Accepted Manuscript Title: Collagen/chitosan film containing biotinylated glycol chitosan nanoparticles for localized drug delivery Author: Ming-Mao Chen Yu-Qing Huang Huan Cao Yan Liu Hao Guo Lillian S. Chen Jian-Hua Wang Qi-Qing Zhang PII: DOI: Reference:
S0927-7765(15)00100-9 http://dx.doi.org/doi:10.1016/j.colsurfb.2015.02.024 COLSUB 6915
To appear in:
Colloids and Surfaces B: Biointerfaces
Received date: Revised date: Accepted date:
3-12-2014 30-1-2015 12-2-2015
Please cite this article as: M.-M. Chen, Y.-Q. Huang, H. Cao, Y. Liu, H. Guo, L.S. Chen, J.-H. Wang, Q.-Q. Zhang, Collagen/chitosan film containing biotinylated glycol chitosan nanoparticles for localized drug delivery, Colloids and Surfaces B: Biointerfaces (2015), http://dx.doi.org/10.1016/j.colsurfb.2015.02.024 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.
Collagen/chitosan film containing biotinylated glycol chitosan nanoparticles for localized drug delivery
Institute of Biomedical and Pharmaceutical Technology, Fuzhou University, Fuzhou
b
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350002, China
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a
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Jian-Hua Wang a, Qi-Qing Zhang a,c,*
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Ming-Mao Chen a, Yu-Qing Huang a, Huan Cao a, Yan Liu b, Hao Guo a, Lillian S. Chen a,
State Key Lab of Structural Chemistry, Fujian Institute of Research on the Structure of
c
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Matter, Chinese Academy of Sciences, Fuzhou 350002, China Key Laboratory of Biomedical Material of Tianjin, Institute of Biomedical Engineering,
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Chinese Academy of Medical Science & Peking Union Medical College, Tianjin 300192, China
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*Corresponding author at: Institute of Biomedical and Pharmaceutical Technology, Fuzhou University, No. 523 Gongye Road, Fuzhou 350002, China.
Tel.: +86 591 83725260; fax: +86 591 83725260. E-mail: [email protected].
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Abstract The objective of this study was to design a drug delivery system consisting of biotinylated cholesterol-modified glycol chitosan (Bio-CHGC) nanoparticles and fish
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collagen/chitosan (Col/Ch) film for localized chemotherapy. Bio-CHGC was synthesized,
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and then its self-assembled nanoparticles were prepared by probe sonication. Doxorubicin (DOX)-loaded Bio-CHGC (DBC) nanoparticles prepared by dialysis had spherical shape,
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and their sizes were in the range of 330-397 nm. Col/Ch/DBC nanoparticle films were
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fabricated by freeze-drying. SEM showed that the DBC nanoparticles were uniformly distributed into the films, and the films retained their structural integrity. A higher
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degradation and swelling rate of the drug films led to a higher diffusion rate of the nanoparticles from the films, resulting in an increase in the drug release from
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nanoparticles. The release of DOX from the films or Bio-CHGC nanoparticles was
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sensitive to the pH value of the release medium. In addition, the DOX release ratio of the
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drug films was lower than that of the nanoparticles alone, suggesting that the drug films had a double-sustained effect on the drug release. MTT assay implied that the DBC nanoparticle film showed a higher inhibitory ratio than the film containing nanoparticles without biotin, indicating that biotin moieties in the nanoparticles played an important role in exerting a cytotoxic effect. These data demonstrate that Col/Ch/DBC nanoparticle film has the potential to be used as a localized delivery system for hydrophobic antitumor drugs. Keywords: Collagen, Drug film, Silver carp skin, Glycol chitosan, Self-assembled nanoparticles 2
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1. Introduction Localized chemotherapy is generally used as an adjuvant to surgery to protect against tumor recurrence. Compared to systemic chemotherapy, localized delivery can supply
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therapeutic levels of drugs at a physical site for a prolonged period, and reduce systemic
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toxicities [1,2]. Implantable films based on the biocompatible and biodegradable polymers have been developed for localized delivery, such as poly(lactic acid) (PLA),
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poly(caprolactone) (PCL), poly(lactide-co-glycolide) (PLGA), collagen and chitosan [3-6].
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Collagen film is a promising delivery system for localized chemotherapy due to its biocompatibility, nontoxicity and biodegradability. However, collagen used in medical
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applications is commonly isolated from bovine and porcine skins and bones, which is associated with a high risk of bovine spongiform encephalopathy or religious issues [7,8].
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Recently, fresh water and marine fishes have been proposed as alternative sources for
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collagen. The investigation of fish collagen focuses on the extraction methods and the
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application in the food field [9-11]. Nevertheless, the application of fish collagen films in localized chemotherapy has seldom been studied so far. Collagen-based films incorporated with drug-loaded nanoparticles have triggered
considerable interest as a carrier for localized drug delivery, because they can sustain drug release, enhance targeted effect, reduce systemic toxicity and increase treatment efficiency. Recently, much attention has been paid to preparing self-assembled polymeric nanoparticles on the basis of natural polysaccharides, such as chitosan, pullulan and curdlan [12-15]. Glycol chitosan, a water-soluble chitosan derivative, is used as a novel drug carrier because of its solubility, biocompatibility, biodegradability and non-toxicity. 3
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Hydrophobically modified glycol chitosan (HGC) derivatives, such as glycol chitosan bearing 5-cholanic acid, deoxycholic acid and cholesterol, have been focusing on due to their amphiphilic nature [16-18]. These polymeric amphiphiles can form self-assembled
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nanoparticles in aqueous media, and the nanoparticles have shown the potential
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application as carriers for hydrophobic drugs and genes. Therefore, a combination of HGC nanoparticles and fish collagen films might be a novel drug delivery system for
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hydrophobic drugs.
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Previously, our group has isolated the collagen from silver carp skin, and prepared fish collagen/chitosan (Col/Ch) composite sponges [19,20]. The results showed that the
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presence of chitosan improved the biostability of collagen sponges, and the composite sponges exhibited noncytotoxicity, biocompatibility, and nonhemolysis. In this study, we
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developed a drug delivery system consisting of biotinylated cholesterol-modified glycol
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chitosan (Bio-CHGC) nanoparticles and fish Col/Ch film. Biotin was introduced into the
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conjugate to enhance tumor-targeted drug delivery. As shown in Fig.1, Bio-CHGC has an amphiphilic property and can form self-assembled nanoparticles by the hydrophobic interactions of cholesterol moieties. Doxorubicin (DOX) was physically loaded into Bio-CHGC nanoparticles, and then DOX-loaded nanoparticles were mixed with Col/Ch solution to prepare the drug films. The characteristics and drug release behavior of the drug films were evaluated. Moreover, the cytotoxicity of the drug films against human hepatoma (HepG2) cells was assayed to demonstrate the antitumor efficacy of this drug delivery system.
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2. Materials and methods 2.1 Materials Collagen was isolated from silver carp skin by the method previously reported [20].
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Cholesterol succinate (CHS) was synthesized following the procedure reported previously
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[21]. DOX hydrochloride (DOX·HCl), glycol chitosan (MW = 2.5 × 105 Da, degree of
deacetylation = 82%), N-hydroxyl succinimide (NHS) and collagenase were obtained
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from Sigma–Aldrich (St. Louis, MO). Chitosan (MW = 5×105 Da, degree of deacetylation
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= 85%) was purchased from Shanghai Qiangshun Chemical Reagent Co.,Ltd. (Shanghai, China). Glutaraldehyde (GA, 25% water solution) was supplied by Sinopharm Chemical
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Reagent Co., Ltd. (Shanghai, China). Dulbecco’s modified Eagle’s medium (DMEM) was purchased from Hyclone (Logan, UT). All other chemical reagents were of analytical
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grade and obtained from commercial sources.
