A novel preparation method of paclitaxcel-loaded folate-modified chitosan microparticles and in vitro evaluation

A new chitosan microparticles loading paclitaxel (PTX) for application as an oral delivery system were developed using a novel double emulsion crosslinking method. To improve the targeted effect, folic acid (FA) was introduced onto the surface of microparticles using chemical method. The method was based on Schiff reaction between amino group of chitosan and carboxyl group of FA, and folate-chitosan (FA-CS) conjugate was characterized using infrared spectrum analysis (FT-IR), and the microparticles were named as FA-CS-PTX/MPs. FA-CS-PTX/MPs had larger size of average diameter 223.6 nm, while PTX-loaded chitosan microparticles (CS-PTX/MPs) had 179.1 nm average diameter. The zeta potential of CS-PTX/MPs and FA-CS-PTX/MPs was 22.3 and 33.1 mV, respectively. SEM and TEM showed both the two microparticles had well-defined spherical structure. The in vitro drug release was studied under different pH conditions, and a two-phase kinetics model was found to be the most adequate kinetic model. Furthermore, the cytotoxicity activities of drug-carriers against L929 cells and the cellular uptake of PTX-loaded microparticles against HepG2 cells were investigated. Results demonstrated that FA-CS-PTX/MPs might be a promising drug carrier for promoting PTX cellular uptake and could be used as a potential tumor-targeted drug vector.     

In vivo evaluation of an oral delivery system for P-gp substrates based on thiolated chitosan

Recently, thiolated polymers, so called thiomers, have been reported to modulate drug absorption by inhibition of intestinal P-glycoprotein (P-gp). The aim of the present study was to provide a proof-of-principle for a delivery system based on thiolated chitosan in vivo in rats, using rhodamine-123 (Rho-123) as representative P-gp substrate. In vitro, the permeation enhancing effect of unmodified chitosan, chitosan-4 thiobutylamidine (Ch-TBA) and the combination of Ch-TBA with reduced glutathione (GSH) was evaluated by using freshly excised rat intestinal mucosa mounted in Ussing-type chambers. In comparison to buffer only, Rho-123 transport in presence of 0.5% (w/v) chitosan, 0.5% (w/v) Ch-TBA and the combination of 0.5% (w/v) Ch-TBA/0.5% (w/v) GSH, was 1.8-fold, 2.6-fold, 3.8-fold improved, respectively. Furthermore, enteric-coated tablets based on unmodified chitosan or Ch-TBA/GSH, were investigated in vivo. In rats, the Ch-TBA/GSH tablets increased the area under the plasma concentration time curve (AUC0-12) of Rho-123 by 217% in comparison to buffer control and by 58% in comparison to unmodified chitosan. This in vivo study showed that a delivery system based on thiolated chitosan significantly increased the oral bioavailability of P-gp substrate Rho-123.     

Synthesis and characterization of thermosensitive chitosan copolymer as a novel biomaterial

Novel water-soluble thermosensitive chitosan copolymers were prepared by graft polymerization of N-isopropylacrylamide (NIPAAm) onto chitosan using cerium ammonium nitrate (CAN) as an initiator. The physicochemical properties of the resulting chitosan-g-NIPAAm copolymers were characterized by Fourier transform infrared (FT-IR) spectroscopy, ¹H-nuclear magnetic resonance, X-ray diffraction measurement, thermogravimetric analysis (TGA) and solubility test. Sol–gel transition behavior was investigated by the cloud point measurement of the chitosan-g-NIPAAm aqueous solution. The gelling temperature was examined using the vial inversion method. The percentage of grafting (%) and efficiency of grafting (%) were investigated according to concentrations of monomer and initiator. The maximum grafted chitosan copolymer was obtained with 0.4 M NIPAAm and 6×10^-3 M CAN. Water-soluble chitosan-g-NIPAAm copolymers were prepared successfully and they formed thermally reversible hydrogel, which exhibits a lower critical solution temperature (LCST) around 32°C in aqueous solutions. A preliminary in vitro cell study showed nontoxic and bio- compatible properties. These results suggest that chitosan-g-NIPAAm copolymer could be very useful in biomedical and pharmaceutical applications as an injectable material for cell and drug delivery.     

