- Email: [email protected]
Novel hyaluronic acid coated hydrophobically modified chitosan polyelectrolyte complex for the delivery of doxorubicin
Accepted Manuscript Novel hyaluronic acid coated hydrophobically modified chitosan polyelectrolyte complex for the delivery of doxorubicin
Lili Chen, Yuanyuan Zheng, Longbao Feng, Zonghua Liu, Rui Guo, Yuanming Zhang PII: DOI: Reference:
S0141-8130(18)35505-3 https://doi.org/10.1016/j.ijbiomac.2018.12.215 BIOMAC 11374
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
15 October 2018 10 December 2018 21 December 2018
Please cite this article as: Lili Chen, Yuanyuan Zheng, Longbao Feng, Zonghua Liu, Rui Guo, Yuanming Zhang , Novel hyaluronic acid coated hydrophobically modified chitosan polyelectrolyte complex for the delivery of doxorubicin. Biomac (2018), https://doi.org/ 10.1016/j.ijbiomac.2018.12.215
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.
ACCEPTED MANUSCRIPT
Novel hyaluronic acid coated hydrophobically modified chitosan polyelectrolyte complex for the delivery of doxorubicin Lili Chen a, b, 1, Yuanyuan Zheng a, 1, Longbao Feng a, Zonghua Liu a, Rui Guo a, *, Yuanming
Key Laboratory of Biomaterials of Guangdong Higher Education Institutes, Guangdong
RI
a
PT
Zhang a, b, *
SC
Provincial Engineering and Technological Research Center for Drug Carrier Development,
Department of Chemistry, Jinan University, Guangzhou 510632, China
MA
b
NU
Department of Biomedical Engineering, Jinan University, Guangzhou 510632, China
* Corresponding authors:
CE
PT E
These authors contributed equally to this work.
AC
1
D
[email protected] (Yuanming Zhang), [email protected] (Rui Guo)
ACCEPTED MANUSCRIPT
Abstract The aim of this work was to examine the formation and properties of a novel polyelectrolyte complex of drug carrier system for the delivery of doxorubicin (DOX), which consists of hyaluronic acid (HA) coated hydrophobically modified chitosan (CS). Various batches of
PT
polyelectrolyte complexes with the molar ratio of deoxycholic acid (DCA) and chitosan (CS) of
RI
0.1, 0.2, 0.3 were prepared, and were termed as CS-DCA10, CS-DCA20, and CS-DCA30
SC
respectively. The samples were characterized by Fourier transform infrared (FTIR) spectroscopy, X-ray diffraction (XRD), Transmission electron microscopy (TEM), nuclear magnetic resonance
NU
hydrogen spectrum (1H NMR) and dynamic light scattering (DLS). Particle sizes of synthesized
MA
polyelectrolyte complex nanoparticles (PCNs) were found to be in the range of 280–310 nm, larger than those of uncoated nanoparticles (~150 nm). The PCNs have large zeta potentials
D
(about 26mV) which make them stable and no sizes change were determined. DOX could be
PT E
easily incorporated into the PCNs with encapsulation efficiency (56%) and kept a sustained release manner without burst effect when exposed to PBS (pH 7.4) at 37 oC. Overall, these
CE
findings confirmed the potential of these PCNs for drug carrier and prolonged and sustained
AC
delivery in the bloodstream.
Keywords: chitosan, deoxycholic acid, hyaluronic acid, polyelectrolyte complex, nanoparticles, drug carrier
ACCEPTED MANUSCRIPT
1. Introduction Polymeric nanoparticles made of natural and synthetic polymers have received the majority of attention due to their stability and easy surface modification [1, 2]. Among natural polymers, polysaccharides tend to be internalized and degraded rapidly, thus enabling a moderate
PT
intracellular release of the drug. Consequently, polysaccharides have been widely used in drug
RI
delivery systems [3, 4], gene delivery and bioimaging [5]. Polysaccharides can be generally
SC
classified into two types, non-polyelectrolyte and polyelectrolyte. Polyelectrolyte contained cationic and anionic polysaccharides, have been paid more and more attention in the last two
NU
decades, due to their outstanding advantages in drug delivery systems [6], probably for the fact
MA
that the spontaneous association of the oppositely-charged polymers leads to the formation of polyelectrolyte complex through strong and reversible electrostatic links; the transient structure
D
obtained avoids the use of toxic covalently bonded chemical cross-linkers, thus allowing them to
PT E
be used in humans [7].
Chitosan (CS) is a natural, non-toxic, biodegradable polysaccharide, which consists of
CE
glucosamine and N-acetylglucosamine units derived from deacetylation of chitin. CS possesses
AC
desirable properties such as biodegradability, biocompatibility and CS scaffolds with high level of porosity that make this biomaterial a good candidate for tissue engineering and drug delivery [8-10]. Recently, the CS has been extensively employed in the development of micro- and nano-carriers in the delivery of anticancer drug, polypeptide, protein and gene [11, 12]. It is worth mentioning that CS is a natural polycationic polysaccharide, but it can only be dissolved in acidic aqueous solution, which is not conducive to the load of some bioactive macromolecular drugs [13, 14]. Hydrophobic modification of polysaccharide makes it become
ACCEPTED MANUSCRIPT amphiphilic and self-assemble to form micelles in aqueous solution by intramolecular and intermolecular hydrophobic effect, which has a broad application prospect in the field of biomedicine [15-18]. Deoxycholic acid (DCA) plays an important role in the emulsification, solubilization, and absorption of cholesterol, fats, and liphophilic vitamins in the body. For the
PT
past decades, DCA has been used to modify CS, which often acting as a promising delivery
RI
system of hydrophobic drugs, peptide and DNA [19-21].
SC
Hyaluronic acid (HA) is one of the most ideal materials due to its high degradability and excellent biocompatibility [22-25]. HA with its derivatives are most commonly used to develop
NU
several carrier systems for cancer diagnosis and treatment. The HA-modified polysaccharides are
MA
able to load gene and peptide/protein for drug delivery. [26-28]. Many studies have reported the overexpression of its main receptor CD44 in several solid tumors [29, 30]. Therefore, a series of
D
HA-based carriers have been developed in order to target tumor cells, e.g. HA-paclitaxel
PT E
conjugates[31, 32], self-assembled HA nanoparticles[33], PEGylated HA nanoparticles, and etc [34, 35].
CE
Probably, the most popular way to produce chitosan nanoparticles is through ionotropic
AC
gelation of chitosan with sodium tripolyphosphate (TPP), a small ion with a triple negative charge throughout the physiologically acceptable pH range. In this study, we designed a targeted PCNs based on hydrophobically modified CS as a novel delivery system, consisting of cationic CS and anionic HA. The PCNs in this work are prominent in stability, and capable of associating DOX. In addition, this type of PCNs doesn’t show cytotoxicity even at high concentrations. From the study above it may be concluded that CS-DCA/HA/TPP/DOX nanoparticles exhibit remarkable biocompatibility and have the
ACCEPTED MANUSCRIPT potential to be used in biomedical applications and treatment of tumor. 2. Materials and methods 2.1. Materials Chitosan (90% deacetylation, 50 kDa Mw) was purchased from Haidebei Marine
Shandong
Freda
Group
(Shandong,
China).
1-ethyl-3-(3-dimethylaminopropyl)
RI
from
PT
Bioengineering Co., LTD (Shandong, China). Hyaluronic acid (1640 kDa Mw) was obtained
SC
carbodiimide hydrochloride (EDC), deoxycholic acid (DCA), tripolyphosphate (TPP), dimethyl sulfoxide (DMSO) and pyrene were purchased from Aladdin reagent Inc. (Shanghai, China).
NU
DOX·HCl was obtained from Beijing Huafeng United Technology Co., LTD (Beijing, China).
MA
Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), Dulbecco’s phosphate-buffered saline (DPBS), penicillin, and streptomycin were acquired from Life
D
Technologies. All other reagents were analytical grade and used without further purification.
PT E
2.2. Synthesis of CS-DCA Conjugates
The CS-DCA conjugates were synthesized based on the mechanism of carbodiimide
CE
chemistry [36]. Briefly, DCA in ethanol was added to 1% (v/v) CS acetic acid solution followed
AC
by the dropwise addition of EDC (1mol/mol DCA) under magnetic stirring at room temperature for 24h. Then the resulting reaction solution was poured into excess amount of the ethanol/ammonia water solution (pH 8). The precipitates were recovered by centrifugation, washed thoroughly with water, methanol and ethanol respectively, followed by freeze-drying. Various batches of polyelectrolyte complex with the molar ratio of deoxycholic acid (DCA) and CS from 0.1 to 0.3 were prepared, and were named CS-DCA10, CS-DCA20, and CS-DCA30 respectively.
ACCEPTED MANUSCRIPT The CS-DCA nanoparticles were prepared according to the dialysis methods described previously by Chae et al [37]. Briefly, after dissolution of CS-DCA (20mg) in 50mL DMSO/water/acetic acid co-solvent (7/3/0.3, by volume, ca 4 mg/mL), the solution was dialyzed against physiological saline for 48h using a dialysis membrane (molecular cut off 3500). After
PT
dialysis, the resulting nanoparticle solutions were sonicated for 2 min (using probe-type solicitor
RI
at 50 W) and were filtered through a 0.45m pore sized syringe filter to remove large aggregates.
SC
Finally, the CS-DCA nanoparticles were obtained by lyophilization. 2.3. Preparations of the CS-DCA/HA/TPP nanoparticles
nanoparticles were prepared using the ionotropic gelation
NU
CS-DCA/HA/TPP
MA
technique[38]. CS-DCA, tripolyphosphate (TPP) and HA were separately dissolved in ultrapure water at 1 mg/ml, 0.5 mg/ml and 2 mg/ml, respectively. Nanoparticles were formed
D
instantaneously upon the drop-wise addition of TPP/HA solution into CS-DCA solution under
PT E
magnetic stirring for 10 min to allow the complete formation of the system. 2.4. Characterization of CS-DCA Conjugates
CE
Degree of substitution (DS) of DCA group of CS-DCA was determined by elemental
AC
analysis using an EA 2400 II elemental analyzer (PE, USA). Since the amount of N was fixed during the reaction, DS of DCA group was calculated by comparing the C and N molar ratio obtained from the element analysis [39]. The equation for DS calculations as follows:
DS
1400 C / N 7526 288
The chemical structure of CS-DCA conjugates was analyzed using a Fourier transform infrared (FT-IR) spectrophotometer (Bruker, Germany) and nuclear magnetic resonance hydrogen spectrum (1H NMR) (Bruker AVANCE 300) with CD3COOD/D2O.
ACCEPTED MANUSCRIPT X-ray diffraction (XRD) diagrams were performed with Bruker D8 FOCUS (Bruker, Germany) powder diffraction meter with Cu Ka radiation in the range of 5-40o (2θ) at 40 kV and 30 mA. 2.5. Characterization of the CS-DCA/HA/TPP nanoparticles The turbidity of solution was monitored as a function of CS-DCA/HA (1mg/mL) mass ratio
PT
at a fixed wavelength of 420 nm with UV-2550 UV-Visible spectrophotometer (Shimadzu,
RI
Japan).
SC
The particle sizes and size distribution of CS-DCA and CS-DCA/HA/TPP nanoparticles in aqueous environment at 1mg/mL were investigated following filtration after ultrasonic dispersion
NU
for 10 minutes by dynamic light scattering (DLS) measurement. The DLS measurements were
MA
carried out using a Zeta-sizer Nano ZS90 (Malvern Instruments, U.K.). The morphology of the nanoparticles was studied by Tecnai 10 TEM (Philips, Netherlands) at 100 kV. Carbon-coated 200
D
mesh copper grids were immersed in CS-DCA solution and dried in air. The stability of the
PT E
CS-DCA/HA/TPP nanoparticles was investigated in PBS (pH 7.4) by DLS at room temperature at the determined time interval. We also studied the serum stability of the complex at 37oC. Briefly，
CE
CS-DCA/HA/TPP complexes (1 mg/mL) was incubated with PBS supplemented with 10% FBS at
AC
37 ºC. At designated time intervals, the size of complexes was monitored by using a Zeta-Sizer Nano ZS.