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2.2 Synthesis of Bio-CHGC conjugate
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Cholesterol-modified glycol chitosan (CHGC) was synthesized according to the previously reported procedure [18,22]. CHS (72.8 mg) in 20 mL of 85% ethanol solution was activated by the addition of EDC and NHS for 10 h. Glycol chitosan (130 mg) was dissolved in 8 mL of distilled water, and then it was added to the above mixture. After 72 h of the reaction at 40℃, it was sequentially dialyzed (MWCO 8-14 kDa) against an excess amount of 80%, 50% ethanol solution and distilled water for 72 h. The dialyzed solution was lyophilized and the CHGC conjugate was obtained. CHGC (248.3 mg) and biotin (122.0 mg) were mixed with EDC and NHS in 40 mL of DMSO. After reacting for 24 h at room temperature, the solution was dialyzed against 5
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distilled water for 48 h. Then the solution was freeze-dried to obtain Bio-CHGC. The chemical structure of Bio-CHGC was characterized by Fourier transform infrared spectroscopy (FTIR, Nicolet AVATAR-360, USA) and 1H-NMR spectroscopy (AVANCE
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III 500, Switzerland) using D2O as the solvent. The degrees of substitution (DS) of the
Germany).
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2.3 Preparation of Bio-CHGC nanoparticles and drug loading
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cholesterol moiety and biotin group were determined by elemental analysis (Vario MICRO,
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Bio-CHGC nanoparticles were made by probe sonication [23]. Briefly, Bio-CHGC was dispersed in distilled water overnight, followed by sonication with a probe type
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sonifier (JY92-IIN, China) in an ice bath at 80 W for 2 min (pulse on 2 s and pulse off 2 s). The solution was filtered through a filter (0.8 μm) to remove dust and impurity.
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DOX-loaded Bio-CHGC (DBC) nanoparticles were prepared using a dialysis method
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[24,25]. DOX·HCl was dissolved in 2 mL of DMSO and trithylamine was added to
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remove hydrochloride. After stirring at room temperature for 12 h in the dark, the mixture was dropped into the above Bio-CHGC nanoparticle solution with ultrasonication at an output power of 80W for 2 min (pulse on 2.0 s and pulse off 2.0 s). After that, the solution was dialyzed in distilled water for 6 h and then freeze-dried. The morphology of the nanoparticles was observed using transmission electron
microscope (TEM, JEM-2010, Japan). The particle size, zeta potential, and size distribution of the nanoparticles were determined by dynamic laser light scattering (DLLS, ZETASIZER 3000, USA). To determine the encapsulation efficiency (EE) and loading content (LC) of DOX, DBC nanoparticles were dissolved in methonal and then measured 6
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with a UV spectrophotometer (UV-2450, Japan) at 480 nm. 2.4 Preparation of Col/Ch/DBC nanoparticle film Collagen and chitosan were co-dissolved in 2% acetic acid (HAc) at different weight
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ratios (1:1, 1:0.25 and 1:0.125) and homogenized to obtain a Col/Ch blend solution.
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Afterward, DBC nanoparticles were added into the solution. After freeze-drying, the drug films were crosslinked at 0.25% glutaraldehyde solution for 6 h, then washed repeatedly
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with a large amount of distilled water and freeze-dried again. The morphology of the drug
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film was observed by scanning electron microscopy (SEM, Nova Nano SEM 230, USA). 2.5 Test of swelling and degradation
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A certain weight of the dry drug film (W1) was incubated in distilled water at 37℃. After 24 h, the wet weight (W2) of the films was measured. The following equation was
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W2 W1 W1
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Swelling fold
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applied to calculate the swelling ratio of the specimens:
The drug films were immersed into 1 mL of PBS containing 0.05 M CaCl2 (pH 7.4),
and incubated for 1 h at 37℃. Then 200 U of bacterial collagenase was added. At different time points, the reaction was stopped by cooling in ice and the addition of 0.2 mL 0.25 M EDTA. Following centrifugation for 15 min at 1500 rpm, the supernatant was hydrolyzed with 6M HCl for 24 h. The content of 4-hydroxyproline was measured with UV spectroscopy at 561 nm to determine the film degradation percentage. 2.6 In vitro drug release study DOX release from Bio-CHGC nanoparticles and the films was studied by a dialysis method. The PBS solutions containing DBC nanoparticles or the drug films were placed 7
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into a dialysis bag (MWCO: 8-14kDa), and immersed in PBS (pH 5.8, 6.5, or 7.4) with collagenase at 37℃ in an air-bath shaker at 100 rpm. At predefined time intervals, the
DOX was measured by the UV method described previously.
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2.7 Cytotoxicity assay
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release media were fully exchanged with the fresh release media. The release amount of
The cytotoxicity of Col/Ch/DBC nanoparticle films, Col/Ch/DOX-loaded CHGC
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(DC) nanoparticle films, and Col/Ch/free DOX films was assessed by a MTT method.
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Briefly, HepG2 cells were seeded on 24-well plates at a density of 1 × 104 cells/well. After incubated at 37℃ in a 5% CO2 atmosphere for 24 h, the DMEM medium was replaced
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with 1 mL of fresh medium, then the films were added and incubated for 48 h. At predefined time, 100 μL of MTT solution (5 mg/mL in PBS) was transferred to each well.
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After 4 h, the supernate was discarded and the formazan crystals were dissolved in 1 mL
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570 nm.
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of DMSO for 30 min. Finally, a microplate reader was used to measure the absorbance at
2.8 Statistical analysis
All experiment data were expressed as means + standard deviations (SD) and
analyzed statistically by an one-way analysis of variance. The level of significance was defined at p < 0.05.
3. Results and discussion 3.1 Synthesis and characterization of Bio-CHGC Fig.1a shows the synthetic route of Bio-CHGC. CHS was firstly grafted on the amino 8
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group of glycol chitosan to obtain cholesterol hydrophobically modified glycol chitosan (CHGC). Then biotin was conjugated to CHGC through coupling its carboxyl group with the amino group of CHGC. FTIR spectra of glycol chitosan and Bio-CHGC are shown in
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Fig.S1. The peak assignment of glycol chitosan (Fig.S1a) were at 1672 cm-1 (amide I band,
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C=O stretch of acetyl group), 1604 cm-1 (amide II band, N–H stretch) and 1070 cm-1 (C–O
stretch). Compared with glycol chitosan, the peaks of Bio-CHGC (Fig.S1b) at 1688 cm-1
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(C=O stretch) observably increased, which indicated the formation of an amide linkage
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between the amino groups of glycol chitosan and the carboxyl groups of CHS or biotin [22]. The chemical structure of Bio-CHGC was further confirmed by 1H NMR (Fig.2).
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The characteristic peaks of glycol chitosan (Fig.2a) appeared at 1.98, 2.63, and 3.2-4.4 ppm. Compared with glycol chitosan, the new peaks in CHGC spectrum (Fig.2b) appeared
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at 1.00-1.20 ppm due to the hydrogen protons of CHS group, which suggested that the
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cholesterol moiety was successfully grafted onto the glycol chitosan. As shown in 1H
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NMR spectrum of Bio-CHGC (Fig.2c), the proton assignment of biotin appeared at 1.30-1.70 and 2.10 ppm, indicating that the biotin group was successfully conjugated to CHGC. DS of the cholesterol moiety and biotin group were measured by elemental analysis and the values were 6.35% and 10.04%, respectively. 3.2 Characterization of Bio-CHGC nanoparticles and drug loading The amphiphilic Bio-CHGC conjugates can form self-assembled nanoparticles in aqueous media (Fig.1b), and the morphology of the nanoparticles observed by TEM was almost spherical shaped (Fig.S2). The mean diameter of Bio-CHGC nanoparticles was 305.2 nm, and the polydispersity index was 0.103. The zeta potential of Bio-CHGC 9
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nanoparticles was +15.02 mv, which suggested that this nanoparticle system would be stable in aqueous phase [21]. DOX-loaded nanoparticles were prepared by dialysis. As shown in Fig.3, DBC
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nanoparticles remained spherical under SEM observations and had relatively narrow size
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distributions (data not shown). The characterizations of DBC nanoparticles are listed in Table 1. The mean diameter increased from 330 to 397 nm with the increase of DOX
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loading content. The DOX loading content increased from 3.75% to 8.94% with the
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weight ratio of DOX to Bio-CHGC nanoparticles at the drug loading stage increasing from 1/20 to 1/5, whereas the DOX encapsulation efficiency decreased from 60.33% to 53.66%.