Synthesis and characterization of thermosensitive chitosan copolymer as a novel biomaterial

Novel water-soluble thermosensitive chitosan copolymers were prepared by graft polymerization of N-isopropylacrylamide (NIPAAm) onto chitosan using cerium ammonium nitrate (CAN) as an initiator. The physicochemical properties of the resulting chitosan-g-NIPAAm copolymers were characterized by Fourier transform infrared (FT-IR) spectroscopy, 1H-nuclear magnetic resonance, X-ray diffraction measurement, thermogravimetric analysis (TGA) and solubility test. Sol-gel transition behavior was investigated by the cloud point measurement of the chitosan-g-NIPAAm aqueous solution. The gelling temperature was examined using the vial inversion method. The percentage of grafting (%) and efficiency of grafting (%) were investigated according to concentrations of monomer and initiator. The maximum grafted chitosan copolymer was obtained with 0.4 M NIPAAm and 6 x 10(-3) M CAN. Water-soluble chitosan-g-NIPAAm copolymers were prepared successfully and they formed thermally reversible hydrogel, which exhibits a lower critical solution temperature (LCST) around 32 degrees C in aqueous solutions. A preliminary in vitro cell study showed nontoxic and biocompatible properties. These results suggest that chitosan-g-NIPAAm copolymer could be very useful in biomedical and pharmaceutical applications as an injectable material for cell and drug delivery.     

In vitro evaluation of a chitosan membrane cross-linked with genipin

The study was to evaluate the characteristics of a chitosan membrane cross-linked with a naturally-occurring cross-linking reagent, genipin. This newly-developed genipin-cross-linked chitosan membrane may be used as an implantable drug-delivery system. The chitosan membrane without cross-linking (fresh) and the glutaraldehyde-cross-linked chitosan membrane were used as controls. The characteristics of test chitosan membranes evaluated were their cross-linking degree, swelling ratio, mechanical properties, antimicrobial activity, cytotoxicity, and degradability. It was found that cross-linking of chitosan membrane using genipin increased its ultimate tensile strength but significantly reduced its strain-at-fracture and swelling ratio. There was no significant difference in antimicrobial activity between the genipin-cross-linked chitosan membrane and its fresh counterpart. Additionally, the results showed that the genipin-cross-linked chitosan membrane had a significantly less cytotoxicity and a slower degradation rate compared to the glutaraldehyde-cross-linked membrane. These results suggested that the genipin-cross-linked chitosan membrane may be a promising carrier for fabricating an implantable drug-delivery system. The drug-release characteristics of the genipin-cross-linked chitosan membrane are currently under investigation.     

壳聚糖基金纳米棒自组装纳米球的制备与表征

目的 通过壳聚糖衍生物表面的巯基与金元素形成非常强的Au-S键,制备表面带有金纳米棒(GNRs)的自组装纳米球CS-GNRs.方法 用透射电子显微镜观察自组装纳米球的形貌特征,动态激光粒度分析仪检测其粒度分布,紫外-可见分光光度计检测其光学特性及其性质改变,同时对其表面增强拉曼光谱(SERS)效应进行检测.结果 该纳米球形态良好、粒径均一、分散性好,并且结晶紫(CV)在壳聚糖纳米金杂化纳米球表面的拉曼增强因子可达2×103.结论 该纳米球在分子检测和拉曼光谱效应检测方面具有潜在的应用价值.     

In vitro evaluation of a chitosan membrane cross-linked with genipin.

