intensity of HA-LPs was slightly weaker than that of HA-NPs. However, DOX-LPs showed very
weak DOX fluorescence in the cell nuclei compared to free DOX.
In HK2 cells, HA-NPs showed relatively weaker fluorescent intensity than in B16F10 cells due to a
lack of CD44 receptors to mediate internalization of nanoparticles. Also, DOX solution, HA-NPs and
HA-LPs showed no dramatic differences in cell uptake efficiency in HK2 cells, which further
demonstrated that the critical role of HA in mediating nanoparticle internalization in cancer cells via
CD44 receptor. These results were consistent with cellular uptake experiments.
Fig. 5. (A) CLSM images of B16F10 cells and HK2 cells incubated with free DOX, HA-NPs,
HA-LPs and DOX-LPs for 2 h and 4 h (40×). The cell nuclei were stained with DAPI (blue), cell
membranes were stained with FITC-labeled phalloidin (green) and DOX fluorescence displayed in red.
The scale bar represents 40 μm. (B) In vitro cell viability of free DOX, HA-NPs, HA-LPs and
DOX-LPs against B16F10 cells, A549 cells, H22 cells and HK2 cells. Data represent mean ± S.D. (n
=
3). *: p < 0.01 vs DOX group. (C) CLSM images of B16F10 MCTs incubated with free DOX,
HA-NPs, HA-LPs and DOX-LPs for 4 h. Z-stack images were obtained from the top toward the tumor
spheroid equatorial plane in 10 μm thickness. The scale bar represents 100 μm.
3
.6. In vitro cytotoxicity
Concentration-related cell viability of DOX solution, HA-NPs, HA-LPs and DOX-LPs against
B16F10, A549 and H22 cells was evaluated by MTT assay (Fig. 5B). At an equivalent drug
concentration, HA-NPs revealed a significantly higher cytotoxicity compared to free DOX (p < 0.01),
whereas HA-LPs induced a less potent effect in the cell lines under investigation. For HK2 cells,
HA-NPs and HA-LPs showed no significant cell killing potency compared with DOX solution. Table
2
showed the 50% inhibitory concentration (IC ) values of free DOX, HA-NPs, HA-LPs and
50
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DOX-LPs in different cell lines. These results agreed well with the cellular uptake experiments and
intracellular DOX release observations (Fig. 4 and Fig. 5A). HA-NPs were likely internalized via
CD44 receptor-mediated endocytosis, thus resulting in the greater cell uptake and higher cytotoxic
effect of HA-NPs. However, HK2 cells did not have CD44 receptor, thus resulting in no differences in
the uptake and cytotoxicity results of free DOX, HA-NPs and HA-LPs. The presence of a
phospholipid bilayer on the surface of HA-LPs had a slight effect on tumor cell targeting.
Table 2
a
IC values of free DOX, HA-NPs and HA-LPs on different cell lines
5
0
IC (μg/mL)
5
0
Free DOX
0.63±0.01
0.96±0.02
5.89±0.11
9.91±0.21
HA-NPs
HA-LPs
DOX-LPs
9.44±0.18
12.16±0.30
66.91±1.24
75.86±1.31
-
3
-2
B16F10
A549
H22
HK2
a
(6.46±0.15)×10 *
(5.80±0.13)×10 *
0.48±0.01*
(2.05±0.03)×10 *
0.12±0.01*
-2
1.06±0.02*
9.82±0.22
9.73±0.19
Data represent mean ± SD (n = 3).
*
: p < 0.01 vs free DOX.