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The zeta potentials of DBC nanoparticles were all positive in PBS (pH 7.4) due to the ionization of amino groups of glycol chitosan. In consideration of the size, drug loading
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content and encapsulation efficiency, 1/10 was determined as the optimal weight ratio in
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preparation for the DBC nanoparticles.
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3.3 Characterization of Col/Ch/DBC nanoparticle films Fig.3 shows the morphology of Col/Ch/DBC nanoparticle films before and after the
release by SEM. In Fig.3a and b, the results revealed that the DBC nanoparticles with spherical shape were uniformly distributed into the Col/Ch films, and the films retained their structural integrity. After the drug released for 120 h (Fig.3 c and d), a few of the nanoparticles were left in the films, indicating that most of the nanoparticles were released from the films. Additionally, the irregular film surface and cross-section were visible, which suggested the erosion and degradation of the films after the release. The swelling ratio of film is an important property for biomedical material. As 10
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shown in Fig.4a, the swelling folds of all drug films were 25- to 33-fold of distilled water and could maintain their morphological stability. The swelling fold of Col1Ch0.125 drug film was higher than those of Col1Ch0.25 and Col1Ch1, suggesting that the swelling fold
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decreased as the proportion of chitosan increased. It could be ascribed to the maintenance
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of three-dimensional structure, the hydrophilicity and the degree of crosslinking [26].
However, the swelling fold of Col1Ch1 drug film was not lower than that of Col1Ch0.25
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because the addition of chitosan provided more amino groups than that was required for
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crosslinking, leading to the decrease of relative crosslinking degree [27]. On the other hand, the swelling fold of drug films was higher than Col/Ch films without DBC
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nanoparticles (data in Ref.11) in the same ratio of collagen to chitosan. It might be due to the integration of DBC nanoparticles with a hydrophilic shell. Besides, the drug films with
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nanoparticles possessed a looser aperture structure, which could provide a shortcut for the
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solutions to enter the internal film, also accounted for the higher swelling fold.
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Fig.4b shows the degradation of Col/Ch/DBC nanoparticle films with different ratios of collagen to chitosan. Generally, the degradation rate of Col/Ch films decreases with the increase of chitosan, because the addition of chitosan could increase the crosslinking degree and biostability of collagen films. However, the drug film of Col1Ch1 showed a relatively higher degradation rate than that of Col1Ch0.25, which was consistent with the result of Col/Ch film without nanoparticles [20]. The reason might be that the higher swelling fold and lower crosslinking degree of films resulted in the higher degradation rate [28]. In addition, the drug film exhibited a little higher degradation rate compared with the Col/Ch film without drug-loaded nanoparticles (data in Ref.11). This 11
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was mainly because the swelling folds of drug films were higher than the films without nanoparticles, leading to the easier penetration of collagenase solution into the films. 3.4 DOX release from Bio-CHGC self-assembled nanoparticles
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DOX release profiles from Bio-CHGC nanoparticles were measured by a dialysis
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method in PBS. As shown in Fig.5, the similar release characteristics for the different drug
loading contents were observed. DOX release from nanoparticles presented a biphasic
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manner, consisting of an initial rapid release within the first 24 hours (phase I) followed
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by a slow and sustained release for more than 120 hours (phase II). Ordinarily, the release rate from polymeric micelles is related with the solubility and diffusivity of drug. In the
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initial stage, the drug diffusion from nanoparticles into the outer aqueous phase was rapid due to the high drug concentration gradient. Then the drug diffusion slowed down with a
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decrease in the drug concentration gradient, resulting in the sustained drug release. From
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Fig.5, the decrease of DOX release rate was observed when the drug loading contents
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increased. The results were consistent with the previously reported phenomena [15,21]. At a higher drug loading content, the hydrophobic drug partially crystallized inside nanoparticles, and phase separation occurred, reducing the drug release rate [29]. The sustained release of DOX from Bio-CHGC nanoparticles was clearly observed for more than 120 h, which suggested that Bio-CHGC self-assembled nanoparticles had a potential as a sustained release carrier for DOX. 3.5 DOX release from Col/Ch/DBC nanoparticle film In vitro DOX release from Col/Ch/DBC nanoparticle films with different ratios of collagen to chitosan was investigated in PBS solutions with pH values of 7.4, 6.5 and 5.8, 12
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respectively. The release profiles are shown in Fig.6. DOX release patterns all consisted of an initial burst release and a slow release. The release rate of DOX from the films increased as the ratio of chitosan decreased at the same pH value. However, the drug film
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of Col1Ch0.25 had a lower release rate than that of Col1Ch1, which agreed with the
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results of swelling and degradation tests. It is commonly believed that the drug release of
the films containing drug-loaded nanoparticles is mainly caused by swelling, erosion and
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diffusion [30]. The higher degradation rate and swelling ratio of the films led to the higher
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diffusion rate of the nanoparticles from the films, resulting in an increase in the drug release from the nanoparticles. The results suggested that the release rate of DOX could be
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controlled by a variation in the ratio of collagen to chitosan. Based on the results of the swelling, degradation and release tests, it could be concluded that the optimal ratio of
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collagen to chitosan in the drug films was 1:0.25. In addition, the cumulative release of
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DOX from the films was evidently lower than that from the nanoparticles alone at the
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same pH value, implying that the drug films had a double-sustained effect on the drug release.
Furthermore, from Fig.6, the release rate increased with a decrease in the pH value of
release medium, suggesting that the release of DOX from the films was sensitive to the pH of the release medium. This phenomenon was similar to the release of DOX from Bio-CHGC nanoparticles (Fig.S3). It might be explained by the following reasons. In general, a lower pH value results in a higher solubility of DOX in release solution, leading to a higher release rate of DOX. Moreover, the release rate of the DOX depended on the swelling of Bio-CHGC nanoparticles and Col/Ch films. The amino groups of Bio-CHGC 13
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nanoparticles and Col/Ch films could ionize in a lower pH value, which caused a higher swelling. The higher swelling of the films led to an increase in the diffusion rate of the nanoparticles from the films, and the higher swelling of the nanoparticles resulted in
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increasing the DOX release rate accordingly [23,31]. It has been reported that the local pH
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of solid tumor was one order of magnitude lower than that of normal tissues, and therefore the pH-sensitive release behavior of DOX from Bio-CHGC nanoparticles and the films
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may be advantageous for the tumor treatment.
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3.6 Cytotoxicity assay
The cytotoxicity of Col/Ch/DBC nanoparticle films, Col/Ch/DC nanoparticle films
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and Col/Ch/free DOX films against HepG2 cells was evaluated using MTT assay. In our previous study, the cell viability of Col/Ch films without drug-loaded nanoparticles was
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above 90% even after 5 days of culturing, which implied the non-cytotoxicity of the films
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against the L02 cells [20]. As shown in Fig.7, the inhibition ratios of all drug films were
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above 85%, indicating that all the films had a high antitumor efficacy against HepG2 cells. The inhibition ratio of free DOX film was higher than others, since free DOX released from Col/Ch film more quickly than Col/Ch/nanoparticle film, suggesting that the nanoparticles exhibited a slow sustained release of DOX. Moreover, the DBC nanoparticle films showed a higher inhibition ratio than DC nanoparticle films. The explanation might be that biotin moieties in Bio-CHGC nanoparticles played an important role in exerting a cytotoxic effect, causing DBC nanoparticles to be localized into HepG2 cells via an endocytic mechanism by a biotin-receptor mediated interaction [32]. The tumor-targeted result of biotin indicated that the nanoparticles had been diffused from the films before the 14
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DOX release. These results demonstrated that Col/Ch/DBC nanoparticle films could be a potential drug delivery system for localized chemotherapy.
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Conclusion
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In this study, the drug films consisting of DBC nanoparticles and fish Col/Ch films
were fabricated using freeze-drying method. SEM showed that the DBC nanoparticles
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with a spherical shape were uniformly distributed into the films. The drug films could
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maintain their morphological stability after swelling. The swelling, degradation and release results suggested that the DOX release rate could be controlled by a variation in
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the ratio of collagen to chitosan and the drug loading content of DBC nanoparticles. In addition, the release of DOX from the films or Bio-CHGC nanoparticles was sensitive to
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the pH value of the release medium, and the drug films had a double-sustained effect on
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the drug release. In cytotoxicity assay, the DBC nanoparticle films showed a higher
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inhibitory effect than DC nanoparticle films. Therefore, this novel drug film system may be useful in the localized delivery for hydrophobic antitumor drugs.