The study was to evaluate the characteristics of a chitosan membrane cross-linked with a naturally-occurring cross-linking reagent, genipin. This newly-developed genipin-cross-linked chitosan membrane may be used as an implantable drug-delivery system. The chitosan membrane without cross-linking (fresh) and the glutaraldehyde-cross-linked chitosan membrane were used as controls. The characteristics of test chitosan membranes evaluated were their cross-linking degree, swelling ratio, mechanical properties. antimicrobial activity, cytotoxicity, and degradability. It was found that cross-linking of chitosan membrane using genipin increased its ultimate tensile strength but significantly reduced its strain-at-fracture and swelling ratio. There was no significant difference in antimicrobial activity between the genipin-cross-linked chitosan membrane and its fresh counterpart. Additionally, the results showed that the genipin-cross-linked chitosan membrane had a significantly less cytotoxicity and a slower degradation rate compared to the glutaraldehyde-cross-linked membrane. These results suggested that the genipin-cross-linked chitosan membrane may be a promising carrier for fabricating an implantable drug-delivery system. The drug-release characteristics of the genipin-cross-linked chitosan membrane are currently under investigation.     

Drug permeability and mucoadhesion properties of thiolated trimethyl chitosan nanoparticles in oral insulin delivery

Trimethyl chitosan-cysteine conjugate (TMC-Cys) was synthesized in an attempt to combine the mucoadhesion and the permeation enhancing effects of TMC and thiolated polymers related to different mechanisms for oral absorption. TMC-Cys with various molecular weights (30, 200, and 500 kDa) and quaternization degrees (15 and 30%) was allowed to form polyelectrolyte nanoparticles with insulin through self-assembly, which demonstrated particle size of 100–200 nm, zeta potential of +12 to +18 mV, and high encapsulation efficiency. TMC-Cys/insulin nanoparticles (TMC-Cys NP) showed a 2.1–4.7-fold increase in mucoadhesion compared to TMC/insulin nanoparticles (TMC NP), which might be partly attributed to disulfide formation between TMC-Cys and mucin as evidenced by DSC measurement. Compared to insulin solution and TMC NP, TMC-Cys NP induced increased insulin transport through rat intestine by 3.3–11.7 and 1.7–2.6 folds, promoted Caco-2 cell internalization by 7.5–12.7 and 1.7–3.0 folds, and augmented uptake in Peyer's patches by 14.7–20.9 and 1.7–5.0 folds, respectively. Such results were further confirmed by in vivo experiment with the optimal TMC-Cys NP. Biocompatibility assessment revealed lack of toxicity of TMC-Cys NP. Therefore, self-assembled nanoparticles between TMC-Cys and protein drugs could be an effective and safe oral delivery system.     

Mucoadhesion mechanism of chitosan and thiolated chitosan-poly(isobutyl cyanoacrylate) core-shell nanoparticles

The study is focused on the evaluation of the potential bioadhesive behaviour of chitosan and thiolated chitosan (chitosan-TBA)-coated poly(isobutyl cyanoacrylates) (PIBCA) nanoparticles. Nanoparticles were obtained by radical emulsion polymerisation with chitosan of different molecular weight and with different proportions of chitosan/chitosan-TBA. Mucoadhesion was ex vivo evaluated under static conditions by applying nanoparticle suspensions on rat intestinal mucosal surfaces and evaluating the amount of nanoparticles remaining attached to the mucosa after incubation. The analysis of the results obtained demonstrated that the presence of either chitosan or thiolated chitosan on the PIBCA nanoparticle surface clearly enhanced the mucoadhesion behaviour thanks to non-covalent interactions (ionic interaction and hydrogen bonds) with mucus chains. Both, the molecular weight of chitosan and the proportion of chitosan-TBA in the formulation influenced the nanoparticle hydrodynamic diameter and hence their transport through the mucus layer. Improved interpenetration ability with the mucus chain during the attachment process was suggested for the chitosan of high molecular weight, enhancing the bioadhesiveness of the system. The presence of thiol groups on the nanoparticle surface at high concentration (200 x 10(-6) micromol SH/cm2) increased the mucoadhesion capacity of nanoparticles by forming covalent bonds with the cysteine residues of the mucus glycoproteins.     