2.6. Doxorubicin Loading and in vitro Release Behavior CS-DCA solution prior to dropwise into HA/TPP solution under magnetic stirring for 10 min to allow the complete formation of the system. Then DOX was incubated in CS-DCA/HA solution under mild agitation at room temperature for 12h. The nanoparticles were isolated by 3K30 ultracentrifugation at 30000×g at 4 °C for 30 min. Then the supernate was placed into a
ACCEPTED MANUSCRIPT dialysis bag and dialyzed against distilled water for 48 h. The water was refreshed at time intervals of 6 h. After 48 h, the aqueous solution in the dialysis bag was lyophilized to obtain the DOX-loaded particles. The encapsulation efficiency (EE) and loading efficiency (LE) of the DOX-loaded nanoparticles were determined by measuring the amount of free DOX in the clear
PT
supernatant after centrifugation as described above. The concentration of DOX was determined
RI
by using the UV-vis spectrophotometer at 481 nm. Furthermore, both LE and EE were calculated
SC
based on the standard curve obtained from DOX.
The EE and LE of DOX in the nanoparticles were calculated with the following equation:
total amount of DOX amount of free DOX in supernatant 100 total amount of DOX total amount of DOX amount of unbound DOX LE (%) 100 nanoparticles weight
MA
NU
EE (%)
The release kinetics of DOX from DOX-loaded particles was conducted through dialysis
D
method using PBS (PH 7.4) containing 0.5% (w/v) Tween 80 as release medium. Briefly, the
PT E
isolated DOX -loaded 0.1 mMol PCNs incubated in 5 mL PBS was placed into a dialysis bag (MWCO 1 kDa, Spectrum Laboratories), and incubated in a tank containing 100 mL of release
CE
medium under gentle shaking at 37 C. At scheduled time points (0, 12, 24, 36, 48, 60, 72, 84,
AC
96h), aliquots of 2mL were taken out of each tube with replenishing of the same volume fresh medium. The suspension was collected and the DOX concentrations were determined by calculation from the standard curve obtained from DOX in PBS. 2.7. Cytotoxicity Assay of CS-DCA/HA/DOX Nanoparticles The cytotoxicity of the CS-DCA/HA and CS-DCA/HA/DOX was assessed using the methyl thiazolyl tetrazolium (MTT) assay. The HeLa cells used for these studies were obtained from Medical College of Jinan University (Guangzhou, China). Both DOX·HCl and DOX-loaded
ACCEPTED MANUSCRIPT nanoparticles were dispersed in PBS (pH 7.4) and then serially diluted to get different drug concentration. Hela cells were seeded in 96-well plate at a density of 1×104cells/well and cultured in 200 μL of DMEM 1640 containing 10% FBS in a humidified 37 °C environment with 5% CO2 overnight. Then, the culture medium was replaced by mixture of 180µL fresh culture
PT
medium and 20 μL DOX·HCl or DOX-loaded nanoparticles with different concentration.
RI
Different concentrations (0-1000 μg/mL) of CS-DCA/HA dissolved in culture medium without
SC
DOX were also added to test the cytotoxicity. The plates were then returned to the incubator and maintained in 5% CO2 at 37 °C for 48 h. Fresh culture medium (180 μL) and 20 μL aliquots of
NU
MTT solutions (5 mg/mL) were used to replace the mixture in each well after 48 h. After being
MA
incubated for 4 h, the culture medium was removed and 200 μL of DMSO was then added to each well to dissolve the internalized purple formazan crystals. Plates were vigorously shaken
D
before measuring the relative color intensity using a microplate reader. A test wavelength of 550
PT E
nm and a reference wavelength of 630 nm were used. All experiments were performed in triplicate.
CE
2.8. Histopathology studies
AC
Six weeks old BALB/c female nude mice (16-20 g) were obtained from the Jinan University Center for Animal Experiment. This experimental design has been approved by the Institutional Administration Panel for Laboratory Animal Care. All the animals were housed and fed in the Experimental Animal Center of Jinan University and were specific pathogen free. The rats were exposed to the drug formulations by intravenous injection on the first day. For the CS-DCA/HA/DOX and DOX groups, rats were injected with an equivalent dose of 1 mg DOX/kg; for the CS-DCA/HA group, rats were injected with a dose of vehicle equivalent to the
ACCEPTED MANUSCRIPT CS-DCA/HA/DOX groups; and for the NS group, rats were injected with normal saline with the same volume as used in the CS-DCA/HA/DOX group. For the histological assay, at 2 days after the first intravenous injection, rats were sacrificed, and the heart, liver, spleen, lung, and kidney were gathered. The tissues were fixed in 4% paraformaldehyde in PBS and embedded in paraffin
PT
wax. After routine processing, paraffin wax was cut into 5-mm-thick sections. Finally, the fixed
RI
sections were stained with hematoxylin and eosin (HE), and observed under a light microscope
SC
(Axio Scope A1 FL; Carl Zeiss, Wetzlar, Germany). 2.9. Statistical analysis
NU
All quantitative data are expressed as mean standard deviation (SD) unless otherwise noted.
MA
Statistical significance was tested using an unpaired, two-tailed Student's t-test. A value of P < 0.05 was considered statistically significant.
D
3. Results and discussion
PT E
3.1. Synthesis and Characterization of CS-DCA Conjugates Hydrophobic DCA with a rigid structure was selected to graft onto CS. The fatty acid
CE
molecule of DCA was covalently grafted onto the main backbone of CS to obtain CS-DCA
AC
conjugated by EDC and NHS. The molar ratio of EDC·HCl and DCA is 4:1. The molar ratio of DCA and CS is adjusted at 0.1:1, 0.2:1, 0.3:1 and the corresponding product is defined as CS-DCA10, CS-DCA20, CS-DCA30, freeze dried and stored. As shown in Table 1, the DS of DCA was calculated with elemental analysis results based on the changes of C/N between CS and CS-DCA and was tuned by adjusting the feed ratio of reagents. With the changing ratio of DCA to aminoglucose units from 10% to 30%, the DS of DCA was changed from 3.2 to 7.8. Compared with the FTIR spectrum of CS (Fig. 1a), the peaks of CS-DCA10 at 1652 cm-1 (amide I band) and
ACCEPTED MANUSCRIPT 1560 cm-1 (amide II band) were obviously intensified, meanwhile the disappearance of the 1600 cm-1 (N-H bending vibration) indicated that hydrogen of the primary amino group was mainly substituted by DCA molecules. It also showed that with the increasing of feed ratio of DCA to CS (Fig. 1a from 10% to 30%), the amide I peak at 1652 cm-1 became sharper, which indirectly
PT
indicated the increase of DS of DCA. Also, the presence of DCA in CS was evaluated by the
RI
characteristic peaks of DCA appearing at 0.6-2.5 ppm in the 1H NMR spectra (Fig. 1b) [40].
SC
Based on the comparison of 1H NMR spectra (Fig. 1b) of CS with CS-DCA, it can be concluded that the small hydrophobic molecule DCA was successfully grafted onto CS due to the appearance
NU
of new peaks from 0.7 to 1.9 ppm. For example, the peak at 0.7 ppm was affiliated to 21-CH3 in
MA
DCA moiety. The X-ray diffraction pattern was conducted for CS, differenced feed ratio of DCA to CS to further evaluate their crystallization behaviors. As shown in Fig. 1c, exhibits a broad peak
D
at 23° due to semi-crystalline nature of CS as reported elsewhere [41]. XRD. In contrast,
PT E
difference feed ratio of DCA to CS still exhibited similar peaks as the CS/DCA, but peaks became weaker. These results are consistent with the FTIR data and 1H NMR date.
CE
3.2. Formation and Characterization of CS-DCA/HA/TPP nanoparticles
AC
The route of self-assembly PCNs was illustrated in Scheme 1. CS was positively charged in acidic solution, while TPP and HA were negatively charged in water solution. Thus, nanoparticles were formed by ionic interaction between positively charged amino groups of CS and negatively charged counterions of TPP and HA. Turbidity measurement is a simple and direct indicator for polyelectrolyte complex formation, accompanied by drastic changes of the system turbidity of the solution [42, 43]. Fig. 2a demonstrated the curve of turbidimetric titration with HA and CS-DCA solutions. Initially, the CS-DCA solution was completely transparent.
ACCEPTED MANUSCRIPT With the increase of the quantity of CS-DCA/HA, the turbidity of the solution gradually ascents, because the complex of CS/HA and CS-DCA/HA are formed by the electrostatic interactions and the CS/HA and CS-DCA/HA with higher HA grafting rate are more difficult to be dissolved in water of PCNs. In the beginning of this process, coacervation layer was formed at the surface of
PT
the CS. Then –P3O105- could penetrate the coacervation layer and reach the core and crosslinked
RI
with it. However, because HA has large molecular size and cannot penetrate the coacervation
SC
layer, it only reacted with –NH3+ at the surface. With the increase of HA and TPP, the CS-DCA underwent a high positive-to-low positive transformation of their zeta potential. The influence of
NU
DS of DCA on the turbidity of CS-DCA was shown in Fig. 2a. The turbidity of CS-DCA
MA
decreased with the increasing DS of a hydrophobic molecule. It could be explained that the higher DS, the higher hydrophobicity, which gave rise to the stronger self-aggregation ability.
D
Thus, the sizes and size distribution of self-aggregated CS-DCA/HA/TPP nanoparticles in PBS
PT E
buffer (pH 7.4) were determined using DLS and were illustrated in Fig. 2b and Table 2. The sizes of the CS-DCA/HA nanoparticles ranged from 160-570 nm, while the diameters of the
CE
HA/CS nanoparticles were 180-680 nm. With the increase of HA, the zeta potentials and the
AC
sizes of the nanoparticles decreased. The sizes of CS-DCA nanoparticles increased from 204 to 240 nm with the decrease of DS of DCA. The zeta potentials of CS-DCA/HA/TPP nanoparticles was further examined (Table 2). The morphology of CS-DCA nanoparticles characterized by TEM is shown in Fig. 3. It indicated that the nano-aggregates were spherical nanoparticles and the CS-DCA30 nanoparticles had the smallest sizes. Furthermore, the sizes of the CS-DCA/HA/TPP kept constant when stored at room temperature for almost 1 month, suggesting the stability of the nanoparticles (Fig. 4A).
ACCEPTED MANUSCRIPT To further confirm the stability of CS-DCA/HA/TPP during circulation, we have introduced experiments of serum stability, the relevant data were shown in Fig. 4B. The particle sizes of (a)
CS/HA, (b) CS-DCA10/HA, (c) CS-DCA20/HA and (d) CS-DCA30/HA nanoparticles maintained the initial size over the experimental time in presence of 10% fetal bovine serum
PT
(FBS), this result indicated that CS-DCA/HA/TPP exhibited excellent stability in FBS at 37 oC.
RI
3.3. Drug Loading and in vitro Release Behavior
DOX, one of the most potent anticancer drugs, was chosen as the drug model. A delivery
SC
system based on CS-DCA/HA nanoparticles was developed for enhancing the efficacy of cancer chemotherapy of DOX. The results as Table 3 shown DOX could be easily loaded into
NU
CS-DCA/HA/TPP nanoparticles, and the drug loading level increased from 6.1% to 7.3% with the increasing DS of DCA while encapsulation efficiency was up to 60%, which was relatively
MA
much higher than traditional drug carriers, such as some micelles [44-46]. The drug loading and encapsulation efficiency for HA-TPP/CS nanoparticles of DOX were 3.7% and 20.75%
D
respectively, which were significantly smaller than that of the CS-DCA/HA/TPP nanoparticles.