Acknowledgments
This research was supported by the National Natural Science Foundation of China
(Grant No. 31271023), the Natural Science Foundation of Fujian Province (Grant No. 2012J01209 and 2013J01388), and the Key Laboratory of Biomedical Materials of Tianjin.
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Appendix A. Supplementary data
References
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[1] J.R. Weiser, W.M. Saltzman, J. Control. Release 190 (2014) 664–673. [2] J.B. Wolinsky, Y.L. Colson, M.W. Grinstaff, J.Control.Release 150 (2012) 14-26.
cr
[3] Y.J. Park , Y. Ku , CP Chung , SJ Lee . J. Control. Release 51 (1998) 201–211.
us
[4] M.D. Blanco, M.V. Bernardo, C. Gomez, E. Muniz, J.M. Teijon. Biomaterials 20 (1999) 1919-1924.
an
[5] D.B. Tada, S. Singh, D. Nagesha, E. Jost, C.O. Levy, E. Gultepe, R. Cormack, G.M. Makrigiorgos, S. Sridhar. Pharm. Res. 27 (2010) 1738–1745.
M
[6] D. Thacharodi, K. Panduranga Rao. Int. J. Pharm. 134 (1996) 239-241.
d
[7] M. D. Fernandez-Diaz, P. Montero, M.C. Gomez-Guillen. Food Chem. 74 (2001)
te
161-167.
Ac ce p
[8] A. Jongjareonrak, S. Benjakul, W. Visessanguan, T. Nagai, M. Tanaka. Food Chem. 93 (2005) 475-484.
[9] Y.R. Huang, C.Y. Shiau, H.H. Chen, B.C. Huang. Food Hydrocolloid. 25 (2011) 1507-1513.
[10] Y. Zhang, W. Liu, G. Li, B. Shi, Y. Miao, X. Wu. Food Chem. 103 (2007) 906-912. [11] A.A. Karim, R. Bhat, Food Hydrocolloids 23 (2009) 563-576. [12] Y.S. Wang, Y. Liu, Y.Y. Liu, Y. Wang, J. Wu, R.S. Li, J.R.Yang, N. Zhang,. Polym. Chem. 5 (2014) 423–432. [13] X.M. Li, M.M. Chen, W.Z. Yang, Z.M. Zhou, L.R. Liu, Q.Q. Zhang, Colloids Surf. B: Biointerf. 92 (2012) 136-141. 16
Page 16 of 30
[14] H.Z. Zhang, F.P. Gao, L.R. Liu, X.M. Li, Z.M. Zhou, X.D. Yang, Q.Q. Zhang. Colloid. Surface. B 71 (2009) 19-26. [15] L. Li, F.P. Gao, H.B. Tang, Y.G. Bai, R.F. Li, X.M. Li, L.R. Liu, Y.S. Wang, Q.Q.
ip t
Zhang. Nanotechnology 21 (2010) 265601 (1-11pp).
cr
[16] S. Kwon, J.H. Park, H. Chung, I.C. Kwon, S.Y. Jeong, Langmuir 19 (2003) 10188-10193.
us
[17] K. Kim, S. Kwon, J.H. Park, H. Chung, S.Y. Jeong, I.C. Kwon. Biomacromolecules 6
an
(2005) 1154-1158.
[18] J.M. Yu, X. Xie, M.R. Zheng, L. Yu, L. Zhang, J.G. Zhao, D.Z. Jiang, X.X. Int. J.
M
Nanomed. 7 (2012) 5079-5090.
(2013) 42-51.
d
[19] J.L. Wu, S.F. Chen, S.Y. Ge, J. Miao, J.H. Li, Q.Q. Zhang, Food Hydrocolloid. 32
te
[20] M.M. Chen, Y.Q. Huang, H. Guo, Y. Liu, J.H. Wang, J.L. Wu, Q.Q. Zhang, J. Appl.
Ac ce p
Polym. Sci. 131 (2014) 40998 (1-8pp).
[21] M.M. Chen, Y. Liu, W.Z. Yang, X.M. Li, L.R. Liu, Z.M. Zhou, Y.S. Wang, R.F. Li, Q.Q. Zhang, Carbohyd. Polym. 84 (2011) 1244-1251. [22] J.M. Yu, Y.J. Li, L.Y. Qiu, Y.Jin, Eur. Polym. J. 44 (2008) 555-565. [23] Y.S. Wang, L.R. Liu, Q. Jiang, Eur. Polym. J. 43 (2007) 43-51. [24] Y.I. Jeong, H.S. Na, J.S. Oh, K.C. Choi, C.E. Song, H.C. Lee. Int. J. Pharm. 322 (2006) 154-60. [25] X.D. Yang, Q.Q. Zhang, Y.S. Wang, H. Chen, H.Z. Zhang, F.P. Gao, L.R. Liu. Colloid. Surface. B 61 (2008) 125-131. 17
Page 17 of 30
[26] L. Ma, C.Y. Gao, Z.W. Mao, J. Zhou, J.C.Shen, X.Q. Hu, C.M. Han, Biomaterials 24 (2003) 4833–4841. [27] X.H. Wang, D.P. Li, W.J. Wang, Q.L. Feng, F.Z. Cui, Y.X. Xu, X.H. Song, M. Werf,
ip t
Biomaterials 24 (2003) 3213-3220.
cr
[28] Y.C. Lin, F.J. Tan, K. G. Marra, S.S. Jan, D.C. Liu, Acta Biomater. 5 (2009) 2591–2600.
us
[29] R. Gref, Y. Minamitake, M.T. Peracchia, V. Trubetskoy, V. Torchilin, R. Langer,
an
Science 263 (1994) 1600–1603.
[30] J. Wu, T.T. Kong, K.W.K. Yeung, H.C. Shum, K.M.C. Cheung, L.Q. Wang, M.K.T.
M
To, Acta Biomater. 9 (2013) 7410–7419.
3193–3201.
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[31] Y. Hu, X.Q. Jiang, Y. Ding, H.X. Ge, Y.Y. Yuan, C.Z. Yang, Biomaterials 23 (2002)
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165–173.
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[32] K. Na, T.B. Lee, K.H. Park, E.K. Shin, Y.B. Lee, Eur. J. of Pharm. Sci. 18 (2003)
18
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Table captions:
Ac ce p
te
d
M
an
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cr
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Table 1 Characterization of DOX-loaded Bio-CHGC nanoparticles.
19
Page 19 of 30
Table 1 Characterization of DOX-loaded Bio-CHGC nanoparticles DOX/carrier a
Mean diameter b (nm)
Zeta potential b (mv)
LC c (%)
EE c (%)
DBC-3.8
1/20
330±14
3.30 ±0.69
3.75±0.26
60.33±6.05
DBC-5.5
1/10
368±15
4.10±0.65
5.48±0.38
60.29±4.27
DBC-8.9
1/5
397±19
4.12±0.69
8.94±0.32
53.66±3.35
cr
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Sample
The feed weight ratio of DOX to Bio-CHGC nanoparticles (mg/mg).
b
The size and zeta potential (mean value ± SD) measured by DLLS with three times.
c
LC and EE
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a
Ac ce p
te
d
M
an
(mean value±SD) determined by UV spectrophotometer with three times.
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Page 20 of 30
Figure captions: Fig.1 Synthetic route of Bio-CHGC (a) and schematic illustration of the assembly of DOX-loaded nanoparticles, and the preparation and release of drug film (b).
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Fig.2 1H NMR spectra of glycol chitosan (a), CHGC (b) and Bio-CHGC (c) in D2O.
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Fig.3 SEM images of Col/Ch/DBC-5.5 nanoparticle film before release (a, b) and after release (c, d) for 120 h: the surface view (a, c) and the cross-section view (b, d).