Manufacture of multimicrotubule chitosan nerve conduits with novel molds and characterization in vitro

Multimicrotubule chitosan conduits (M-conduits) were fabricated using novel molds and a thermal-induced phase-separation technique. Hollow chitosan conduits (H-conduits) with an inner diameter of 1-5 mm and a wall thickness of 0.2-1.0 mm were made, and then a novel mold composed of a styrofoam insulating pedestal with several holes and a stainless steel cover plate was used to make M-conduits. In brief, corresponding H-conduits were inserted upright into the holes of the styrofoam pedestal, and filled with chitosan solution, then rapidly covered with the precooled stainless steel cover plate, and then placed in a freezer. The styrofoam insulating pedestal enclosing the conduits could reduce the heat transfer through the side wall of the conduits. Gradual phase separation then occurred uniaxially in the presence of a unidirectional temperature gradient from the top end to the bottom end of the chitosan conduits. The phase-separated polymer/solvent systems were then dried in a freeze-dryer. The microtubule diameters were controlled by adjusting the polymer concentration and cooling temperature. In vitro characterization demonstrated that the mold-based multimicrotubule chitosan conduits possessed suitable mechanical strength, microtubule diameter distribution, porosity, swelling, biodegradability, and nerve cell affinity, and so they showed potential for application as nerve tissue engineering scaffolds.     

Synthesis and efficient hepatocyte targeting of galactosylated chitosan as a gene carrier in vitro and in vivo

While chitosan (CS) has been researched widely as a non-viral vector, its usefulness has been limited by its low cell specificity and transfection efficiency. Therefore, we successfully synthesized galactosylated chitosan (GC) and complexed it with an enhanced green fluorescent protein plasmid (pIRES-EGFP) for transfection into cultured H22 cells (murine hepatic cancer cell line) using various GC/EGFP (N/P) charge ratios. Maximal gene transfection rates detected by flow cytometry occurred at an N/P ratio 5:1. Compared with those of lipofectin/EGFP and naked pIRES-EGFP, GC/EGFP complexes show a very efficient cell-selective transfection to hepatocytes. The MTT assay detected relatively low cytotoxicity in cells transfected with GC. A recombinant plasmid granulocyte-macrophage colony-stimulating factor (GM-SCF) and interleukin (IL) 21 (pIRES/GM-CSF-IL21) was successfully constructed and GC/GM-CSF-IL21 nanoparticles (average diameter, 82.1 nm) were administered via the tail vein of mice with liver metastasis of colon cancer model, for 5 consecutive days. The GC/GM-CSF-IL21 nanoparticles exhibited hepatocyte and passive tumor specificity, increased therapeutic efficacy compared to control groups, promoted leukocytes to aggregate in tumor tissues, and activated the cytotoxicity of natural killer (NK) cells and cytolytic T lymphocyte (CTL). Our results indicate that GC can be used in gene therapy to improve transfection efficiency and can be used as an immunological stimulant in vivo.     

一种新型体外血浆脂类吸附过滤器的性能评价

目的对一种新型体外血浆脂类吸附过滤器JX—DELP进行性能评价。方法①通过体外静态吸附实验检验过滤器滤膜对血脂的吸附效果。②模拟过滤器产品结构组成，设计并加工了小型血浆过滤装置。使用该装置模拟临床应用条件，考察了血浆用量、过滤速度和时间对过滤吸附效果的影响；考察了循环过滤方式对高脂血症患者血浆的综合过滤吸附效果。③考察滤膜与全血直接接触条件下材料的血液相容性。结果过滤器的滤膜在体外对血脂的清除率分别为：总胆固醇（TC）16．7％，甘油三酯（TG）9．7％，低密度脂蛋白（LDL）13．5％。在体外循环实验中，在一定的血浆处理体积、过滤速度和时间内，对TC、TG和LDL的清除率分别为52．0％、50．1％和38．4％。血浆其他组分的浓度变化不显著。过滤器滤膜对全血各组分无显著破坏作用，溶血率仅为0．19％，具有良好的血液相容性。结论体外血浆脂类吸附过滤装置，JX—DELP，能快速有效地去除高脂血症患者血浆中含量过高的血脂，而不会对血液其他组分产生破坏作用，同时滤膜本身有很好的血液相容性。该装置可用于降低血脂和血黏度，临床治疗某些高血脂相关疾病。     