3
.7. Penetration in MCTs
3
-D multicellular tumor spheroids (MCTs) of B16F10 cells were generated to evaluate the
penetration efficiency of HA-NPs and HA-LPs. As shown in Fig. 5C, HA-NPs showed excellent
penetration across different focal planes of the MCTs, while HA-LPs treated MCTs showed slightly
weaker fluorescence intensity. Free DOX have weaker capacity of penetration in the MCTs and
DOX-LPs could hardly penetrate into the MCTs. Most evidently, the fluorescence of HA-NPs and
HA-LPs was stronger than free DOX and DOX-LPs. The stronger fluorescence and deeper penetration
of HA-NPs and HA-LPs appear in the B16F10 MCTs compared to free DOX, indicating that our
HA-NPs and HA-LPs can actively penetrate into the MCTs. The presence of a phospholipid bilayer
on the surface of HA-LPs showed some effect on MCTs penetration but not obvious.
3
.8. Pharmacokinetics
Once entering circulation after intravenous administration, nanoparticles could be diluted and this
dilution effect can be enlarged since most of nanoparticles were distributed to different organs (e.g.
lung, spleen and liver), leaving a small percentage of nanoparticles retaining in the blood [48, 49].
Therefore, retarding blood clearance and prolonging blood circulation of nanoparticles have been
proven critical for drug redistribution to the target tumor site [31, 50]. The prolonged blood circulation
of nanoparticles can increase accumulation of nanoparticles in the tumor site through EPR effect [51].
The pharmacokinetic result is shown in Fig. 6 and Table 3. The release of DOX from HA-NPs and
HA-LPs continued for 6 h, whereas serum DOX concentration of free drug group dropped to baseline
level within 3 h. The blood circulation times of HA-NPs and HA-LPs were greatly extended compared
with free DOX. The plasma DOX concentration of HA-NPs was higher than that of free DOX,
indicating an obvious retardation effect in clearance. Also, the in vitro release rate of DOX from
HA-LPs was slower than that from HA-NPs, which was likely due to the liposomal bilayer delaying
the release of the drug. A direct correlation between in vivo and in vitro tests was observed and the
results in the in vitro tests could be supported by the pharmacokinetic study. However, DOX release
1
2

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of all three groups was very fast in a very short period of time after injection and it was not a
satisfactory result. This might be caused by two reasons. One reason was that plasma contained a lot
of proteins. These plasma proteins could be combined with drugs and then removed them, leading to
plasma drug concentration decreased rapidly. Another reason was that the existence of inorganic salts
in plasma caused the change of surface charge of HA-NPs and HA-LPs, this could lead to the
sedimentation and flocculation of the nanoparticles and liposomes. In our further study, if small
amounts of DSPE-mPEG2000 were added to phospholipids, such HA-LPs would have better
sustained-release effect, however the sudden release still existed. In our future research, improved the
sudden release of HA-NPs was an important work.
Fig. 6. In vivo pharmacokinetic profiles after intravenous injection of free DOX, HA-NPs and
HA-LPs in rats. Data represent mean ± S.D. (n = 5).
Table 3
Pharmacokinetic parameters of free DOX, HA-NPs and HA-LPs
Sample
Dose (mg/kg)
Free DOX
2.0
467.7±42.3
5
HA-NPs
2.0
850.2±60.4
5
HA-LPs
2.0
1633.8±100.2*
5
a
Cmax (ng/ml)
b
Tmax (min)
c
AUC (min·ng/ml)
11546
5.77×10
16.4
23722
1.19×10
56599*
-3
-2
-2
AUC/dose (min·kg/ml)
t (min)
2.83×10 *
65.5
152.1*
1
/2
AUC, area under the curve.
a
Average maximum serum DOX concentrations. All values are mean ± standard deviation (n=5).
Tmax was determined from the average serum DOX concentrations for five rats at the predetermined
b
sampling time.
c
AUC for free DOX was determined from the curve area of average serum DOX concentrations
between 0 and 3 h; AUC for HA-NPs and HA-LPs between 0 and 6 h.
*
: p < 0.01 vs free DOX.