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Fig.4 The swelling ratio (a) and in vitro degradation (b) of Col/Ch/DBC-5.5 nanoparticle
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film with different ratios of collagen to chitosan. The values are the means and standard deviations (n=3, * p
S0927-7765(15)00100-9 http://dx.doi.org/doi:10.1016/j.colsurfb.2015.02.024 COLSUB 6915
To appear in:
Colloids and Surfaces B: Biointerfaces
Received date: Revised date: Accepted date:
3-12-2014 30-1-2015 12-2-2015
Please cite this article as: M.-M. Chen, Y.-Q. Huang, H. Cao, Y. Liu, H. Guo, L.S. Chen, J.-H. Wang, Q.-Q. Zhang, Collagen/chitosan film containing biotinylated glycol chitosan nanoparticles for localized drug delivery, Colloids and Surfaces B: Biointerfaces (2015), http://dx.doi.org/10.1016/j.colsurfb.2015.02.024 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.
Collagen/chitosan film containing biotinylated glycol chitosan nanoparticles for localized drug delivery
Institute of Biomedical and Pharmaceutical Technology, Fuzhou University, Fuzhou
b
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350002, China
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a
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Jian-Hua Wang a, Qi-Qing Zhang a,c,*
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Ming-Mao Chen a, Yu-Qing Huang a, Huan Cao a, Yan Liu b, Hao Guo a, Lillian S. Chen a,
State Key Lab of Structural Chemistry, Fujian Institute of Research on the Structure of
c
M
Matter, Chinese Academy of Sciences, Fuzhou 350002, China Key Laboratory of Biomedical Material of Tianjin, Institute of Biomedical Engineering,
te
d
Chinese Academy of Medical Science & Peking Union Medical College, Tianjin 300192, China
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*Corresponding author at: Institute of Biomedical and Pharmaceutical Technology, Fuzhou University, No. 523 Gongye Road, Fuzhou 350002, China.
Tel.: +86 591 83725260; fax: +86 591 83725260. E-mail: [email protected].
1
Page 1 of 30
Abstract The objective of this study was to design a drug delivery system consisting of biotinylated cholesterol-modified glycol chitosan (Bio-CHGC) nanoparticles and fish
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collagen/chitosan (Col/Ch) film for localized chemotherapy. Bio-CHGC was synthesized,
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and then its self-assembled nanoparticles were prepared by probe sonication. Doxorubicin (DOX)-loaded Bio-CHGC (DBC) nanoparticles prepared by dialysis had spherical shape,
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and their sizes were in the range of 330-397 nm. Col/Ch/DBC nanoparticle films were
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fabricated by freeze-drying. SEM showed that the DBC nanoparticles were uniformly distributed into the films, and the films retained their structural integrity. A higher
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degradation and swelling rate of the drug films led to a higher diffusion rate of the nanoparticles from the films, resulting in an increase in the drug release from
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nanoparticles. The release of DOX from the films or Bio-CHGC nanoparticles was
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sensitive to the pH value of the release medium. In addition, the DOX release ratio of the
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drug films was lower than that of the nanoparticles alone, suggesting that the drug films had a double-sustained effect on the drug release. MTT assay implied that the DBC nanoparticle film showed a higher inhibitory ratio than the film containing nanoparticles without biotin, indicating that biotin moieties in the nanoparticles played an important role in exerting a cytotoxic effect. These data demonstrate that Col/Ch/DBC nanoparticle film has the potential to be used as a localized delivery system for hydrophobic antitumor drugs. Keywords: Collagen, Drug film, Silver carp skin, Glycol chitosan, Self-assembled nanoparticles 2
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1. Introduction Localized chemotherapy is generally used as an adjuvant to surgery to protect against tumor recurrence. Compared to systemic chemotherapy, localized delivery can supply
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therapeutic levels of drugs at a physical site for a prolonged period, and reduce systemic
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toxicities [1,2]. Implantable films based on the biocompatible and biodegradable polymers have been developed for localized delivery, such as poly(lactic acid) (PLA),
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poly(caprolactone) (PCL), poly(lactide-co-glycolide) (PLGA), collagen and chitosan [3-6].
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Collagen film is a promising delivery system for localized chemotherapy due to its biocompatibility, nontoxicity and biodegradability. However, collagen used in medical
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applications is commonly isolated from bovine and porcine skins and bones, which is associated with a high risk of bovine spongiform encephalopathy or religious issues [7,8].
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Recently, fresh water and marine fishes have been proposed as alternative sources for
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collagen. The investigation of fish collagen focuses on the extraction methods and the
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application in the food field [9-11]. Nevertheless, the application of fish collagen films in localized chemotherapy has seldom been studied so far. Collagen-based films incorporated with drug-loaded nanoparticles have triggered
considerable interest as a carrier for localized drug delivery, because they can sustain drug release, enhance targeted effect, reduce systemic toxicity and increase treatment efficiency. Recently, much attention has been paid to preparing self-assembled polymeric nanoparticles on the basis of natural polysaccharides, such as chitosan, pullulan and curdlan [12-15]. Glycol chitosan, a water-soluble chitosan derivative, is used as a novel drug carrier because of its solubility, biocompatibility, biodegradability and non-toxicity. 3
Page 3 of 30
Hydrophobically modified glycol chitosan (HGC) derivatives, such as glycol chitosan bearing 5-cholanic acid, deoxycholic acid and cholesterol, have been focusing on due to their amphiphilic nature [16-18]. These polymeric amphiphiles can form self-assembled
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nanoparticles in aqueous media, and the nanoparticles have shown the potential
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application as carriers for hydrophobic drugs and genes. Therefore, a combination of HGC nanoparticles and fish collagen films might be a novel drug delivery system for
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hydrophobic drugs.
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Previously, our group has isolated the collagen from silver carp skin, and prepared fish collagen/chitosan (Col/Ch) composite sponges [19,20]. The results showed that the
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presence of chitosan improved the biostability of collagen sponges, and the composite sponges exhibited noncytotoxicity, biocompatibility, and nonhemolysis. In this study, we
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developed a drug delivery system consisting of biotinylated cholesterol-modified glycol
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chitosan (Bio-CHGC) nanoparticles and fish Col/Ch film. Biotin was introduced into the
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conjugate to enhance tumor-targeted drug delivery. As shown in Fig.1, Bio-CHGC has an amphiphilic property and can form self-assembled nanoparticles by the hydrophobic interactions of cholesterol moieties. Doxorubicin (DOX) was physically loaded into Bio-CHGC nanoparticles, and then DOX-loaded nanoparticles were mixed with Col/Ch solution to prepare the drug films. The characteristics and drug release behavior of the drug films were evaluated. Moreover, the cytotoxicity of the drug films against human hepatoma (HepG2) cells was assayed to demonstrate the antitumor efficacy of this drug delivery system.
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2. Materials and methods 2.1 Materials Collagen was isolated from silver carp skin by the method previously reported [20].
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Cholesterol succinate (CHS) was synthesized following the procedure reported previously
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[21]. DOX hydrochloride (DOX·HCl), glycol chitosan (MW = 2.5 × 105 Da, degree of
deacetylation = 82%), N-hydroxyl succinimide (NHS) and collagenase were obtained
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from Sigma–Aldrich (St. Louis, MO). Chitosan (MW = 5×105 Da, degree of deacetylation
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= 85%) was purchased from Shanghai Qiangshun Chemical Reagent Co.,Ltd. (Shanghai, China). Glutaraldehyde (GA, 25% water solution) was supplied by Sinopharm Chemical
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Reagent Co., Ltd. (Shanghai, China). Dulbecco’s modified Eagle’s medium (DMEM) was purchased from Hyclone (Logan, UT). All other chemical reagents were of analytical
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grade and obtained from commercial sources.