In vitro evaluation of a multi-layer radial-flow bioreactor based on galactosylated chitosan nanofiber scaffolds

Clinical use of bioartificial livers (BAL) strongly relies on the development of bioreactors. In this study, we developed a multi-layer radial-flow bioreactor based on galactosylated chitosan nanofiber scaffolds and evaluated its efficacy in vitro. The bioreactor contains 65 layers of stacked flat plates, on which the nanofiber scaffolds were electrospinned for hepatocyte immobilization and aggregation. Culture medium containing pig red blood cells (RBCs) was perfused from the center to periphery, so that exchange materials are sufficient to afford enough oxygen. We determined the parameters for hepatocyte-specific function and general metabolism and also measured the oxygen consumption rate (OCR). Microscope and scanned electron microscopy observation showed a tight adhesion between cells and scaffolds. Compared with the control (bioreactors without nanofiber scaffolds), the number of adhered cells in our bioreactor was 1.59-fold; the protein-synthesis capacity of hepatocytes was 1.73-fold and urea was 2.86-fold. Moreover, the OCR of bioreactors with RBCs was about 1.91-fold that of bioreactors without RBCs. The galactosylated chitosan nanofiber scaffolds introduced into our new bioreactor greatly enhanced cell adhesion and function, and the RBCs added into the culture medium were able to afford enough oxygen for hepatocytes. Importantly, our new bioreactor showed an exciting efficiency, and it may afford the short-term support of patients with hepatic failure.     

In vitro and in vivo evaluation of mucoadhensiveness of chitosan/cellulose acetate multimicrospheres

Chitosan/cellulose acetate multimicrospheres (CCAM) with or without ranitidine (RT) were prepared by the method of W/O/W emulsion with no toxic reagents and had the size interval of 200-280 microm. The angles of repose were only a little more than 30 degrees and the maximum angles of one-plane-critical-stability (OPCS phi) were about 20 degrees . The CCAM had good suspension ability for the tapped density of CCAM was less than 0.127g/mL. The pH value affected the swelling ability of CCAM and the relative humidity had little effect on the characteristics of CCAM when it was not more than 75%. The CCAM system had good effect on the controlled release of RT in vitro and the release rate was almost 60% during 48 h. Furthermore the release of RT was not affected by pH value of release medium. The mucoadhesive tests showed that CCAM could retain in gastrointestinal tract for an extended period of time. There were 53.7% of CCAM remained in stomach after administered for 2(1/2) h and 98.9% of CCAM remained in stomach and small intestine after administered for 3(1/2) h. These results suggest that CCAM is a useful dosage form targeting the gastric mucosa or prolonging gastric residence time as a multiple-unit mucoadhesive system.     

Synthesis and in vitro antimicrobial and antioxidant activities of quaternary ammonium chitosan modified with nisin