PT E
Furthermore, the sizes of DOX loaded nanoparticles increased about 70 nm while PDI remained low. The hydrophobic group of DCA formed a hydrophobic micro-area, on the outside of which the hydrophilic skeleton of CS coiled to form a hydrophilic shell. Thus, the surface energy of the
CE
aqueous solution was decreased to the lowest. The intermolecular and intramolecular hydrogen bonding of the CS hydrophilic skeleton promoted the CS-DCA to form nanoparticles. The unique
AC
super molecular structure is well suited for capture of hydrophobic drugs. In vitro release evaluation results were shown in Fig.5. DOX was released slowly from CS-DCA/HA/TPP/DOX nanoparticles without burst release phenomenon in the first 12 h. Meanwhile, it had to be mentioned that the higher DS of DCA, the higher release rate of drugs due to the CS-DCA is one of the key factors of the DOX load. The accumulative amount of DOX released from CS-DCA10/HA/TPP, CS-DCA20/HA/TPP, and CS-DCA30/HA/TPP nanoparticles was 28.5, 33.9, and 39.8%, respectively, within 48 h and the HA/CS also have the slower release
ACCEPTED MANUSCRIPT rate due to CS main chain contains hydrophobic micro area, small molecules can by hydrophobic effect load hydrophobic drug doxorubicin. The slow release rate of DOX from CS-DCA nanoparticles may be ascribed to the strong interaction between hydrophobic DCA and DOX. 3.4 Cytotoxicity Assay of CS-DCA/HA/DOX Nanoparticles
PT
In order to confirm whether the ability to deliver DOX by CS-DCA/HA/TPP nanoparticles
RI
could really work, the toxicity of CS-DCA/HA/TPP nanoparticles to HeLa cells was carried out.
SC
We got the toxicity results by MTT assay. The cytotoxicity of DOX loaded CS-DCA/HA/TPP nanoparticles was compared with that of free DOX dissolved in PBS. The cytotoxicity of blank
NU
CS-DCA/HA/TPP nanoparticles was also investigated to ensure that the cytotoxicity was caused
MA
by DOX alone. As shown in Fig.6a, the cytotoxicity results of blank CS-DCA/HA/TPP nanoparticles, when the concentrations below 1000 µg/mL CS-DCA/HA/TPP will not cause
D
significant cytotoxicity, all of the Cell activity in more than 80% [37]. Based on this result, the
PT E
detection concentration of CS-DC/HA/DOX were lower than 1000 µg/mL, excluding the factors of cytotoxicity caused by carrier. It was found from Fig. 6b that CS-DCA/HA/DOX can cause
CE
obvious cytotoxicity. When DOX was loaded in CS-DCA/HA/TPP nanoparticles, the cell viability
AC
decreased significantly from almost 100% to less than 10% as the DOX concentration increased from 1 to 50 μg/mL, while the cell viability decreased to about 10% when free DOX with the same concentration was used in cells for 48 h. The possible reason is that DOX loaded CS-DCA/HA/TPP nanoparticles with positive charge facilitate the entrance into the tumor cells and then into the nucleus to take effect. CS-DCA/HA/TPP/DOX nanoparticles with higher DS of DCA showed higher antitumor efficiency than that with lower DS of DCA, which may be also the results that drug release speed increased with the increase of DS of DCA and that DOX-loaded
ACCEPTED MANUSCRIPT CS-DCA/HA/TPP nanoparticles with higher DS of DCA have smaller sizes which were favorable for the entrance into cells. In summary, the drug loading and releasing ability of CS-DC/HA had obvious effect in vitro. 3.5. Histological analysis of organs
PT
Through H&E staining analysis of rat were performed. The images of the heart, liver, spleen,
RI
lung and kidney were obtained using light microscopy (Fig. 7). In the treatment group of CS,
SC
CS-DCA, and CS-DCA/HA/DOX, no visible morphological changes were observed in corresponding organs compared with the DOX control group [47]. However, the tubular dailation
NU
with flattening of the epithelium cells were observed in the kidneys under the free drug treatment
effects on important organ cytotoxicity.
D
4. Conclusion
MA
of animals. In conclusion, these results suggest that PCNs enhance drug delivery and reduce the
PT E
In this study, novel PCNs based on hydrophobically modified CS and hyaluronic acid were produced. The PCNs in this work are prominent in stability, which keep stable up to 1week in PBS
CE
(pH 7.4) with no obvious sizes change and capable of associating DOX, with EE up to 56% and
AC
keep a sustained release manner for design time (96h) without burst release at 37 oC. In addition, this type of PCNs doesn’t show cytotoxicity even at high concentrations. From the study above it may be concluded that CS-DCA/HA/TPP/DOX nanoparticles exhibit remarkable biocompatibility and have the potential to be used in biomedical applications and treatment of tumor. Acknowledgements This study was supported financially by the Natural Science Foundation of China (51303064), the Science and Technology Program of Guangzhou (201508020115, 201601010270,
ACCEPTED MANUSCRIPT 2017010160489, 201704030083), the Pearl River S&T Nova Program of Guangzhou
(201710010155, 201806010072), the Science and Technology Project of Guangdong province
AC
CE
PT E
D
MA
NU
SC
RI
PT
(2015A010101313, 2017A050506011, 2017B090911012).
ACCEPTED MANUSCRIPT
References [1] S. Xu, R. Zhang, W. Zhao, Y. Zhu, W. Wei, X. Liu, J. Luo, Self-assembled polymeric nanoparticles film stabilizing gold nanoparticles as a versatile platform for ultrasensitive detection of carcino-embryonic antigen, Biosensors & bioelectronics 92 (2016) 570-6.
PT
[2] C.L. Oliveira, F. Veiga, C. Varela, F. Roleira, E. Tavares, I. Silveira, A.J. Ribeiro, Characterization
RI
of polymeric nanoparticles for intravenous delivery: Focus on stability, Colloids and surfaces. B,
SC
Biointerfaces 150 (2016) 126-133.
[3] M. Prabaharan, J.F. Mano, Chitosan-based particles as controlled drug delivery systems, Drug
NU
delivery 12(1) (2005) 41-57.
MA
[4] M. Lee, A.C. Lo, P.T. Cheung, D. Wong, B.P. Chan, Drug carrier systems based on collagen-alginate composite structures for improving the performance of GDNF-secreting HEK293
D
cells, Biomaterials 30(6) (2009) 1214-21.
PT E
[5] P. Zhang, J.H. Yang, W.C. Li, W. Wang, C.J. Liu, M. Griffith, W.G. Liu, Cationic polymer brush grafted-nanodiamond via atom transfer radical polymerization for enhanced gene delivery and
CE
bioimaging, J Mater Chem 21(21) (2011) 7755-64.
AC
[6] Y. Luo, Q. Wang, Recent development of chitosan-based polyelectrolyte complexes with natural polysaccharides for drug delivery, International journal of biological macromolecules 64 (2014) 353-67.
[7] S.P. Thakker, A.P. Rokhade, S.S. Abbigerimath, S.R. Iliger, V.H. Kulkarni, U.A. More, T.M. Aminabhavi, Inter-polymer complex microspheres of chitosan and cellulose acetate phthalate for oral delivery of 5-fluorouracil, Polym Bull 71(8) (2014) 2113-31. [8] J. Venkatesan, S. Anil, S.K. Kim, M.S. Shim, Chitosan as a vehicle for growth factor delivery:
ACCEPTED MANUSCRIPT
Various preparations and their applications in bone tissue regeneration, International journal of biological macromolecules 104 (2017) 1383-97. [9] D.P. Biswas, P.A. Tran, C. Tallon, A.J. O'Connor, Combining mechanical foaming and thermally induced phase separation to generate chitosan scaffolds for soft tissue engineering, Journal of
PT
biomaterials science. Polymer edition 28(2) (2017) 207-226.
RI
[10] M. Rodriguez-Vazquez, B. Vega-Ruiz, R. Ramos-Zuniga, D.A. Saldana-Koppel, L.F.
SC
Quinones-Olvera, Chitosan and Its Potential Use as a Scaffold for Tissue Engineering in Regenerative Medicine, Biomed Res Int (2015) Article ID 821279, 15 pages.
NU
[11] Y. Li, J. Yang, B. Xu, F. Gao, W. Wang, W. Liu, Enhanced Therapeutic siRNA to Tumor Cells by
MA
a pH-Sensitive Agmatine-Chitosan Bioconjugate, ACS applied materials & interfaces 7(15) (2015) 8114-24.
D
[12] L.H. Peng, J. Niu, C.Z. Zhang, W. Yu, J.H. Wu, Y.H. Shan, X.R. Wang, Y.Q. Shen, Z.W. Mao,
PT E
W.Q. Liang, J.Q. Gao, TAT conjugated cationic noble metal nanoparticles for gene delivery to epidermal stem cells, Biomaterials 35(21) (2014) 5605-18.
CE
[13] W. Xiong, L. Li, Y. Wang, Y. Yu, S. Wang, Y. Gao, Y. Liang, G. Zhang, W. Pan, X. Yang, Design
AC
and evaluation of a novel potential carrier for a hydrophilic antitumor drug: Auricularia auricular polysaccharide-chitosan nanoparticles as a delivery system for doxorubicin hydrochloride, International journal of pharmaceutics 511(1) (2016) 267-75. [14] D. Nayak, A.P. Minz, S. Ashe, P.R. Rauta, M. Kumari, P. Chopra, B. Nayak, Synergistic combination of antioxidants, silver nanoparticles and chitosan in a nanoparticle based formulation: Characterization and cytotoxic effect on MCF-7 breast cancer cell lines, Journal of colloid and interface science 470 (2016) 142-52.
ACCEPTED MANUSCRIPT
[15] M. Vincekovic, M. Curlin, D. Jurasin, Impact of cationic surfactant on the self-assembly of sodium caseinate, Journal of agricultural and food chemistry 62(34) (2014) 8543-54. [16] S. Wang, R.G. Larson, Coarse-grained molecular dynamics simulation of self-assembly and surface adsorption of ionic surfactants using an implicit water model, Langmuir : the ACS journal of
PT
surfaces and colloids 31(4) (2015) 1262-71.
RI
[17] B. Xu, W. Yao, Y. Li, S. Zhang, X. Huang, Perfluorocyclobutyl Aryl Ether-Based ABC
SC
Amphiphilic Triblock Copolymer, Scientific reports 6 (2016) 39504.
[18] D. Niu, Y. Li, J. Shi, Silica/organosilica cross-linked block copolymer micelles: a versatile
NU
theranostic platform, Chemical Society reviews 46(3) (2017) 569-585.
Aminabhavi,
Oral
insulin
MA
[19] K. Chaturvedi, K. Ganguly, A.R. Kulkarni, W.E. Rudzinski, L. Krauss, M.N. Nadagouda, T.M. delivery
using
deoxycholic
acid
conjugated
PEGylated
D
polyhydroxybutyrate co-polymeric nanoparticles, Nanomedicine (Lond) 10(10) (2015) 1569-83.
PT E
[20] Y.I. Jeong, K.D. Chung, D.H. Kim, Y.H. Kim, Y.S. Lee, K.C. Choi, All-trans retinoic acid-incorporated nanoparticles of deoxycholic acid-conjugated dextran for treatment of CT26
CE
colorectal carcinoma cells, International journal of nanomedicine 8 (2013) 485-93.
AC
[21] J.Y. Lee, C. Crake, B. Teo, D. Carugo, M. de Saint Victor, A. Seth, E. Stride, Ultrasound-Enhanced siRNA Delivery Using Magnetic Nanoparticle-Loaded Chitosan-Deoxycholic Acid Nanodroplets, Advanced healthcare materials 6(8) (2017) 1601246. [22] A. Almalik, S. Karimi, S. Ouasti, R. Donno, C. Wandrey, P.J. Day, N. Tirelli, Hyaluronic acid (HA) presentation as a tool to modulate and control the receptor-mediated uptake of HA-coated nanoparticles, Biomaterials 34(21) (2013) 5369-80. [23] E. Auzenne, S.C. Ghosh, M. Khodadadian, B. Rivera, D. Farquhar, R.E. Price, M. Ravoori, V.