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2.2 Synthesis of Bio-CHGC conjugate
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Cholesterol-modified glycol chitosan (CHGC) was synthesized according to the previously reported procedure [18,22]. CHS (72.8 mg) in 20 mL of 85% ethanol solution was activated by the addition of EDC and NHS for 10 h. Glycol chitosan (130 mg) was dissolved in 8 mL of distilled water, and then it was added to the above mixture. After 72 h of the reaction at 40℃, it was sequentially dialyzed (MWCO 8-14 kDa) against an excess amount of 80%, 50% ethanol solution and distilled water for 72 h. The dialyzed solution was lyophilized and the CHGC conjugate was obtained. CHGC (248.3 mg) and biotin (122.0 mg) were mixed with EDC and NHS in 40 mL of DMSO. After reacting for 24 h at room temperature, the solution was dialyzed against 5
Page 5 of 30
distilled water for 48 h. Then the solution was freeze-dried to obtain Bio-CHGC. The chemical structure of Bio-CHGC was characterized by Fourier transform infrared spectroscopy (FTIR, Nicolet AVATAR-360, USA) and 1H-NMR spectroscopy (AVANCE
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III 500, Switzerland) using D2O as the solvent. The degrees of substitution (DS) of the
Germany).
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2.3 Preparation of Bio-CHGC nanoparticles and drug loading
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cholesterol moiety and biotin group were determined by elemental analysis (Vario MICRO,
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Bio-CHGC nanoparticles were made by probe sonication [23]. Briefly, Bio-CHGC was dispersed in distilled water overnight, followed by sonication with a probe type
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sonifier (JY92-IIN, China) in an ice bath at 80 W for 2 min (pulse on 2 s and pulse off 2 s). The solution was filtered through a filter (0.8 μm) to remove dust and impurity.
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DOX-loaded Bio-CHGC (DBC) nanoparticles were prepared using a dialysis method
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[24,25]. DOX·HCl was dissolved in 2 mL of DMSO and trithylamine was added to
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remove hydrochloride. After stirring at room temperature for 12 h in the dark, the mixture was dropped into the above Bio-CHGC nanoparticle solution with ultrasonication at an output power of 80W for 2 min (pulse on 2.0 s and pulse off 2.0 s). After that, the solution was dialyzed in distilled water for 6 h and then freeze-dried. The morphology of the nanoparticles was observed using transmission electron
microscope (TEM, JEM-2010, Japan). The particle size, zeta potential, and size distribution of the nanoparticles were determined by dynamic laser light scattering (DLLS, ZETASIZER 3000, USA). To determine the encapsulation efficiency (EE) and loading content (LC) of DOX, DBC nanoparticles were dissolved in methonal and then measured 6
Page 6 of 30
with a UV spectrophotometer (UV-2450, Japan) at 480 nm. 2.4 Preparation of Col/Ch/DBC nanoparticle film Collagen and chitosan were co-dissolved in 2% acetic acid (HAc) at different weight
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ratios (1:1, 1:0.25 and 1:0.125) and homogenized to obtain a Col/Ch blend solution.
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Afterward, DBC nanoparticles were added into the solution. After freeze-drying, the drug films were crosslinked at 0.25% glutaraldehyde solution for 6 h, then washed repeatedly
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with a large amount of distilled water and freeze-dried again. The morphology of the drug
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film was observed by scanning electron microscopy (SEM, Nova Nano SEM 230, USA). 2.5 Test of swelling and degradation
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A certain weight of the dry drug film (W1) was incubated in distilled water at 37℃. After 24 h, the wet weight (W2) of the films was measured. The following equation was
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W2 W1 W1
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Swelling fold
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applied to calculate the swelling ratio of the specimens:
The drug films were immersed into 1 mL of PBS containing 0.05 M CaCl2 (pH 7.4),
and incubated for 1 h at 37℃. Then 200 U of bacterial collagenase was added. At different time points, the reaction was stopped by cooling in ice and the addition of 0.2 mL 0.25 M EDTA. Following centrifugation for 15 min at 1500 rpm, the supernatant was hydrolyzed with 6M HCl for 24 h. The content of 4-hydroxyproline was measured with UV spectroscopy at 561 nm to determine the film degradation percentage. 2.6 In vitro drug release study DOX release from Bio-CHGC nanoparticles and the films was studied by a dialysis method. The PBS solutions containing DBC nanoparticles or the drug films were placed 7
Page 7 of 30
into a dialysis bag (MWCO: 8-14kDa), and immersed in PBS (pH 5.8, 6.5, or 7.4) with collagenase at 37℃ in an air-bath shaker at 100 rpm. At predefined time intervals, the
DOX was measured by the UV method described previously.
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2.7 Cytotoxicity assay
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release media were fully exchanged with the fresh release media. The release amount of
The cytotoxicity of Col/Ch/DBC nanoparticle films, Col/Ch/DOX-loaded CHGC
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(DC) nanoparticle films, and Col/Ch/free DOX films was assessed by a MTT method.
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Briefly, HepG2 cells were seeded on 24-well plates at a density of 1 × 104 cells/well. After incubated at 37℃ in a 5% CO2 atmosphere for 24 h, the DMEM medium was replaced
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with 1 mL of fresh medium, then the films were added and incubated for 48 h. At predefined time, 100 μL of MTT solution (5 mg/mL in PBS) was transferred to each well.
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After 4 h, the supernate was discarded and the formazan crystals were dissolved in 1 mL
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570 nm.
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of DMSO for 30 min. Finally, a microplate reader was used to measure the absorbance at
2.8 Statistical analysis
All experiment data were expressed as means + standard deviations (SD) and
analyzed statistically by an one-way analysis of variance. The level of significance was defined at p < 0.05.
3. Results and discussion 3.1 Synthesis and characterization of Bio-CHGC Fig.1a shows the synthetic route of Bio-CHGC. CHS was firstly grafted on the amino 8
Page 8 of 30
group of glycol chitosan to obtain cholesterol hydrophobically modified glycol chitosan (CHGC). Then biotin was conjugated to CHGC through coupling its carboxyl group with the amino group of CHGC. FTIR spectra of glycol chitosan and Bio-CHGC are shown in
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Fig.S1. The peak assignment of glycol chitosan (Fig.S1a) were at 1672 cm-1 (amide I band,
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C=O stretch of acetyl group), 1604 cm-1 (amide II band, N–H stretch) and 1070 cm-1 (C–O
stretch). Compared with glycol chitosan, the peaks of Bio-CHGC (Fig.S1b) at 1688 cm-1
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(C=O stretch) observably increased, which indicated the formation of an amide linkage
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between the amino groups of glycol chitosan and the carboxyl groups of CHS or biotin [22]. The chemical structure of Bio-CHGC was further confirmed by 1H NMR (Fig.2).
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The characteristic peaks of glycol chitosan (Fig.2a) appeared at 1.98, 2.63, and 3.2-4.4 ppm. Compared with glycol chitosan, the new peaks in CHGC spectrum (Fig.2b) appeared
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at 1.00-1.20 ppm due to the hydrogen protons of CHS group, which suggested that the
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cholesterol moiety was successfully grafted onto the glycol chitosan. As shown in 1H
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NMR spectrum of Bio-CHGC (Fig.2c), the proton assignment of biotin appeared at 1.30-1.70 and 2.10 ppm, indicating that the biotin group was successfully conjugated to CHGC. DS of the cholesterol moiety and biotin group were measured by elemental analysis and the values were 6.35% and 10.04%, respectively. 3.2 Characterization of Bio-CHGC nanoparticles and drug loading The amphiphilic Bio-CHGC conjugates can form self-assembled nanoparticles in aqueous media (Fig.1b), and the morphology of the nanoparticles observed by TEM was almost spherical shaped (Fig.S2). The mean diameter of Bio-CHGC nanoparticles was 305.2 nm, and the polydispersity index was 0.103. The zeta potential of Bio-CHGC 9
Page 9 of 30
nanoparticles was +15.02 mv, which suggested that this nanoparticle system would be stable in aqueous phase [21]. DOX-loaded nanoparticles were prepared by dialysis. As shown in Fig.3, DBC
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nanoparticles remained spherical under SEM observations and had relatively narrow size
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distributions (data not shown). The characterizations of DBC nanoparticles are listed in Table 1. The mean diameter increased from 330 to 397 nm with the increase of DOX
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loading content. The DOX loading content increased from 3.75% to 8.94% with the
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weight ratio of DOX to Bio-CHGC nanoparticles at the drug loading stage increasing from 1/20 to 1/5, whereas the DOX encapsulation efficiency decreased from 60.33% to 53.66%.