Nisin had been grafted onto quaternary ammonium chitosan (QCS) through an enzyme-catalyzed reaction to enhance its limited antimicrobial activity. QCS was synthesized by incorporating N-(3-chloro-2-hydroxypropyl) trimethyl ammonium chloride (CHPTAC) onto chitosan’s primary amine group. The modification had been confirmed by FT-IR and ¹H NMR spectroscopy. Degree of substitution (DS) of QCS–nisin could be controlled by adjusting the reaction conditions. The synthesized compounds were screened in vitro to evaluate their antimicrobial and antioxidant activities. The results suggested that QCS–nisin significantly suppressed the growth of both gram-positive bacteria and gram-negative bacteria; The antioxidant effects on 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical, hydroxyl radical and hydrogen peroxide (H₂O₂) proved to be enhanced with increasing DS and concentration. In addition, QCS–nisin showed excellent moisture absorption and retention properties; MTT assay exhibited that QCS–nisin revealed low cytotoxicity effects on cultured NIH-3T3 fibroblasts. These results suggest that QCS–nisin would appear to be a promising candidate for wound dressing application.     

In vitro evaluation of degradation and cytotoxicity of a novel composite as a bone substitute

The purpose of this study was to prepare and evaluate in vitro the feasibility and cytocompatibility of a novel composite (GGT) as a large defect bone substitute. The composite is tricalcium phosphate ceramic particles combined with genipin crosslinked gelatin. After soaking the GGT composites in Ringer solutions at 37 degrees C for 7, 14, 28, 42, 56, and 84 days, the in vitro biologic degradation rate and biocompatibility were determined. Substances released from soaked GGT composites were analyzed with an ultraviolet visible light spectrophotometer. In addition, the solution soaking the GGT was co-cultured with osteoblasts to determine whether or not the released substances from GGT could facilitate the growth of bone cells. After they had been cultured for 2 days, the osteoblasts were tested for differentiation and proliferation by alkaline phosphatase (ALP) activity and a MTT assay. Results indicate that the concentration of the genipin solution is a critical factor in deciding the crosslinking degree of the GGT composite. Complete crosslinking reaction in the GGT composite occurred when 0.5 wt % of genipin had been added. Cytotoxic testing revealed that 80 ppm of the genipin in the culture medium served as the level over which cytotoxicity to osteoblasts could be produced. In addition, we found that gelatin and calcium continuously were released from the GGT composite in the soaking solution, which promoted differentiation and proliferation of the osteoblasts.     

A novel osteotropic biomaterial OG-PLG: Synthesis and in vitro release

Statins (e.g., simvastatin) have shown to induce expression of the bone morphogenic protein-2 gene in bone cells, but they are not used clinically because of a lack of a suitable delivery device. The overall objective is to develop optimized statin delivery devices for bone regeneration. The specific objective was to determine the effect of grafting statins to biodegradable poly[lactide-co-glycolide] (PLG) on release kinetics. Simvastatin was grafted to PLG (OG-PLG) and characterized using contact-angle measurements, attenuated total reflectance-Fourier transform infrared, and ultraviolet-visible spectroscopy to determine success of the synthesis. An ultraviolet-visible assay for measuring release of statins and degraded OG-PLG in media was also developed. In vitro release studies using films and scaffolds made with PLG, PLG blended with simvastatin (PLG + Sim), and OG-PLG (simvastatin grafted to PLG) blended into PLG at different concentrations showed that release rate of OG-PLG from films was significantly greater than that of PLG + Sim. However, release rate from scaffolds showed PLG + Sim to be significantly higher than that of OG-PLG. The diffusion-controlled release kinetics of simvastatin from PLG + Sim seems to be more heavily affected by device morphology, whereas the degradation-controlled release kinetics seem to be less affected. In short, release kinetics can be modulated by grafting statins to PLG.     