3
.9. Biodistribution
Imaging of the major organs (heart, lung, spleen, liver and kidney) and tumors at 0.25, 1 and 2 h
after administration were obtained in mice bearing H22 tumors for biodistribution studies (Fig. 7A),
and the fluorescence intensity was quantitatively analyzed by LC-MS (Fig. 7B). For free DOX group,
1
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liver and kidney showed strong fluorescence, indicating that DOX were mainly captured and
metabolized by kidney and liver [52]. A favorable biodistribution with increased accumulation in
tumor for the injection of HA-NPs and HA-LPs could be observed. In addition, the fairly weaker
fluorescence in heart for the injection of HA-NPs and HA-LPs was observed compared to free DOX,
which could be concluded by the significantly reduced the cardiac toxicity of DOX. These results
indicated that HA-NPs and HA-LPs were able to change the biodistribution of DOX and provide
significant benefits to enhance the tumor accumulation while reducing the systemic toxicity of the
DOX.
Fig. 7. (A) Ex vivo DOX fluorescence images showing the drug bio-distribution of free DOX,
HA-NPs and HA-LPs in mice bearing H22 tumor at 0.25, 1 and 2 h post-injection. (B) The mean
concentrations of DOX in different tissues of H22 tumor-bearing mice after intravenous
administration of free DOX, HA-NPs and HA-LPs at (a) 0.25 h, (b) 1 h and (c) 2 h. Data represent
mean ± S.D. (n = 3).
Notably, compared with free DOX, a higher intensity of HA-NPs and HA-LPs in the liver and
kidney was measured. This might be caused by two reasons. One reason was that the
pharmacokinetics of HA-NPs and HA-LPs improved significantly, leading to their longer blood
retention time and more difficult to be cleared quickly. Another reason was likely due to the existence
of CD44 receptor in liver and kidney, consequently leading to a higher accumulation in kidney and
liver compared to free DOX [22, 53, 54]. Compared with free DOX, HA-NPs and HA-LPs increased
DOX accumulation in tumor site by 3.7-fold and 4.3-fold, respectively at 1 h after administration.
HA-NPs and HA-LPs delivered DOX to the tumor with around 2.7 and 3.7-fold higher concentration
than free DOX group, respectively at 2 h after administration. That might be attribute to the
combination of passive targeting (EPR effect) and active targeting (CD44 receptor-mediated
endocytosis) [55]. Interestingly, compared with HA-NPs, HA-LPs slightly increased DOX
accumulation in the tumor at each time point. This indicated that the presence of a phospholipid
bilayer on the surface of HA-LPs could enhance the stability of HA-LPs in vivo compared to
HA-NPs.
3
.10. In vivo toxicity and tolerability
Chemotherapeutic agents have many advantages in tumor therapy, but their clinical applications
were often limited by systemic toxicity. In order to determine the toxicity and tolerability of HA-NPs
and HA-LPs, we estimated the MTD by intravenous administration to Kunming mice. Survival details
and body weight of the mice were monitored for 14 days after administration of free DOX, HA-NPs
and HA-LPs (Fig. 8). As we expected, an obvious weight loss was observed at 10, 15, 20 and 25
1
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mg/kg of free DOX, meanwhile the groups treated with dosage of free DOX higher than 10 mg/kghad
dead mice. In contrast, HA-NPs and HA-LPs could reduce the systemic toxicity effectively with a
significant prolongation of survival time, less weight loss at all doses and gradual recovery of body
weight. The MTD was evaluated based on the threshold at which the loss of body weight was below
2
0%, meanwhile all animals survived [56]. The MTD of free DOX was 5 mg/kg, which was
consistent with literature [57]. However, HA-NPs and HA-LPs could increase the MTD of DOX to 10
and 15 mg/kg (2 and 3 fold of free DOX), respectively. The increase in the dose of DOX in a clinical
setting is significant as it may allow patients to obtain a higher dose of chemotherapy without the dose
limiting toxicities. This result suggested that HA-NPs and HA-LPs could increase the MTD of DOX
and reduce systemic toxicity simultaneously. The toxicity of HA-LPs was weaker than HA-NPs, and
the tolerability was better.