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The zeta potentials of DBC nanoparticles were all positive in PBS (pH 7.4) due to the ionization of amino groups of glycol chitosan. In consideration of the size, drug loading
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content and encapsulation efficiency, 1/10 was determined as the optimal weight ratio in
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preparation for the DBC nanoparticles.
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3.3 Characterization of Col/Ch/DBC nanoparticle films Fig.3 shows the morphology of Col/Ch/DBC nanoparticle films before and after the
release by SEM. In Fig.3a and b, the results revealed that the DBC nanoparticles with spherical shape were uniformly distributed into the Col/Ch films, and the films retained their structural integrity. After the drug released for 120 h (Fig.3 c and d), a few of the nanoparticles were left in the films, indicating that most of the nanoparticles were released from the films. Additionally, the irregular film surface and cross-section were visible, which suggested the erosion and degradation of the films after the release. The swelling ratio of film is an important property for biomedical material. As 10
Page 10 of 30
shown in Fig.4a, the swelling folds of all drug films were 25- to 33-fold of distilled water and could maintain their morphological stability. The swelling fold of Col1Ch0.125 drug film was higher than those of Col1Ch0.25 and Col1Ch1, suggesting that the swelling fold
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decreased as the proportion of chitosan increased. It could be ascribed to the maintenance
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of three-dimensional structure, the hydrophilicity and the degree of crosslinking [26].
However, the swelling fold of Col1Ch1 drug film was not lower than that of Col1Ch0.25
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because the addition of chitosan provided more amino groups than that was required for
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crosslinking, leading to the decrease of relative crosslinking degree [27]. On the other hand, the swelling fold of drug films was higher than Col/Ch films without DBC
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nanoparticles (data in Ref.11) in the same ratio of collagen to chitosan. It might be due to the integration of DBC nanoparticles with a hydrophilic shell. Besides, the drug films with
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nanoparticles possessed a looser aperture structure, which could provide a shortcut for the
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solutions to enter the internal film, also accounted for the higher swelling fold.
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Fig.4b shows the degradation of Col/Ch/DBC nanoparticle films with different ratios of collagen to chitosan. Generally, the degradation rate of Col/Ch films decreases with the increase of chitosan, because the addition of chitosan could increase the crosslinking degree and biostability of collagen films. However, the drug film of Col1Ch1 showed a relatively higher degradation rate than that of Col1Ch0.25, which was consistent with the result of Col/Ch film without nanoparticles [20]. The reason might be that the higher swelling fold and lower crosslinking degree of films resulted in the higher degradation rate [28]. In addition, the drug film exhibited a little higher degradation rate compared with the Col/Ch film without drug-loaded nanoparticles (data in Ref.11). This 11
Page 11 of 30
was mainly because the swelling folds of drug films were higher than the films without nanoparticles, leading to the easier penetration of collagenase solution into the films. 3.4 DOX release from Bio-CHGC self-assembled nanoparticles
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DOX release profiles from Bio-CHGC nanoparticles were measured by a dialysis
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method in PBS. As shown in Fig.5, the similar release characteristics for the different drug
loading contents were observed. DOX release from nanoparticles presented a biphasic
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manner, consisting of an initial rapid release within the first 24 hours (phase I) followed
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by a slow and sustained release for more than 120 hours (phase II). Ordinarily, the release rate from polymeric micelles is related with the solubility and diffusivity of drug. In the
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initial stage, the drug diffusion from nanoparticles into the outer aqueous phase was rapid due to the high drug concentration gradient. Then the drug diffusion slowed down with a
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decrease in the drug concentration gradient, resulting in the sustained drug release. From
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Fig.5, the decrease of DOX release rate was observed when the drug loading contents
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increased. The results were consistent with the previously reported phenomena [15,21]. At a higher drug loading content, the hydrophobic drug partially crystallized inside nanoparticles, and phase separation occurred, reducing the drug release rate [29]. The sustained release of DOX from Bio-CHGC nanoparticles was clearly observed for more than 120 h, which suggested that Bio-CHGC self-assembled nanoparticles had a potential as a sustained release carrier for DOX. 3.5 DOX release from Col/Ch/DBC nanoparticle film In vitro DOX release from Col/Ch/DBC nanoparticle films with different ratios of collagen to chitosan was investigated in PBS solutions with pH values of 7.4, 6.5 and 5.8, 12
Page 12 of 30
respectively. The release profiles are shown in Fig.6. DOX release patterns all consisted of an initial burst release and a slow release. The release rate of DOX from the films increased as the ratio of chitosan decreased at the same pH value. However, the drug film
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of Col1Ch0.25 had a lower release rate than that of Col1Ch1, which agreed with the
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results of swelling and degradation tests. It is commonly believed that the drug release of
the films containing drug-loaded nanoparticles is mainly caused by swelling, erosion and
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diffusion [30]. The higher degradation rate and swelling ratio of the films led to the higher
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diffusion rate of the nanoparticles from the films, resulting in an increase in the drug release from the nanoparticles. The results suggested that the release rate of DOX could be
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controlled by a variation in the ratio of collagen to chitosan. Based on the results of the swelling, degradation and release tests, it could be concluded that the optimal ratio of
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collagen to chitosan in the drug films was 1:0.25. In addition, the cumulative release of
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DOX from the films was evidently lower than that from the nanoparticles alone at the
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same pH value, implying that the drug films had a double-sustained effect on the drug release.
Furthermore, from Fig.6, the release rate increased with a decrease in the pH value of
release medium, suggesting that the release of DOX from the films was sensitive to the pH of the release medium. This phenomenon was similar to the release of DOX from Bio-CHGC nanoparticles (Fig.S3). It might be explained by the following reasons. In general, a lower pH value results in a higher solubility of DOX in release solution, leading to a higher release rate of DOX. Moreover, the release rate of the DOX depended on the swelling of Bio-CHGC nanoparticles and Col/Ch films. The amino groups of Bio-CHGC 13
Page 13 of 30
nanoparticles and Col/Ch films could ionize in a lower pH value, which caused a higher swelling. The higher swelling of the films led to an increase in the diffusion rate of the nanoparticles from the films, and the higher swelling of the nanoparticles resulted in
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increasing the DOX release rate accordingly [23,31]. It has been reported that the local pH
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of solid tumor was one order of magnitude lower than that of normal tissues, and therefore the pH-sensitive release behavior of DOX from Bio-CHGC nanoparticles and the films
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may be advantageous for the tumor treatment.
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3.6 Cytotoxicity assay
The cytotoxicity of Col/Ch/DBC nanoparticle films, Col/Ch/DC nanoparticle films
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and Col/Ch/free DOX films against HepG2 cells was evaluated using MTT assay. In our previous study, the cell viability of Col/Ch films without drug-loaded nanoparticles was
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above 90% even after 5 days of culturing, which implied the non-cytotoxicity of the films
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against the L02 cells [20]. As shown in Fig.7, the inhibition ratios of all drug films were
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above 85%, indicating that all the films had a high antitumor efficacy against HepG2 cells. The inhibition ratio of free DOX film was higher than others, since free DOX released from Col/Ch film more quickly than Col/Ch/nanoparticle film, suggesting that the nanoparticles exhibited a slow sustained release of DOX. Moreover, the DBC nanoparticle films showed a higher inhibition ratio than DC nanoparticle films. The explanation might be that biotin moieties in Bio-CHGC nanoparticles played an important role in exerting a cytotoxic effect, causing DBC nanoparticles to be localized into HepG2 cells via an endocytic mechanism by a biotin-receptor mediated interaction [32]. The tumor-targeted result of biotin indicated that the nanoparticles had been diffused from the films before the 14
Page 14 of 30
DOX release. These results demonstrated that Col/Ch/DBC nanoparticle films could be a potential drug delivery system for localized chemotherapy.
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Conclusion
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In this study, the drug films consisting of DBC nanoparticles and fish Col/Ch films
were fabricated using freeze-drying method. SEM showed that the DBC nanoparticles
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with a spherical shape were uniformly distributed into the films. The drug films could
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maintain their morphological stability after swelling. The swelling, degradation and release results suggested that the DOX release rate could be controlled by a variation in
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the ratio of collagen to chitosan and the drug loading content of DBC nanoparticles. In addition, the release of DOX from the films or Bio-CHGC nanoparticles was sensitive to
d
the pH value of the release medium, and the drug films had a double-sustained effect on
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the drug release. In cytotoxicity assay, the DBC nanoparticle films showed a higher
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inhibitory effect than DC nanoparticle films. Therefore, this novel drug film system may be useful in the localized delivery for hydrophobic antitumor drugs.