ACCEPTED MANUSCRIPT
Kundra, R.S. Freedman, J. Klostergaard, Hyaluronic acid-paclitaxel: antitumor efficacy against CD44(+) human ovarian carcinoma xenografts, Neoplasia (New York, N.Y 9(6) (2007) 479-86. [24] K.Y. Choi, H. Chung, K.H. Min, H.Y. Yoon, K. Kim, J.H. Park, I.C. Kwon, S.Y. Jeong, Self-assembled hyaluronic acid nanoparticles for active tumor targeting, Biomaterials 31(1) (2010)
PT
106-14.
RI
[25] K.Y. Choi, K.H. Min, H.Y. Yoon, K. Kim, J.H. Park, I.C. Kwon, K. Choi, S.Y. Jeong, PEGylation
SC
of hyaluronic acid nanoparticles improves tumor targetability in vivo, Biomaterials 32(7) (2011) 1880-9.
NU
[26] G. Pitarresi, F.S. Palumbo, A. Albanese, C. Fiorica, P. Picone, G. Giammona, Self-assembled
MA
amphiphilic hyaluronic acid graft copolymers for targeted release of antitumoral drug, Journal of drug targeting 18(4) (2010) 264-76.
D
[27] F.S. Palumbo, R. Puleio, C. Fiorica, G. Pitarresi, G.R. Loria, G. Cassata, G. Giammona, Matrices
PT E
of a hydrophobically functionalized hyaluronic acid derivative for the locoregional tumour treatment, Acta biomaterialia 25 (2015) 205-15.
CE
[28] G. Tripodo, A. Trapani, M.L. Torre, G. Giammona, G. Trapani, D. Mandracchia, Hyaluronic acid
AC
and its derivatives in drug delivery and imaging: Recent advances and challenges, European journal of pharmaceutics and biopharmaceutics : official journal of Arbeitsgemeinschaft fur Pharmazeutische Verfahrenstechnik e.V 97(Pt B) (2015) 400-16. [29] S. Ganesh, A.K. Iyer, D.V. Morrissey, M.M. Amiji, Hyaluronic acid based self-assembling nanosystems for CD44 target mediated siRNA delivery to solid tumors, Biomaterials 34(13) (2013) 3489-502. [30] A. Sharma, R.A. Gadkari, S.V. Ramakanth, K. Padmanabhan, D.S. Madhumathi, L. Devi, L.
ACCEPTED MANUSCRIPT
Appaji, J.C. Aster, A. Rangarajan, R.R. Dighe, A novel Monoclonal Antibody against Notch1 Targets Leukemia-associated Mutant Notch1 and Depletes Therapy Resistant Cancer Stem Cells in Solid Tumors, Scientific reports 5 (2015) 11012. [31] C.E. Galer, D. Sano, S.C. Ghosh, J.H. Hah, E. Auzenne, A.N. Hamir, J.N. Myers, J. Klostergaard,
PT
Hyaluronic acid-paclitaxel conjugate inhibits growth of human squamous cell carcinomas of the head
RI
and neck via a hyaluronic acid-mediated mechanism, Oral oncology 47(11) (2011) 1039-47.
SC
[32] H. Lee, K. Lee, T.G. Park, Hyaluronic acid-paclitaxel conjugate micelles: synthesis, characterization, and antitumor activity, Bioconjugate chemistry 19(6) (2008) 1319-25.
NU
[33] H.J. Cho, H.Y. Yoon, H. Koo, S.H. Ko, J.S. Shim, J.H. Lee, K. Kim, I.C. Kwon, D.D. Kim,
MA
Self-assembled nanoparticles based on hyaluronic acid-ceramide (HA-CE) and Pluronic(R) for tumor-targeted delivery of docetaxel, Biomaterials 32(29) (2011) 7181-90.
D
[34] S. Siegman, N.F. Truong, T. Segura, Encapsulation of PEGylated low-molecular-weight PEI
PT E
polyplexes in hyaluronic acid hydrogels reduces aggregation, Acta biomaterialia 28 (2015) 45-54. [35] R. Ran, Y. Liu, H. Gao, Q. Kuang, Q. Zhang, J. Tang, K. Huang, X. Chen, Z. Zhang, Q. He,
CE
Enhanced gene delivery efficiency of cationic liposomes coated with PEGylated hyaluronic acid for
AC
anti P-glycoprotein siRNA: a potential candidate for overcoming multi-drug resistance, International journal of pharmaceutics 477(1-2) (2014) 590-600. [36] K.Y. Lee, I.C. Kwon, Y.H. Kim, W.H. Jo, S.Y. Jeong, Preparation of chitosan self-aggregates as a gene delivery system, J Control Release 51(2-3) (1998) 213-20. [37] S.Y. Chae, S. Son, M. Lee, M.K. Jang, J.W. Nah, Deoxycholic acid-conjugated chitosan oligosaccharide nanoparticles for efficient gene carrier, Journal of controlled release : official journal of the Controlled Release Society 109(1-3) (2005) 330-44.
ACCEPTED MANUSCRIPT
[38] M. de la Fuente, B. Seijo, M.J. Alonso, Bioadhesive hyaluronan-chitosan nanoparticles can transport genes across the ocular mucosa and transfect ocular tissue, Gene therapy 15(9) (2008) 668-76. [39] A. Miwa, A. Ishibe, M. Nakano, T. Yamahira, S. Itai, S. Jinno, H. Kawahara, Development of
PT
novel chitosan derivatives as micellar carriers of taxol, Pharmaceutical research 15(12) (1998)
RI
1844-50.
SC
[40] K. Kim, S. Kwon, J.H. Park, H. Chung, S.Y. Jeong, I.C. Kwon, I.S. Kim, Physicochemical characterizations of self-assembled nanoparticles of glycol chitosan-deoxycholic acid conjugates,
NU
Biomacromolecules 6(2) (2005) 1154-8.
MA
[41] S. Hajji, I. Younes, O. Ghorbel-Bellaaj, R. Hajji, M. Rinaudo, M. Nasri, K. Jellouli, Structural differences between chitin and chitosan extracted from three different marine sources, International
D
journal of biological macromolecules 65 (2014) 298-306.
based
on
methoxy
PT E
[42] K. Luo, J. Yin, Z. Song, L. Cui, B. Cao, X. Chen, Biodegradable interpolyelectrolyte complexes poly(ethylene
glycol)-b-poly(alpha,L-glutamic
acid)
and
chitosan,
CE
Biomacromolecules 9(10) (2008) 2653-61.
AC
[43] A. Drogoz, L. David, C. Rochas, A. Domard, T. Delair, Polyelectrolyte complexes from polysaccharides: formation and stoichiometry monitoring, Langmuir 23(22) (2007) 10950-8. [44] X. Zeng, X. Zhou, M. Li, C. Wang, J. Xu, D. Ma, W. Xue, Redox poly(ethylene glycol)-b-poly(L-lactide) micelles containing diselenide bonds for effective drug delivery, Journal of materials science. Materials in medicine 26(9) (2015) 234. [45] W. Chen, P. Zhong, F. Meng, R. Cheng, C. Deng, J. Feijen, Z. Zhong, Redox and pH-responsive degradable micelles for dually activated intracellular anticancer drug release, Journal of controlled
ACCEPTED MANUSCRIPT
release : official journal of the Controlled Release Society 169(3) (2013) 171-9. [46] L.Y. Qiu, Y.H. Bae, Self-assembled polyethylenimine-graft-poly(epsilon-caprolactone) micelles as potential dual carriers of genes and anticancer drugs, Biomaterials 28(28) (2007) 4132-42. [47] Y.F. Mo, H.W. Wang, J.H. Liu, Y. Lan, R. Guo, Y. Zhang, W. Xue, Y.M. Zhang, Controlled
PT
release and targeted delivery to cancer cells of doxorubicin from polysaccharide-functionalised
AC
CE
PT E
D
MA
NU
SC
RI
single-walled carbon nanotubes, J Mater Chem B 3(9) (2015) 1846-1855.
ACCEPTED MANUSCRIPT
Figure Captions Fig.1. FT-IR spectra of (a) CS and CS-DCA derivatives; 1H-NMR spectra of (b) CS and CS-DCA derivatives; XRD diffraction patterns of (c) CS and CS-DCA derivatives. Fig.2. (a) Turbidimetric titration curves for HA and CS-DCA derivatives solutions as a function
PT
of CS-DCA/HA mass ratio; (b) Size of HA and CS-DCA derivatives solutions as a function of
RI
CS-DCA/HA mass ratio.
SC
Fig.3. TEM images of (a) CS/HA, (b) CS-DCA10/HA, (c) CS-DCA20/HA and (d) CS-DCA30/HA nanoparticles.
NU
Fig.4. Size of (a) CS/HA, (b) CS-DCA10/HA, (c) CS-DCA20/HA and (d) CS-DCA30/HA
MA
nanoparticles in PBS at (A) room temperature and (B) containing10% fetal bovine serum (FBS) at 37 oC as a function of time.
D
Fig.5. In vitro release profile of DOX from nanoparticles incubated in 0.02 M PBS (pH 7.4) at 37 o
PT E
C: (a) CS/HA, (b) CS-DCA10/HA, (c) CS-DCA20/HA and (d) CS-DCA30/HA.
Fig. 6. (a) Effects of CS-DCA derivatives on the Hela cell viability at different concentration
CE
after incubation for 48h; (b) Viability of Hela cells difference nanoparticles for 48 h with
AC
different concentration. The results represent the means SD (n 6). Fig.7. Morphological change of SD rat heart, liver, spleen, lung, and kidney, exposed to (a) CS, (b) CS-DCA, (c) CS-DCA/HA/DOX, and (d) DOX. The bar corresponds to 50 mm.
ACCEPTED MANUSCRIPT Data images-Tables Table 1. Degree of substitution of deoxycholic acid. CS-DCA10
CS-DCA20
CS-DCA30
sugar unit/DA
100/0
100/10
100/20
100/30
C%
44.03
41.41
38.63
42.81
H%
6.05
11.04
8.05
11.00
N%
8.25
6.83
5.76
6.14
C/N
5.33
6.06
DA DS%
0
3.18
RI
PT
CS
CE
PT E
D
MA
6.71
6.97
6.44
7.75
SC
NU
All values are averaged from three measurements.
AC
a
Samples
ACCEPTED MANUSCRIPT Table 2. Size, PDI and Zeta Potential of CS-DCA nanoparticles with a different DS of DCA. CS-DCA10
CS
Zeta Potential (mV)
1.0
0.6
0.8
0.6
0.8
0.6
384.6 ± 1.2 0.52 ± 0.01 33.5 ± 0.2
313.3 ± 0.4 0.5 ± 0.01 27.1 ± 0.5
179.7 ± 1.3 0.19 ± 0.01 24.9 ± 0.3
243.5 ± 0.3 0.18 ± 0.01 41.5 ± 0.6
160.5 ± 0.2 0.11 ± 0.01 26.7 ± 0.5
220.0 ± 0.3 0.15 ± 0.01 28.3 ± 0.7
205.6 ± 0.1 0.02 ± 0.01 16.9 ± 0.1
203.6 ± 1.3
CE
PT E
D
MA
NU
SC
PDI represent the polydispersity index.
AC
a
0.8
PT
PDI a
CS-DCA30
0.6
RI
HA/CS-DCA mass ratio Size (nm)
CS-DCA20
0.13 ± 0.01 21.7± 0.1
ACCEPTED MANUSCRIPT Table 3. Characterization of HA and CS derivatives. PDIa
Zeta potential (mV)
EE% b
LE% c
CS/HA/DOX
438.6 ± 1.7
0.53 ± 0.01
31.3 ± 0.2
20.75
3.7
CS-DCA10/HA/DOX
306.5 ± 0.2
0.31± 0.01
39.7 ± 0.6
38.18
6.1
CA-DCA20/HA/DOX
290.0 ± 0.8
0.25± 0.01
25.5 ± 0.7
47.46
6.7
CS-DCA30/HA/DOX
283.6 ± 1.2
0.23 ± 0.01
18.2 ± 0.1
56.62
7.3
PT
Size (nm)
PDI represent the polydispersity index.