Fig. 8. Survival rate and body weight change of Kunming mice treated with free DOX, HA-NPs and
HA- LPs at a dose of (a) 5, (b) 10, (c) 15, (d) 20 and (e) 25 mg/kg DOX.
3
.11. In vivo antitumor efficacy
Owing to the prolonged circulation, enhanced tolerability and good tumor localization, HA-NPs
and HA-LPs might own excellent antitumor efficacy without unexpected toxic and side effects. To
provide the evidence for the antitumor potentiality of HA-NPs and HA-LPs in vivo, the antitumor
effect was further researched in Kunming mice bearing murine hepatoma cells (H22). The experiment
was done by intravenously physiological saline, free DOX, HA-NPs and HA-LPs, respectively, into
tumor-bearing mice.
The tumor volume of mice injected with free DOX, HA-NPs and HA-LPs was 56.9%, 20.0% and
1
2.0% of the saline-treated mice, respectively for 21 days (Fig. 9a). The tumor growth was effectively
inhibited in the groups injected with free DOX and DOX-loaded formulations compared to the control
group (physiological saline group), which proved the excellent antitumor efficacy of DOX. Compared
with free DOX, intravenous injection of HA-NPs and HA-LPs was more efficient in inhibiting tumor
growth (P < 0.01). TIR values of HA-NPs and HA-LPs were calculated to be 67.7% and 78.9%,
which was 1.78-fold and 2.08-fold higher than that of free DOX, respectively (Fig. 9c). As shown in
1
5

ACCEPTED MANUSCRIPT
Fig. 9b, the body weight of free DOX group decreased significantly, which may be due to the fact that
repeated injections of free DOX induced severe off-target toxicity. Increase in body weight of
HA-NPs and HA-LPs indicated that repeated injection of HA-NPs and HA-LPs had weaker toxicity to
the body than free DOX. Moreover, the weight obtained from the sacrificed mice injected with
different DOX formulation and the tumor image (Fig. 9d) also indicated that HA-NPs and HA-LPs
showed remarkable antitumor activity.
These results indicated that HA-NPs and HA-LPs had higher antitumor activity and lower systemic
toxicities than free DOX. These differences indicated that the encapsulation of DOX molecules in
HA-NPs reduced the distribution of drug to normal organs, increased the passive (EPR effect) and
active (CD44 receptor-mediated endocytosis) accumulation of the nanoparticles to tumor sites, and
thus enhanced antitumor efficacy and led to lower undesirable systemic toxicities. HA-LPs showed
slightly higher antitumor efficacy and lower systemic toxicities than HA-NPs, which was attributed to
the liposomal bilayer delaying the release of DOX.
Fig. 9. (a) Changes of tumor volume after intravenous injection of saline, free DOX, HA-NPs and
HA-LPs in H22 tumor-bearing mice. (b) Body weight change of H22-bearing mice treated with saline,
free DOX, HA-NPs and HA-LPs on the indicated days (shown by arrows). (c) Tumor weight and TIR
of H22-bearing mice treated with saline, free DOX, HA-NP and HA-LP at day 21 after the mice were
euthanized. (d) Photograph of tumors stripped from mice administrated intravenously with saline, free
DOX, HA-NP and HA-LP. Data represent mean ± S.D. (n=5). *: p < 0.05; **: p < 0.01.
3
.12. Safety evaluation
Bone marrow suppression and cardiac toxicity have been the most serious side effects of DOX. To
research the effect of bone marrow suppression, RBC, WBC and Plt counts were determined 3 days
after administration. Changes in the counts of RBC, WBC and Plt might reflect the appearance of
bone marrow suppression and the abnormality of the immune system. Compared with physiological
saline group, free DOX group induced a significant reduction in RBC, WBC and Plt counts (Fig. 10a),
indicating strong bone marrow suppression effect of DOX. HA-NPs and HA-LPs groups showed
higher RBC, WBC and Plt counts than free DOX group, which indicated that encapsulating DOX in
nanoparticles may contribute to reduce the systemic toxicity. The WBC, RBC and Plt counts of
HA-LPs group were higher than that of HA-NPs group and closed to normal level. This further
1
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confirmed that encapsulating nanoparticles into liposomes could delay the release of DOX and reduce
the systemic toxicity.