Acknowledgments
This research was supported by the National Natural Science Foundation of China
(Grant No. 31271023), the Natural Science Foundation of Fujian Province (Grant No. 2012J01209 and 2013J01388), and the Key Laboratory of Biomedical Materials of Tianjin.
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Appendix A. Supplementary data
References
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[1] J.R. Weiser, W.M. Saltzman, J. Control. Release 190 (2014) 664–673. [2] J.B. Wolinsky, Y.L. Colson, M.W. Grinstaff, J.Control.Release 150 (2012) 14-26.
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[3] Y.J. Park , Y. Ku , CP Chung , SJ Lee . J. Control. Release 51 (1998) 201–211.
us
[4] M.D. Blanco, M.V. Bernardo, C. Gomez, E. Muniz, J.M. Teijon. Biomaterials 20 (1999) 1919-1924.
an
[5] D.B. Tada, S. Singh, D. Nagesha, E. Jost, C.O. Levy, E. Gultepe, R. Cormack, G.M. Makrigiorgos, S. Sridhar. Pharm. Res. 27 (2010) 1738–1745.
M
[6] D. Thacharodi, K. Panduranga Rao. Int. J. Pharm. 134 (1996) 239-241.
d
[7] M. D. Fernandez-Diaz, P. Montero, M.C. Gomez-Guillen. Food Chem. 74 (2001)
te
161-167.
Ac ce p
[8] A. Jongjareonrak, S. Benjakul, W. Visessanguan, T. Nagai, M. Tanaka. Food Chem. 93 (2005) 475-484.
[9] Y.R. Huang, C.Y. Shiau, H.H. Chen, B.C. Huang. Food Hydrocolloid. 25 (2011) 1507-1513.
[10] Y. Zhang, W. Liu, G. Li, B. Shi, Y. Miao, X. Wu. Food Chem. 103 (2007) 906-912. [11] A.A. Karim, R. Bhat, Food Hydrocolloids 23 (2009) 563-576. [12] Y.S. Wang, Y. Liu, Y.Y. Liu, Y. Wang, J. Wu, R.S. Li, J.R.Yang, N. Zhang,. Polym. Chem. 5 (2014) 423–432. [13] X.M. Li, M.M. Chen, W.Z. Yang, Z.M. Zhou, L.R. Liu, Q.Q. Zhang, Colloids Surf. B: Biointerf. 92 (2012) 136-141. 16
Page 16 of 30
[14] H.Z. Zhang, F.P. Gao, L.R. Liu, X.M. Li, Z.M. Zhou, X.D. Yang, Q.Q. Zhang. Colloid. Surface. B 71 (2009) 19-26. [15] L. Li, F.P. Gao, H.B. Tang, Y.G. Bai, R.F. Li, X.M. Li, L.R. Liu, Y.S. Wang, Q.Q.
ip t
Zhang. Nanotechnology 21 (2010) 265601 (1-11pp).
cr
[16] S. Kwon, J.H. Park, H. Chung, I.C. Kwon, S.Y. Jeong, Langmuir 19 (2003) 10188-10193.
us
[17] K. Kim, S. Kwon, J.H. Park, H. Chung, S.Y. Jeong, I.C. Kwon. Biomacromolecules 6
an
(2005) 1154-1158.
[18] J.M. Yu, X. Xie, M.R. Zheng, L. Yu, L. Zhang, J.G. Zhao, D.Z. Jiang, X.X. Int. J.
M
Nanomed. 7 (2012) 5079-5090.
(2013) 42-51.
d
[19] J.L. Wu, S.F. Chen, S.Y. Ge, J. Miao, J.H. Li, Q.Q. Zhang, Food Hydrocolloid. 32
te
[20] M.M. Chen, Y.Q. Huang, H. Guo, Y. Liu, J.H. Wang, J.L. Wu, Q.Q. Zhang, J. Appl.
Ac ce p
Polym. Sci. 131 (2014) 40998 (1-8pp).
[21] M.M. Chen, Y. Liu, W.Z. Yang, X.M. Li, L.R. Liu, Z.M. Zhou, Y.S. Wang, R.F. Li, Q.Q. Zhang, Carbohyd. Polym. 84 (2011) 1244-1251. [22] J.M. Yu, Y.J. Li, L.Y. Qiu, Y.Jin, Eur. Polym. J. 44 (2008) 555-565. [23] Y.S. Wang, L.R. Liu, Q. Jiang, Eur. Polym. J. 43 (2007) 43-51. [24] Y.I. Jeong, H.S. Na, J.S. Oh, K.C. Choi, C.E. Song, H.C. Lee. Int. J. Pharm. 322 (2006) 154-60. [25] X.D. Yang, Q.Q. Zhang, Y.S. Wang, H. Chen, H.Z. Zhang, F.P. Gao, L.R. Liu. Colloid. Surface. B 61 (2008) 125-131. 17
Page 17 of 30
[26] L. Ma, C.Y. Gao, Z.W. Mao, J. Zhou, J.C.Shen, X.Q. Hu, C.M. Han, Biomaterials 24 (2003) 4833–4841. [27] X.H. Wang, D.P. Li, W.J. Wang, Q.L. Feng, F.Z. Cui, Y.X. Xu, X.H. Song, M. Werf,
ip t
Biomaterials 24 (2003) 3213-3220.
cr
[28] Y.C. Lin, F.J. Tan, K. G. Marra, S.S. Jan, D.C. Liu, Acta Biomater. 5 (2009) 2591–2600.
us
[29] R. Gref, Y. Minamitake, M.T. Peracchia, V. Trubetskoy, V. Torchilin, R. Langer,
an
Science 263 (1994) 1600–1603.
[30] J. Wu, T.T. Kong, K.W.K. Yeung, H.C. Shum, K.M.C. Cheung, L.Q. Wang, M.K.T.
M
To, Acta Biomater. 9 (2013) 7410–7419.
3193–3201.
d
[31] Y. Hu, X.Q. Jiang, Y. Ding, H.X. Ge, Y.Y. Yuan, C.Z. Yang, Biomaterials 23 (2002)
Ac ce p
165–173.
te
[32] K. Na, T.B. Lee, K.H. Park, E.K. Shin, Y.B. Lee, Eur. J. of Pharm. Sci. 18 (2003)
18
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Table captions:
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d
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Table 1 Characterization of DOX-loaded Bio-CHGC nanoparticles.
19
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Table 1 Characterization of DOX-loaded Bio-CHGC nanoparticles DOX/carrier a
Mean diameter b (nm)
Zeta potential b (mv)
LC c (%)
EE c (%)
DBC-3.8
1/20
330±14
3.30 ±0.69
3.75±0.26
60.33±6.05
DBC-5.5
1/10
368±15
4.10±0.65
5.48±0.38
60.29±4.27
DBC-8.9
1/5
397±19
4.12±0.69
8.94±0.32
53.66±3.35
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Sample
The feed weight ratio of DOX to Bio-CHGC nanoparticles (mg/mg).
b
The size and zeta potential (mean value ± SD) measured by DLLS with three times.
c
LC and EE
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a
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d
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(mean value±SD) determined by UV spectrophotometer with three times.
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Figure captions: Fig.1 Synthetic route of Bio-CHGC (a) and schematic illustration of the assembly of DOX-loaded nanoparticles, and the preparation and release of drug film (b).
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Fig.2 1H NMR spectra of glycol chitosan (a), CHGC (b) and Bio-CHGC (c) in D2O.
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Fig.3 SEM images of Col/Ch/DBC-5.5 nanoparticle film before release (a, b) and after release (c, d) for 120 h: the surface view (a, c) and the cross-section view (b, d).
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Fig.4 The swelling ratio (a) and in vitro degradation (b) of Col/Ch/DBC-5.5 nanoparticle
an
film with different ratios of collagen to chitosan. The values are the means and standard deviations (n=3, * p