PT E
D
MA
NU
SC
RI
mean encapsulation efficiency (EE) and loading efficiency (LE), respectively.
CE
b and c
AC
a
Samples
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Lili Chen, Yuanyuan Zheng, Longbao Feng, Zonghua Liu, Rui Guo, Yuanming Zhang PII: DOI: Reference:
S0141-8130(18)35505-3 https://doi.org/10.1016/j.ijbiomac.2018.12.215 BIOMAC 11374
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
15 October 2018 10 December 2018 21 December 2018
Please cite this article as: Lili Chen, Yuanyuan Zheng, Longbao Feng, Zonghua Liu, Rui Guo, Yuanming Zhang , Novel hyaluronic acid coated hydrophobically modified chitosan polyelectrolyte complex for the delivery of doxorubicin. Biomac (2018), https://doi.org/ 10.1016/j.ijbiomac.2018.12.215
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.
ACCEPTED MANUSCRIPT
Novel hyaluronic acid coated hydrophobically modified chitosan polyelectrolyte complex for the delivery of doxorubicin Lili Chen a, b, 1, Yuanyuan Zheng a, 1, Longbao Feng a, Zonghua Liu a, Rui Guo a, *, Yuanming
Key Laboratory of Biomaterials of Guangdong Higher Education Institutes, Guangdong
RI
a
PT
Zhang a, b, *
SC
Provincial Engineering and Technological Research Center for Drug Carrier Development,
Department of Chemistry, Jinan University, Guangzhou 510632, China
MA
b
NU
Department of Biomedical Engineering, Jinan University, Guangzhou 510632, China
* Corresponding authors:
CE
PT E
These authors contributed equally to this work.
AC
1
D
[email protected] (Yuanming Zhang), [email protected] (Rui Guo)
ACCEPTED MANUSCRIPT
Abstract The aim of this work was to examine the formation and properties of a novel polyelectrolyte complex of drug carrier system for the delivery of doxorubicin (DOX), which consists of hyaluronic acid (HA) coated hydrophobically modified chitosan (CS). Various batches of
PT
polyelectrolyte complexes with the molar ratio of deoxycholic acid (DCA) and chitosan (CS) of
RI
0.1, 0.2, 0.3 were prepared, and were termed as CS-DCA10, CS-DCA20, and CS-DCA30
SC
respectively. The samples were characterized by Fourier transform infrared (FTIR) spectroscopy, X-ray diffraction (XRD), Transmission electron microscopy (TEM), nuclear magnetic resonance
NU
hydrogen spectrum (1H NMR) and dynamic light scattering (DLS). Particle sizes of synthesized
MA
polyelectrolyte complex nanoparticles (PCNs) were found to be in the range of 280–310 nm, larger than those of uncoated nanoparticles (~150 nm). The PCNs have large zeta potentials
D
(about 26mV) which make them stable and no sizes change were determined. DOX could be
PT E
easily incorporated into the PCNs with encapsulation efficiency (56%) and kept a sustained release manner without burst effect when exposed to PBS (pH 7.4) at 37 oC. Overall, these
CE
findings confirmed the potential of these PCNs for drug carrier and prolonged and sustained
AC
delivery in the bloodstream.
Keywords: chitosan, deoxycholic acid, hyaluronic acid, polyelectrolyte complex, nanoparticles, drug carrier
ACCEPTED MANUSCRIPT
1. Introduction Polymeric nanoparticles made of natural and synthetic polymers have received the majority of attention due to their stability and easy surface modification [1, 2]. Among natural polymers, polysaccharides tend to be internalized and degraded rapidly, thus enabling a moderate
PT
intracellular release of the drug. Consequently, polysaccharides have been widely used in drug
RI
delivery systems [3, 4], gene delivery and bioimaging [5]. Polysaccharides can be generally
SC
classified into two types, non-polyelectrolyte and polyelectrolyte. Polyelectrolyte contained cationic and anionic polysaccharides, have been paid more and more attention in the last two
NU
decades, due to their outstanding advantages in drug delivery systems [6], probably for the fact
MA
that the spontaneous association of the oppositely-charged polymers leads to the formation of polyelectrolyte complex through strong and reversible electrostatic links; the transient structure
D
obtained avoids the use of toxic covalently bonded chemical cross-linkers, thus allowing them to
PT E
be used in humans [7].
Chitosan (CS) is a natural, non-toxic, biodegradable polysaccharide, which consists of
CE
glucosamine and N-acetylglucosamine units derived from deacetylation of chitin. CS possesses
AC
desirable properties such as biodegradability, biocompatibility and CS scaffolds with high level of porosity that make this biomaterial a good candidate for tissue engineering and drug delivery [8-10]. Recently, the CS has been extensively employed in the development of micro- and nano-carriers in the delivery of anticancer drug, polypeptide, protein and gene [11, 12]. It is worth mentioning that CS is a natural polycationic polysaccharide, but it can only be dissolved in acidic aqueous solution, which is not conducive to the load of some bioactive macromolecular drugs [13, 14]. Hydrophobic modification of polysaccharide makes it become
ACCEPTED MANUSCRIPT amphiphilic and self-assemble to form micelles in aqueous solution by intramolecular and intermolecular hydrophobic effect, which has a broad application prospect in the field of biomedicine [15-18]. Deoxycholic acid (DCA) plays an important role in the emulsification, solubilization, and absorption of cholesterol, fats, and liphophilic vitamins in the body. For the
PT
past decades, DCA has been used to modify CS, which often acting as a promising delivery
RI
system of hydrophobic drugs, peptide and DNA [19-21].
SC
Hyaluronic acid (HA) is one of the most ideal materials due to its high degradability and excellent biocompatibility [22-25]. HA with its derivatives are most commonly used to develop
NU
several carrier systems for cancer diagnosis and treatment. The HA-modified polysaccharides are
MA
able to load gene and peptide/protein for drug delivery. [26-28]. Many studies have reported the overexpression of its main receptor CD44 in several solid tumors [29, 30]. Therefore, a series of
D
HA-based carriers have been developed in order to target tumor cells, e.g. HA-paclitaxel
PT E
conjugates[31, 32], self-assembled HA nanoparticles[33], PEGylated HA nanoparticles, and etc [34, 35].
CE
Probably, the most popular way to produce chitosan nanoparticles is through ionotropic
AC
gelation of chitosan with sodium tripolyphosphate (TPP), a small ion with a triple negative charge throughout the physiologically acceptable pH range. In this study, we designed a targeted PCNs based on hydrophobically modified CS as a novel delivery system, consisting of cationic CS and anionic HA. The PCNs in this work are prominent in stability, and capable of associating DOX. In addition, this type of PCNs doesn’t show cytotoxicity even at high concentrations. From the study above it may be concluded that CS-DCA/HA/TPP/DOX nanoparticles exhibit remarkable biocompatibility and have the
ACCEPTED MANUSCRIPT potential to be used in biomedical applications and treatment of tumor. 2. Materials and methods 2.1. Materials Chitosan (90% deacetylation, 50 kDa Mw) was purchased from Haidebei Marine
Shandong
Freda
Group
(Shandong,
China).
1-ethyl-3-(3-dimethylaminopropyl)
RI
from
PT
Bioengineering Co., LTD (Shandong, China). Hyaluronic acid (1640 kDa Mw) was obtained
SC
carbodiimide hydrochloride (EDC), deoxycholic acid (DCA), tripolyphosphate (TPP), dimethyl sulfoxide (DMSO) and pyrene were purchased from Aladdin reagent Inc. (Shanghai, China).
NU
DOX·HCl was obtained from Beijing Huafeng United Technology Co., LTD (Beijing, China).
MA
Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), Dulbecco’s phosphate-buffered saline (DPBS), penicillin, and streptomycin were acquired from Life
D
Technologies. All other reagents were analytical grade and used without further purification.
PT E
2.2. Synthesis of CS-DCA Conjugates
The CS-DCA conjugates were synthesized based on the mechanism of carbodiimide
CE
chemistry [36]. Briefly, DCA in ethanol was added to 1% (v/v) CS acetic acid solution followed
AC
by the dropwise addition of EDC (1mol/mol DCA) under magnetic stirring at room temperature for 24h. Then the resulting reaction solution was poured into excess amount of the ethanol/ammonia water solution (pH 8). The precipitates were recovered by centrifugation, washed thoroughly with water, methanol and ethanol respectively, followed by freeze-drying. Various batches of polyelectrolyte complex with the molar ratio of deoxycholic acid (DCA) and CS from 0.1 to 0.3 were prepared, and were named CS-DCA10, CS-DCA20, and CS-DCA30 respectively.
ACCEPTED MANUSCRIPT The CS-DCA nanoparticles were prepared according to the dialysis methods described previously by Chae et al [37]. Briefly, after dissolution of CS-DCA (20mg) in 50mL DMSO/water/acetic acid co-solvent (7/3/0.3, by volume, ca 4 mg/mL), the solution was dialyzed against physiological saline for 48h using a dialysis membrane (molecular cut off 3500). After
PT
dialysis, the resulting nanoparticle solutions were sonicated for 2 min (using probe-type solicitor
RI
at 50 W) and were filtered through a 0.45m pore sized syringe filter to remove large aggregates.
SC
Finally, the CS-DCA nanoparticles were obtained by lyophilization. 2.3. Preparations of the CS-DCA/HA/TPP nanoparticles
nanoparticles were prepared using the ionotropic gelation
NU
CS-DCA/HA/TPP
MA
technique[38]. CS-DCA, tripolyphosphate (TPP) and HA were separately dissolved in ultrapure water at 1 mg/ml, 0.5 mg/ml and 2 mg/ml, respectively. Nanoparticles were formed
D
instantaneously upon the drop-wise addition of TPP/HA solution into CS-DCA solution under
PT E
magnetic stirring for 10 min to allow the complete formation of the system. 2.4. Characterization of CS-DCA Conjugates
CE
Degree of substitution (DS) of DCA group of CS-DCA was determined by elemental
AC
analysis using an EA 2400 II elemental analyzer (PE, USA). Since the amount of N was fixed during the reaction, DS of DCA group was calculated by comparing the C and N molar ratio obtained from the element analysis [39]. The equation for DS calculations as follows:
DS
1400 C / N 7526 288
The chemical structure of CS-DCA conjugates was analyzed using a Fourier transform infrared (FT-IR) spectrophotometer (Bruker, Germany) and nuclear magnetic resonance hydrogen spectrum (1H NMR) (Bruker AVANCE 300) with CD3COOD/D2O.
ACCEPTED MANUSCRIPT X-ray diffraction (XRD) diagrams were performed with Bruker D8 FOCUS (Bruker, Germany) powder diffraction meter with Cu Ka radiation in the range of 5-40o (2θ) at 40 kV and 30 mA. 2.5. Characterization of the CS-DCA/HA/TPP nanoparticles The turbidity of solution was monitored as a function of CS-DCA/HA (1mg/mL) mass ratio
PT
at a fixed wavelength of 420 nm with UV-2550 UV-Visible spectrophotometer (Shimadzu,
RI
Japan).
SC
The particle sizes and size distribution of CS-DCA and CS-DCA/HA/TPP nanoparticles in aqueous environment at 1mg/mL were investigated following filtration after ultrasonic dispersion
NU
for 10 minutes by dynamic light scattering (DLS) measurement. The DLS measurements were
MA
carried out using a Zeta-sizer Nano ZS90 (Malvern Instruments, U.K.). The morphology of the nanoparticles was studied by Tecnai 10 TEM (Philips, Netherlands) at 100 kV. Carbon-coated 200
D
mesh copper grids were immersed in CS-DCA solution and dried in air. The stability of the
PT E
CS-DCA/HA/TPP nanoparticles was investigated in PBS (pH 7.4) by DLS at room temperature at the determined time interval. We also studied the serum stability of the complex at 37oC. Briefly，
CE
CS-DCA/HA/TPP complexes (1 mg/mL) was incubated with PBS supplemented with 10% FBS at
AC
37 ºC. At designated time intervals, the size of complexes was monitored by using a Zeta-Sizer Nano ZS.