DOX was reported to lead to dose-limiting systemic toxicities especially cardiac toxicity when used
in clinic [58-60]. So, it is important to evaluate the damage to heart caused by HA-NPs and HA-LPs.
Free DOX group showed severe cardiac toxicity such as myocardial fiber hyperemia, necrosis and
breakage (Fig. 10b). However, the histological sections from HA-NPs treated group exhibited
significantly reduced cardiac toxicity, and the HA-LPs group did not show obvious cardiac toxicity.
Therefore, HA-NPs and HA-LPs were testified effective in alleviating the cardiac toxicity induced by
DOX.
Fig. 10. (a) WBC, RBC and Plt cell counts of physiological saline, free DOX, HA-NPs and HA-LPs
after 3 days of treatment. Data represent mean ± S.D. (n=5). *: p < 0.05; **: p < 0.01. (b) Heart
morphology of free DOX, HA-NPs and HA-LPs treated groups. Hearts were isolated and stained with
hematoxylin and eosin (H&E) for histopathological analysis. Scale bar represents 200 μm. These are
representative sections from five rats analyzed for each condition.
Interestingly, HA-NPs showed superiority in vitro compared with HA-LPs while it was opposite in
vivo from the above results. For example, in vitro, HA-NPs showed better tumor cell uptake
efficiency, higher cytotoxic efficiency to tumor cells and deeper penetration in MCTs compared with
HA-LPs. However, in vivo, HA-LPs showed better long circulation effect, stronger tumor targeting
ability, better antitumor efficacy and lower toxicity than HA-NPs. This was because the liposomal
bilayer of HA-LPs could delay the release of the drug and protect nanoparticles from being destroyed
in the blood until HA-NPs reached the target site, in vivo. However, in vitro, HA-NPs would not be
destroyed by proteins and inorganic salts which present in the blood and the liposomal bilayer of
HA-LPs would affect the exposure of HA, and then influence the uptake of nanoparticles by tumor
cells.
4
. Conclusions
In this study, we successfully prepared HA-NPs by forming ion-pairs between HA and DOX via
electrostatic interactions. The preparation process was simple and unique which did not involve the
use of organic solvents and harsh conditions such as heating and sonication. Furthermore, HA-NPs
were coated with a phospholipid bilayer (HA-LPs) to reduce premature release of DOX from HA-NPs.
1
7

ACCEPTED MANUSCRIPT
HA-LPs displayed a desirable sustained-release profile in vitro and significantly higher AUC values in
+
vivo compared to free DOX solution. HA-NPs and HA-LPs displayed high selectivity to CD44 tumor
cells to induce potent cytotoxic effects. In addition, DOX can be released intracellularly and delivered
to the nuclei of cancer cells. Importantly, HA-NPs and HA-LPs significantly improved the penetration
of DOX in MCTs, the biodistribution and antitumor efficacy in vivo, and reduced DOX-related
multiorgan toxicity. Furthermore, the systemic delivery of the HA-NPs and HA-LPs carrying DOX
showed remarkable antitumor activity in H22 xenograft murine model. In sum, HA-NPs and HA-LPs
represent a novel nanoparticulate system with excellent biocompatibility, sustained release profile and
+
high selectivity to CD44 tumor cells, which may show great potentiality in tumor targeted drug
delivery.
Acknowledgments
The work was financially supported by National Natural Science Foundation of China (81273443)
and the National Basic Research Program of China (2013CB932504).
Supplementary data
The following is the supplementary data related to this article:
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