新型壳聚糖对血浆胆红素和细胞因子吸附性能的体外筛选初步研究

目的观察各类新型吸附剂对重型肝炎患者血浆中的胆红素和细胞因子的吸附性能。方法第一步,收集第一组重型肝炎患者的血浆各3ml,以8种不同的吸附剂(1#～3#为致孔剂浓度分别为3%、1%、5%的壳聚糖,4#为胺基化壳聚糖,5#为苯乙烯/二乙烯苯聚合物,6#为后交连苯乙烯/二乙烯苯聚合物,7#为壳聚糖-苯乙烯/二乙烯苯聚合物,8#为壳聚糖-后交连苯乙烯/二乙烯苯聚合物)各1ml进行吸附,检测对胆红素的吸附能力,做吸附剂筛选实验和吸附实验。第二步,用以上筛选出吸附率较好的两种吸附剂各1ml对第二组患者血浆(各3ml)进行胆红素和细胞因子的吸附实验。结果第一步实验显示胺基壳聚糖(4#)和苯乙烯/二乙烯苯聚合物(5#)有较好的吸附效果,4#吸附剂对总胆红素(TBiL)、直接胆红素(DBiL)、间接胆红素(IBiL)的吸附率分别为50.5%±3.4%、57.0%±11.3%、39.0%±7.2%;5#分别为30.1%±2.5%、32.6%±3.0%、27.6%±2.9%。第二步实验显示4#胺基化壳聚糖吸附后血浆中TBiL、DBiL、IBiL、白细胞介素6(IL-6)和肿瘤坏死因子α(TNF-α)水平下降比5#明显,有统计学意义(P<0.001)。结论胺基化壳聚糖(4#)体外吸附重型肝炎患者血浆中胆红素、细胞因子效果有明显优势。     

Characterization and ornidazole release in vitro of a novel composite film prepared with chitosan/poly(vinyl alcohol)/alginate

To create a moist environment for rapid wound healing, a new C-P-A film with sustained antibacterial capacity had been developed by the casting/solvent evaporation method. This new type of C-P-A film consists of a chitosan top layer and sodium alginate sublayer separated by an ornidazole-incorporated poly(vinyl alcohol) layer, exhibited perfect binding characteristics among the three layers. Physical characterization of the C-P-A film showed that the triple-layerd film had excellent light transmittance, control of water vapor transmission rate, and fluid drainage ability promotion, compared with the single-layer film. From the in vitro release studies, about 90% of OD was released from the composite films within 60 min, and no significant difference was observed in cumulative release percentage with increases in the drug content. The composite film at low concentration of OD (1.0 mg/cm2) showed effective antimicrobial activity in the cultures of Staphylococcus aureus and Escherichia coli in agar plates. The results obtained in this work indicated that the new type of C-P-A composite film incorporated with ornidazole has the potential for wound dressing application.     

Characterization and evaluation of chitosan matrix for in vitro growth of MCF-7 breast cancer cell lines

Biodegradable polymer scaffolds were prepared from chitosan with varying degree of deacetylation for in vitro culture of human breast cancer MCF-7 cell lines. These polymers were characterized in terms of functional groups by FTIR and swelling properties. Polymers having high degree of deacetylation showed better swelling properties irrespective of the molecular weight. These polymers were biocompatible and non-toxic towards human epithelial MCF-7 cell lines. Attachment kinetics of MCF-7 cell lines on to polymer scaffold was investigated and it was observed that polymer having high degree of deacetylation favored better cell attachment. In CPIII polymer scaffold having 80% degree of deacetylation, a maximum of 1 millions cells per mg pf polymer were adsorbed within 1h. It appears that high swelling and high degree of deacetylation of chitosan helped in better adsorption of cancer cell lines. The cellular morphology of the attached cells on chitosan matrix was similar to that observed with regular plastic culture with the difference that, cells grew as three-dimensional clumps on chitosan matrix. Polymer having high degree of deacetylation not only favored better adsorption but also showed improved cell growth kinetics. Maximum cell concentration of 6.5 x 10(5) cells/ml was achieved in 5 days culture on CPIII polymer scaffold. The glucose consumption and lactate production pattern of the MCF-7 cell lines on chitosan polymer matrix were similar to that observed on cell growth on tissue culture flask. These results indicate that chitosan scaffold having high degree of deacetylation can be used for three-dimensional growth of MCF-7 cancer cell lines. Such in vitro 3D culture of cancer cells can thus be used as a model for the cytotoxic evaluation of anticancer drugs.