2.6. Doxorubicin Loading and in vitro Release Behavior CS-DCA solution prior to dropwise into HA/TPP solution under magnetic stirring for 10 min to allow the complete formation of the system. Then DOX was incubated in CS-DCA/HA solution under mild agitation at room temperature for 12h. The nanoparticles were isolated by 3K30 ultracentrifugation at 30000×g at 4 °C for 30 min. Then the supernate was placed into a
ACCEPTED MANUSCRIPT dialysis bag and dialyzed against distilled water for 48 h. The water was refreshed at time intervals of 6 h. After 48 h, the aqueous solution in the dialysis bag was lyophilized to obtain the DOX-loaded particles. The encapsulation efficiency (EE) and loading efficiency (LE) of the DOX-loaded nanoparticles were determined by measuring the amount of free DOX in the clear
PT
supernatant after centrifugation as described above. The concentration of DOX was determined
RI
by using the UV-vis spectrophotometer at 481 nm. Furthermore, both LE and EE were calculated
SC
based on the standard curve obtained from DOX.
The EE and LE of DOX in the nanoparticles were calculated with the following equation:
total amount of DOX amount of free DOX in supernatant 100 total amount of DOX total amount of DOX amount of unbound DOX LE (%) 100 nanoparticles weight
MA
NU
EE (%)
The release kinetics of DOX from DOX-loaded particles was conducted through dialysis
D
method using PBS (PH 7.4) containing 0.5% (w/v) Tween 80 as release medium. Briefly, the
PT E
isolated DOX -loaded 0.1 mMol PCNs incubated in 5 mL PBS was placed into a dialysis bag (MWCO 1 kDa, Spectrum Laboratories), and incubated in a tank containing 100 mL of release
CE
medium under gentle shaking at 37 C. At scheduled time points (0, 12, 24, 36, 48, 60, 72, 84,
AC
96h), aliquots of 2mL were taken out of each tube with replenishing of the same volume fresh medium. The suspension was collected and the DOX concentrations were determined by calculation from the standard curve obtained from DOX in PBS. 2.7. Cytotoxicity Assay of CS-DCA/HA/DOX Nanoparticles The cytotoxicity of the CS-DCA/HA and CS-DCA/HA/DOX was assessed using the methyl thiazolyl tetrazolium (MTT) assay. The HeLa cells used for these studies were obtained from Medical College of Jinan University (Guangzhou, China). Both DOX·HCl and DOX-loaded
ACCEPTED MANUSCRIPT nanoparticles were dispersed in PBS (pH 7.4) and then serially diluted to get different drug concentration. Hela cells were seeded in 96-well plate at a density of 1×104cells/well and cultured in 200 μL of DMEM 1640 containing 10% FBS in a humidified 37 °C environment with 5% CO2 overnight. Then, the culture medium was replaced by mixture of 180µL fresh culture
PT
medium and 20 μL DOX·HCl or DOX-loaded nanoparticles with different concentration.
RI
Different concentrations (0-1000 μg/mL) of CS-DCA/HA dissolved in culture medium without
SC
DOX were also added to test the cytotoxicity. The plates were then returned to the incubator and maintained in 5% CO2 at 37 °C for 48 h. Fresh culture medium (180 μL) and 20 μL aliquots of
NU
MTT solutions (5 mg/mL) were used to replace the mixture in each well after 48 h. After being
MA
incubated for 4 h, the culture medium was removed and 200 μL of DMSO was then added to each well to dissolve the internalized purple formazan crystals. Plates were vigorously shaken
D
before measuring the relative color intensity using a microplate reader. A test wavelength of 550
PT E
nm and a reference wavelength of 630 nm were used. All experiments were performed in triplicate.
CE
2.8. Histopathology studies
AC
Six weeks old BALB/c female nude mice (16-20 g) were obtained from the Jinan University Center for Animal Experiment. This experimental design has been approved by the Institutional Administration Panel for Laboratory Animal Care. All the animals were housed and fed in the Experimental Animal Center of Jinan University and were specific pathogen free. The rats were exposed to the drug formulations by intravenous injection on the first day. For the CS-DCA/HA/DOX and DOX groups, rats were injected with an equivalent dose of 1 mg DOX/kg; for the CS-DCA/HA group, rats were injected with a dose of vehicle equivalent to the
ACCEPTED MANUSCRIPT CS-DCA/HA/DOX groups; and for the NS group, rats were injected with normal saline with the same volume as used in the CS-DCA/HA/DOX group. For the histological assay, at 2 days after the first intravenous injection, rats were sacrificed, and the heart, liver, spleen, lung, and kidney were gathered. The tissues were fixed in 4% paraformaldehyde in PBS and embedded in paraffin
PT
wax. After routine processing, paraffin wax was cut into 5-mm-thick sections. Finally, the fixed
RI
sections were stained with hematoxylin and eosin (HE), and observed under a light microscope
SC
(Axio Scope A1 FL; Carl Zeiss, Wetzlar, Germany). 2.9. Statistical analysis
NU
All quantitative data are expressed as mean standard deviation (SD) unless otherwise noted.
MA
Statistical significance was tested using an unpaired, two-tailed Student's t-test. A value of P < 0.05 was considered statistically significant.
D
3. Results and discussion
PT E
3.1. Synthesis and Characterization of CS-DCA Conjugates Hydrophobic DCA with a rigid structure was selected to graft onto CS. The fatty acid
CE
molecule of DCA was covalently grafted onto the main backbone of CS to obtain CS-DCA
AC
conjugated by EDC and NHS. The molar ratio of EDC·HCl and DCA is 4:1. The molar ratio of DCA and CS is adjusted at 0.1:1, 0.2:1, 0.3:1 and the corresponding product is defined as CS-DCA10, CS-DCA20, CS-DCA30, freeze dried and stored. As shown in Table 1, the DS of DCA was calculated with elemental analysis results based on the changes of C/N between CS and CS-DCA and was tuned by adjusting the feed ratio of reagents. With the changing ratio of DCA to aminoglucose units from 10% to 30%, the DS of DCA was changed from 3.2 to 7.8. Compared with the FTIR spectrum of CS (Fig. 1a), the peaks of CS-DCA10 at 1652 cm-1 (amide I band) and
ACCEPTED MANUSCRIPT 1560 cm-1 (amide II band) were obviously intensified, meanwhile the disappearance of the 1600 cm-1 (N-H bending vibration) indicated that hydrogen of the primary amino group was mainly substituted by DCA molecules. It also showed that with the increasing of feed ratio of DCA to CS (Fig. 1a from 10% to 30%), the amide I peak at 1652 cm-1 became sharper, which indirectly
PT
indicated the increase of DS of DCA. Also, the presence of DCA in CS was evaluated by the
RI
characteristic peaks of DCA appearing at 0.6-2.5 ppm in the 1H NMR spectra (Fig. 1b) [40].
SC
Based on the comparison of 1H NMR spectra (Fig. 1b) of CS with CS-DCA, it can be concluded that the small hydrophobic molecule DCA was successfully grafted onto CS due to the appearance
NU
of new peaks from 0.7 to 1.9 ppm. For example, the peak at 0.7 ppm was affiliated to 21-CH3 in
MA
DCA moiety. The X-ray diffraction pattern was conducted for CS, differenced feed ratio of DCA to CS to further evaluate their crystallization behaviors. As shown in Fig. 1c, exhibits a broad peak
D
at 23° due to semi-crystalline nature of CS as reported elsewhere [41]. XRD. In contrast,
PT E
difference feed ratio of DCA to CS still exhibited similar peaks as the CS/DCA, but peaks became weaker. These results are consistent with the FTIR data and 1H NMR date.
CE
3.2. Formation and Characterization of CS-DCA/HA/TPP nanoparticles
AC
The route of self-assembly PCNs was illustrated in Scheme 1. CS was positively charged in acidic solution, while TPP and HA were negatively charged in water solution. Thus, nanoparticles were formed by ionic interaction between positively charged amino groups of CS and negatively charged counterions of TPP and HA. Turbidity measurement is a simple and direct indicator for polyelectrolyte complex formation, accompanied by drastic changes of the system turbidity of the solution [42, 43]. Fig. 2a demonstrated the curve of turbidimetric titration with HA and CS-DCA solutions. Initially, the CS-DCA solution was completely transparent.
ACCEPTED MANUSCRIPT With the increase of the quantity of CS-DCA/HA, the turbidity of the solution gradually ascents, because the complex of CS/HA and CS-DCA/HA are formed by the electrostatic interactions and the CS/HA and CS-DCA/HA with higher HA grafting rate are more difficult to be dissolved in water of PCNs. In the beginning of this process, coacervation layer was formed at the surface of
PT
the CS. Then –P3O105- could penetrate the coacervation layer and reach the core and crosslinked
RI
with it. However, because HA has large molecular size and cannot penetrate the coacervation
SC
layer, it only reacted with –NH3+ at the surface. With the increase of HA and TPP, the CS-DCA underwent a high positive-to-low positive transformation of their zeta potential. The influence of
NU
DS of DCA on the turbidity of CS-DCA was shown in Fig. 2a. The turbidity of CS-DCA
MA
decreased with the increasing DS of a hydrophobic molecule. It could be explained that the higher DS, the higher hydrophobicity, which gave rise to the stronger self-aggregation ability.
D
Thus, the sizes and size distribution of self-aggregated CS-DCA/HA/TPP nanoparticles in PBS
PT E
buffer (pH 7.4) were determined using DLS and were illustrated in Fig. 2b and Table 2. The sizes of the CS-DCA/HA nanoparticles ranged from 160-570 nm, while the diameters of the
CE
HA/CS nanoparticles were 180-680 nm. With the increase of HA, the zeta potentials and the
AC
sizes of the nanoparticles decreased. The sizes of CS-DCA nanoparticles increased from 204 to 240 nm with the decrease of DS of DCA. The zeta potentials of CS-DCA/HA/TPP nanoparticles was further examined (Table 2). The morphology of CS-DCA nanoparticles characterized by TEM is shown in Fig. 3. It indicated that the nano-aggregates were spherical nanoparticles and the CS-DCA30 nanoparticles had the smallest sizes. Furthermore, the sizes of the CS-DCA/HA/TPP kept constant when stored at room temperature for almost 1 month, suggesting the stability of the nanoparticles (Fig. 4A).
ACCEPTED MANUSCRIPT To further confirm the stability of CS-DCA/HA/TPP during circulation, we have introduced experiments of serum stability, the relevant data were shown in Fig. 4B. The particle sizes of (a)
CS/HA, (b) CS-DCA10/HA, (c) CS-DCA20/HA and (d) CS-DCA30/HA nanoparticles maintained the initial size over the experimental time in presence of 10% fetal bovine serum
PT
(FBS), this result indicated that CS-DCA/HA/TPP exhibited excellent stability in FBS at 37 oC.
RI
3.3. Drug Loading and in vitro Release Behavior
DOX, one of the most potent anticancer drugs, was chosen as the drug model. A delivery
SC
system based on CS-DCA/HA nanoparticles was developed for enhancing the efficacy of cancer chemotherapy of DOX. The results as Table 3 shown DOX could be easily loaded into
NU
CS-DCA/HA/TPP nanoparticles, and the drug loading level increased from 6.1% to 7.3% with the increasing DS of DCA while encapsulation efficiency was up to 60%, which was relatively
MA
much higher than traditional drug carriers, such as some micelles [44-46]. The drug loading and encapsulation efficiency for HA-TPP/CS nanoparticles of DOX were 3.7% and 20.75%
D
respectively, which were significantly smaller than that of the CS-DCA/HA/TPP nanoparticles.
PT E
Furthermore, the sizes of DOX loaded nanoparticles increased about 70 nm while PDI remained low. The hydrophobic group of DCA formed a hydrophobic micro-area, on the outside of which the hydrophilic skeleton of CS coiled to form a hydrophilic shell. Thus, the surface energy of the
CE
aqueous solution was decreased to the lowest. The intermolecular and intramolecular hydrogen bonding of the CS hydrophilic skeleton promoted the CS-DCA to form nanoparticles. The unique
AC
super molecular structure is well suited for capture of hydrophobic drugs. In vitro release evaluation results were shown in Fig.5. DOX was released slowly from CS-DCA/HA/TPP/DOX nanoparticles without burst release phenomenon in the first 12 h. Meanwhile, it had to be mentioned that the higher DS of DCA, the higher release rate of drugs due to the CS-DCA is one of the key factors of the DOX load. The accumulative amount of DOX released from CS-DCA10/HA/TPP, CS-DCA20/HA/TPP, and CS-DCA30/HA/TPP nanoparticles was 28.5, 33.9, and 39.8%, respectively, within 48 h and the HA/CS also have the slower release
ACCEPTED MANUSCRIPT rate due to CS main chain contains hydrophobic micro area, small molecules can by hydrophobic effect load hydrophobic drug doxorubicin. The slow release rate of DOX from CS-DCA nanoparticles may be ascribed to the strong interaction between hydrophobic DCA and DOX. 3.4 Cytotoxicity Assay of CS-DCA/HA/DOX Nanoparticles
PT
In order to confirm whether the ability to deliver DOX by CS-DCA/HA/TPP nanoparticles
RI
could really work, the toxicity of CS-DCA/HA/TPP nanoparticles to HeLa cells was carried out.
SC
We got the toxicity results by MTT assay. The cytotoxicity of DOX loaded CS-DCA/HA/TPP nanoparticles was compared with that of free DOX dissolved in PBS. The cytotoxicity of blank
NU
CS-DCA/HA/TPP nanoparticles was also investigated to ensure that the cytotoxicity was caused
MA
by DOX alone. As shown in Fig.6a, the cytotoxicity results of blank CS-DCA/HA/TPP nanoparticles, when the concentrations below 1000 µg/mL CS-DCA/HA/TPP will not cause
D
significant cytotoxicity, all of the Cell activity in more than 80% [37]. Based on this result, the
PT E
detection concentration of CS-DC/HA/DOX were lower than 1000 µg/mL, excluding the factors of cytotoxicity caused by carrier. It was found from Fig. 6b that CS-DCA/HA/DOX can cause
CE
obvious cytotoxicity. When DOX was loaded in CS-DCA/HA/TPP nanoparticles, the cell viability
AC
decreased significantly from almost 100% to less than 10% as the DOX concentration increased from 1 to 50 μg/mL, while the cell viability decreased to about 10% when free DOX with the same concentration was used in cells for 48 h. The possible reason is that DOX loaded CS-DCA/HA/TPP nanoparticles with positive charge facilitate the entrance into the tumor cells and then into the nucleus to take effect. CS-DCA/HA/TPP/DOX nanoparticles with higher DS of DCA showed higher antitumor efficiency than that with lower DS of DCA, which may be also the results that drug release speed increased with the increase of DS of DCA and that DOX-loaded
ACCEPTED MANUSCRIPT CS-DCA/HA/TPP nanoparticles with higher DS of DCA have smaller sizes which were favorable for the entrance into cells. In summary, the drug loading and releasing ability of CS-DC/HA had obvious effect in vitro. 3.5. Histological analysis of organs
PT
Through H&E staining analysis of rat were performed. The images of the heart, liver, spleen,
RI
lung and kidney were obtained using light microscopy (Fig. 7). In the treatment group of CS,
SC
CS-DCA, and CS-DCA/HA/DOX, no visible morphological changes were observed in corresponding organs compared with the DOX control group [47]. However, the tubular dailation
NU
with flattening of the epithelium cells were observed in the kidneys under the free drug treatment
effects on important organ cytotoxicity.
D
4. Conclusion
MA
of animals. In conclusion, these results suggest that PCNs enhance drug delivery and reduce the
PT E
In this study, novel PCNs based on hydrophobically modified CS and hyaluronic acid were produced. The PCNs in this work are prominent in stability, which keep stable up to 1week in PBS
CE
(pH 7.4) with no obvious sizes change and capable of associating DOX, with EE up to 56% and
AC
keep a sustained release manner for design time (96h) without burst release at 37 oC. In addition, this type of PCNs doesn’t show cytotoxicity even at high concentrations. From the study above it may be concluded that CS-DCA/HA/TPP/DOX nanoparticles exhibit remarkable biocompatibility and have the potential to be used in biomedical applications and treatment of tumor. Acknowledgements This study was supported financially by the Natural Science Foundation of China (51303064), the Science and Technology Program of Guangzhou (201508020115, 201601010270,
ACCEPTED MANUSCRIPT 2017010160489, 201704030083), the Pearl River S&T Nova Program of Guangzhou
(201710010155, 201806010072), the Science and Technology Project of Guangdong province
AC
CE
PT E
D
MA
NU
SC
RI
PT
(2015A010101313, 2017A050506011, 2017B090911012).
ACCEPTED MANUSCRIPT
References [1] S. Xu, R. Zhang, W. Zhao, Y. Zhu, W. Wei, X. Liu, J. Luo, Self-assembled polymeric nanoparticles film stabilizing gold nanoparticles as a versatile platform for ultrasensitive detection of carcino-embryonic antigen, Biosensors & bioelectronics 92 (2016) 570-6.
PT
[2] C.L. Oliveira, F. Veiga, C. Varela, F. Roleira, E. Tavares, I. Silveira, A.J. Ribeiro, Characterization
RI
of polymeric nanoparticles for intravenous delivery: Focus on stability, Colloids and surfaces. B,
SC
Biointerfaces 150 (2016) 126-133.
[3] M. Prabaharan, J.F. Mano, Chitosan-based particles as controlled drug delivery systems, Drug
NU
delivery 12(1) (2005) 41-57.
MA
[4] M. Lee, A.C. Lo, P.T. Cheung, D. Wong, B.P. Chan, Drug carrier systems based on collagen-alginate composite structures for improving the performance of GDNF-secreting HEK293
D
cells, Biomaterials 30(6) (2009) 1214-21.
PT E
[5] P. Zhang, J.H. Yang, W.C. Li, W. Wang, C.J. Liu, M. Griffith, W.G. Liu, Cationic polymer brush grafted-nanodiamond via atom transfer radical polymerization for enhanced gene delivery and
CE
bioimaging, J Mater Chem 21(21) (2011) 7755-64.
AC
[6] Y. Luo, Q. Wang, Recent development of chitosan-based polyelectrolyte complexes with natural polysaccharides for drug delivery, International journal of biological macromolecules 64 (2014) 353-67.
[7] S.P. Thakker, A.P. Rokhade, S.S. Abbigerimath, S.R. Iliger, V.H. Kulkarni, U.A. More, T.M. Aminabhavi, Inter-polymer complex microspheres of chitosan and cellulose acetate phthalate for oral delivery of 5-fluorouracil, Polym Bull 71(8) (2014) 2113-31. [8] J. Venkatesan, S. Anil, S.K. Kim, M.S. Shim, Chitosan as a vehicle for growth factor delivery:
ACCEPTED MANUSCRIPT
Various preparations and their applications in bone tissue regeneration, International journal of biological macromolecules 104 (2017) 1383-97. [9] D.P. Biswas, P.A. Tran, C. Tallon, A.J. O'Connor, Combining mechanical foaming and thermally induced phase separation to generate chitosan scaffolds for soft tissue engineering, Journal of
PT
biomaterials science. Polymer edition 28(2) (2017) 207-226.
RI
[10] M. Rodriguez-Vazquez, B. Vega-Ruiz, R. Ramos-Zuniga, D.A. Saldana-Koppel, L.F.
SC
Quinones-Olvera, Chitosan and Its Potential Use as a Scaffold for Tissue Engineering in Regenerative Medicine, Biomed Res Int (2015) Article ID 821279, 15 pages.
NU
[11] Y. Li, J. Yang, B. Xu, F. Gao, W. Wang, W. Liu, Enhanced Therapeutic siRNA to Tumor Cells by
MA
a pH-Sensitive Agmatine-Chitosan Bioconjugate, ACS applied materials & interfaces 7(15) (2015) 8114-24.
D
[12] L.H. Peng, J. Niu, C.Z. Zhang, W. Yu, J.H. Wu, Y.H. Shan, X.R. Wang, Y.Q. Shen, Z.W. Mao,
PT E
W.Q. Liang, J.Q. Gao, TAT conjugated cationic noble metal nanoparticles for gene delivery to epidermal stem cells, Biomaterials 35(21) (2014) 5605-18.
CE
[13] W. Xiong, L. Li, Y. Wang, Y. Yu, S. Wang, Y. Gao, Y. Liang, G. Zhang, W. Pan, X. Yang, Design
AC
and evaluation of a novel potential carrier for a hydrophilic antitumor drug: Auricularia auricular polysaccharide-chitosan nanoparticles as a delivery system for doxorubicin hydrochloride, International journal of pharmaceutics 511(1) (2016) 267-75. [14] D. Nayak, A.P. Minz, S. Ashe, P.R. Rauta, M. Kumari, P. Chopra, B. Nayak, Synergistic combination of antioxidants, silver nanoparticles and chitosan in a nanoparticle based formulation: Characterization and cytotoxic effect on MCF-7 breast cancer cell lines, Journal of colloid and interface science 470 (2016) 142-52.
ACCEPTED MANUSCRIPT
[15] M. Vincekovic, M. Curlin, D. Jurasin, Impact of cationic surfactant on the self-assembly of sodium caseinate, Journal of agricultural and food chemistry 62(34) (2014) 8543-54. [16] S. Wang, R.G. Larson, Coarse-grained molecular dynamics simulation of self-assembly and surface adsorption of ionic surfactants using an implicit water model, Langmuir : the ACS journal of
PT
surfaces and colloids 31(4) (2015) 1262-71.
RI
[17] B. Xu, W. Yao, Y. Li, S. Zhang, X. Huang, Perfluorocyclobutyl Aryl Ether-Based ABC
SC
Amphiphilic Triblock Copolymer, Scientific reports 6 (2016) 39504.
[18] D. Niu, Y. Li, J. Shi, Silica/organosilica cross-linked block copolymer micelles: a versatile
NU
theranostic platform, Chemical Society reviews 46(3) (2017) 569-585.
Aminabhavi,
Oral
insulin
MA
[19] K. Chaturvedi, K. Ganguly, A.R. Kulkarni, W.E. Rudzinski, L. Krauss, M.N. Nadagouda, T.M. delivery
using
deoxycholic
acid
conjugated
PEGylated
D
polyhydroxybutyrate co-polymeric nanoparticles, Nanomedicine (Lond) 10(10) (2015) 1569-83.
PT E
[20] Y.I. Jeong, K.D. Chung, D.H. Kim, Y.H. Kim, Y.S. Lee, K.C. Choi, All-trans retinoic acid-incorporated nanoparticles of deoxycholic acid-conjugated dextran for treatment of CT26
CE
colorectal carcinoma cells, International journal of nanomedicine 8 (2013) 485-93.
AC
[21] J.Y. Lee, C. Crake, B. Teo, D. Carugo, M. de Saint Victor, A. Seth, E. Stride, Ultrasound-Enhanced siRNA Delivery Using Magnetic Nanoparticle-Loaded Chitosan-Deoxycholic Acid Nanodroplets, Advanced healthcare materials 6(8) (2017) 1601246. [22] A. Almalik, S. Karimi, S. Ouasti, R. Donno, C. Wandrey, P.J. Day, N. Tirelli, Hyaluronic acid (HA) presentation as a tool to modulate and control the receptor-mediated uptake of HA-coated nanoparticles, Biomaterials 34(21) (2013) 5369-80. [23] E. Auzenne, S.C. Ghosh, M. Khodadadian, B. Rivera, D. Farquhar, R.E. Price, M. Ravoori, V.
ACCEPTED MANUSCRIPT
Kundra, R.S. Freedman, J. Klostergaard, Hyaluronic acid-paclitaxel: antitumor efficacy against CD44(+) human ovarian carcinoma xenografts, Neoplasia (New York, N.Y 9(6) (2007) 479-86. [24] K.Y. Choi, H. Chung, K.H. Min, H.Y. Yoon, K. Kim, J.H. Park, I.C. Kwon, S.Y. Jeong, Self-assembled hyaluronic acid nanoparticles for active tumor targeting, Biomaterials 31(1) (2010)
PT
106-14.
RI
[25] K.Y. Choi, K.H. Min, H.Y. Yoon, K. Kim, J.H. Park, I.C. Kwon, K. Choi, S.Y. Jeong, PEGylation
SC
of hyaluronic acid nanoparticles improves tumor targetability in vivo, Biomaterials 32(7) (2011) 1880-9.
NU
[26] G. Pitarresi, F.S. Palumbo, A. Albanese, C. Fiorica, P. Picone, G. Giammona, Self-assembled
MA
amphiphilic hyaluronic acid graft copolymers for targeted release of antitumoral drug, Journal of drug targeting 18(4) (2010) 264-76.
D
[27] F.S. Palumbo, R. Puleio, C. Fiorica, G. Pitarresi, G.R. Loria, G. Cassata, G. Giammona, Matrices
PT E
of a hydrophobically functionalized hyaluronic acid derivative for the locoregional tumour treatment, Acta biomaterialia 25 (2015) 205-15.
CE
[28] G. Tripodo, A. Trapani, M.L. Torre, G. Giammona, G. Trapani, D. Mandracchia, Hyaluronic acid
AC
and its derivatives in drug delivery and imaging: Recent advances and challenges, European journal of pharmaceutics and biopharmaceutics : official journal of Arbeitsgemeinschaft fur Pharmazeutische Verfahrenstechnik e.V 97(Pt B) (2015) 400-16. [29] S. Ganesh, A.K. Iyer, D.V. Morrissey, M.M. Amiji, Hyaluronic acid based self-assembling nanosystems for CD44 target mediated siRNA delivery to solid tumors, Biomaterials 34(13) (2013) 3489-502. [30] A. Sharma, R.A. Gadkari, S.V. Ramakanth, K. Padmanabhan, D.S. Madhumathi, L. Devi, L.
ACCEPTED MANUSCRIPT
Appaji, J.C. Aster, A. Rangarajan, R.R. Dighe, A novel Monoclonal Antibody against Notch1 Targets Leukemia-associated Mutant Notch1 and Depletes Therapy Resistant Cancer Stem Cells in Solid Tumors, Scientific reports 5 (2015) 11012. [31] C.E. Galer, D. Sano, S.C. Ghosh, J.H. Hah, E. Auzenne, A.N. Hamir, J.N. Myers, J. Klostergaard,
PT
Hyaluronic acid-paclitaxel conjugate inhibits growth of human squamous cell carcinomas of the head
RI
and neck via a hyaluronic acid-mediated mechanism, Oral oncology 47(11) (2011) 1039-47.
SC
[32] H. Lee, K. Lee, T.G. Park, Hyaluronic acid-paclitaxel conjugate micelles: synthesis, characterization, and antitumor activity, Bioconjugate chemistry 19(6) (2008) 1319-25.
NU
[33] H.J. Cho, H.Y. Yoon, H. Koo, S.H. Ko, J.S. Shim, J.H. Lee, K. Kim, I.C. Kwon, D.D. Kim,
MA
Self-assembled nanoparticles based on hyaluronic acid-ceramide (HA-CE) and Pluronic(R) for tumor-targeted delivery of docetaxel, Biomaterials 32(29) (2011) 7181-90.
D
[34] S. Siegman, N.F. Truong, T. Segura, Encapsulation of PEGylated low-molecular-weight PEI
PT E
polyplexes in hyaluronic acid hydrogels reduces aggregation, Acta biomaterialia 28 (2015) 45-54. [35] R. Ran, Y. Liu, H. Gao, Q. Kuang, Q. Zhang, J. Tang, K. Huang, X. Chen, Z. Zhang, Q. He,
CE
Enhanced gene delivery efficiency of cationic liposomes coated with PEGylated hyaluronic acid for
AC
anti P-glycoprotein siRNA: a potential candidate for overcoming multi-drug resistance, International journal of pharmaceutics 477(1-2) (2014) 590-600. [36] K.Y. Lee, I.C. Kwon, Y.H. Kim, W.H. Jo, S.Y. Jeong, Preparation of chitosan self-aggregates as a gene delivery system, J Control Release 51(2-3) (1998) 213-20. [37] S.Y. Chae, S. Son, M. Lee, M.K. Jang, J.W. Nah, Deoxycholic acid-conjugated chitosan oligosaccharide nanoparticles for efficient gene carrier, Journal of controlled release : official journal of the Controlled Release Society 109(1-3) (2005) 330-44.
ACCEPTED MANUSCRIPT
[38] M. de la Fuente, B. Seijo, M.J. Alonso, Bioadhesive hyaluronan-chitosan nanoparticles can transport genes across the ocular mucosa and transfect ocular tissue, Gene therapy 15(9) (2008) 668-76. [39] A. Miwa, A. Ishibe, M. Nakano, T. Yamahira, S. Itai, S. Jinno, H. Kawahara, Development of
PT
novel chitosan derivatives as micellar carriers of taxol, Pharmaceutical research 15(12) (1998)
RI
1844-50.
SC
[40] K. Kim, S. Kwon, J.H. Park, H. Chung, S.Y. Jeong, I.C. Kwon, I.S. Kim, Physicochemical characterizations of self-assembled nanoparticles of glycol chitosan-deoxycholic acid conjugates,
NU
Biomacromolecules 6(2) (2005) 1154-8.
MA
[41] S. Hajji, I. Younes, O. Ghorbel-Bellaaj, R. Hajji, M. Rinaudo, M. Nasri, K. Jellouli, Structural differences between chitin and chitosan extracted from three different marine sources, International
D
journal of biological macromolecules 65 (2014) 298-306.
based
on
methoxy
PT E
[42] K. Luo, J. Yin, Z. Song, L. Cui, B. Cao, X. Chen, Biodegradable interpolyelectrolyte complexes poly(ethylene
glycol)-b-poly(alpha,L-glutamic
acid)
and
chitosan,
CE
Biomacromolecules 9(10) (2008) 2653-61.
AC
[43] A. Drogoz, L. David, C. Rochas, A. Domard, T. Delair, Polyelectrolyte complexes from polysaccharides: formation and stoichiometry monitoring, Langmuir 23(22) (2007) 10950-8. [44] X. Zeng, X. Zhou, M. Li, C. Wang, J. Xu, D. Ma, W. Xue, Redox poly(ethylene glycol)-b-poly(L-lactide) micelles containing diselenide bonds for effective drug delivery, Journal of materials science. Materials in medicine 26(9) (2015) 234. [45] W. Chen, P. Zhong, F. Meng, R. Cheng, C. Deng, J. Feijen, Z. Zhong, Redox and pH-responsive degradable micelles for dually activated intracellular anticancer drug release, Journal of controlled
ACCEPTED MANUSCRIPT
release : official journal of the Controlled Release Society 169(3) (2013) 171-9. [46] L.Y. Qiu, Y.H. Bae, Self-assembled polyethylenimine-graft-poly(epsilon-caprolactone) micelles as potential dual carriers of genes and anticancer drugs, Biomaterials 28(28) (2007) 4132-42. [47] Y.F. Mo, H.W. Wang, J.H. Liu, Y. Lan, R. Guo, Y. Zhang, W. Xue, Y.M. Zhang, Controlled
PT
release and targeted delivery to cancer cells of doxorubicin from polysaccharide-functionalised
AC
CE
PT E
D
MA
NU
SC
RI
single-walled carbon nanotubes, J Mater Chem B 3(9) (2015) 1846-1855.
ACCEPTED MANUSCRIPT
Figure Captions Fig.1. FT-IR spectra of (a) CS and CS-DCA derivatives; 1H-NMR spectra of (b) CS and CS-DCA derivatives; XRD diffraction patterns of (c) CS and CS-DCA derivatives. Fig.2. (a) Turbidimetric titration curves for HA and CS-DCA derivatives solutions as a function
PT
of CS-DCA/HA mass ratio; (b) Size of HA and CS-DCA derivatives solutions as a function of
RI
CS-DCA/HA mass ratio.
SC
Fig.3. TEM images of (a) CS/HA, (b) CS-DCA10/HA, (c) CS-DCA20/HA and (d) CS-DCA30/HA nanoparticles.
NU
Fig.4. Size of (a) CS/HA, (b) CS-DCA10/HA, (c) CS-DCA20/HA and (d) CS-DCA30/HA
MA
nanoparticles in PBS at (A) room temperature and (B) containing10% fetal bovine serum (FBS) at 37 oC as a function of time.
D
Fig.5. In vitro release profile of DOX from nanoparticles incubated in 0.02 M PBS (pH 7.4) at 37 o
PT E
C: (a) CS/HA, (b) CS-DCA10/HA, (c) CS-DCA20/HA and (d) CS-DCA30/HA.
Fig. 6. (a) Effects of CS-DCA derivatives on the Hela cell viability at different concentration
CE
after incubation for 48h; (b) Viability of Hela cells difference nanoparticles for 48 h with
AC
different concentration. The results represent the means SD (n 6). Fig.7. Morphological change of SD rat heart, liver, spleen, lung, and kidney, exposed to (a) CS, (b) CS-DCA, (c) CS-DCA/HA/DOX, and (d) DOX. The bar corresponds to 50 mm.
ACCEPTED MANUSCRIPT Data images-Tables Table 1. Degree of substitution of deoxycholic acid. CS-DCA10
CS-DCA20
CS-DCA30
sugar unit/DA
100/0
100/10
100/20
100/30
C%
44.03
41.41
38.63
42.81
H%
6.05
11.04
8.05
11.00
N%
8.25
6.83
5.76
6.14
C/N
5.33
6.06
DA DS%
0
3.18
RI
PT
CS
CE
PT E
D
MA
6.71
6.97
6.44
7.75
SC
NU
All values are averaged from three measurements.
AC
a
Samples
ACCEPTED MANUSCRIPT Table 2. Size, PDI and Zeta Potential of CS-DCA nanoparticles with a different DS of DCA. CS-DCA10
CS
Zeta Potential (mV)
1.0
0.6
0.8
0.6
0.8
0.6
384.6 ± 1.2 0.52 ± 0.01 33.5 ± 0.2
313.3 ± 0.4 0.5 ± 0.01 27.1 ± 0.5
179.7 ± 1.3 0.19 ± 0.01 24.9 ± 0.3
243.5 ± 0.3 0.18 ± 0.01 41.5 ± 0.6
160.5 ± 0.2 0.11 ± 0.01 26.7 ± 0.5
220.0 ± 0.3 0.15 ± 0.01 28.3 ± 0.7
205.6 ± 0.1 0.02 ± 0.01 16.9 ± 0.1
203.6 ± 1.3
CE
PT E
D
MA
NU
SC
PDI represent the polydispersity index.
AC
a
0.8
PT
PDI a
CS-DCA30
0.6
RI
HA/CS-DCA mass ratio Size (nm)
CS-DCA20
0.13 ± 0.01 21.7± 0.1
ACCEPTED MANUSCRIPT Table 3. Characterization of HA and CS derivatives. PDIa
Zeta potential (mV)
EE% b
LE% c
CS/HA/DOX
438.6 ± 1.7
0.53 ± 0.01
31.3 ± 0.2
20.75
3.7
CS-DCA10/HA/DOX
306.5 ± 0.2
0.31± 0.01
39.7 ± 0.6
38.18
6.1
CA-DCA20/HA/DOX
290.0 ± 0.8
0.25± 0.01
25.5 ± 0.7
47.46
6.7
CS-DCA30/HA/DOX
283.6 ± 1.2
0.23 ± 0.01
18.2 ± 0.1
56.62
7.3
PT
Size (nm)
PDI represent the polydispersity index.
PT E
D
MA
NU
SC
RI
mean encapsulation efficiency (EE) and loading efficiency (LE), respectively.
CE
b and c
AC
a
Samples
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7