毕业论文外文翻译-壳聚糖-茶多酚纳米颗粒的合成和性质及细胞毒性的研究.doc

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1、 Synthesis, characterization and cytotoxicity studies of chitosan-coated tea polyphenols nanoparticlesa b s t r a c t：Chitosan nanoparticles (CS-NPs) were prepared by ionic gelation method using carboxymethyl chitosan and chitosan hydrochloride as carriers of tea polyphenols. The characteristics of

2、chitosan-coated tea polyphenols nanoparticles (CS-TP NPs) were determined by using transmission electron microscopy (TEM) and FT-IR spectroscopy. It was found that the synthesized CS-TP NPs were non-spherical in shape with an average size of 40750 nm. Meanwhile, the drug content and encapsulation ra

3、te of the nanoparticles was 816% and 4483%, respectively. These CS-TP NPs also demonstrated sustained release of tea polyphenols in PBS. The antitumor of CS-TP NPs towards HepG2 cancer cells was investigated. The result showed that CS-TP NPs retained significant antitumor activities.1. IntroductionN

4、owadays, cancer is the major public health problem and the existing treatment approaches and surgical techniques have not been able to cope effectively with this dreaded disease. Because of this, chemoprevention is a valid approach to reduce the incidence of cancer 1. As an effective cancer chemopre

5、ventive agent, tea polyphenols are known to be strong antioxidants and anticarcinogenic activities 2. In vitro and animal studies provide strong evidence that polyphenols derived from tea may possess the bioactivity to inhibit tumorigenesis in a variety of animal models of carcinogenesis 3. Tea poly

6、phenols has been shown to inhibit the development of cancer in animal models of oral, esophageal, forestomach, stomach, intestinal, colon, skin, liver,bladder, prostate, and breast cancer 4,5. Although tea polyphenols are widely used for the prevention and treatment of cancer, its therapeutic effect

7、s are always limited by severe adverse effects, such as the stability of biological activity in tissue and bioavailability in vivo, etc. 6. There have been reported that the biological activity of tea polyphenols might depend on the form of their administration7. To overcome these disadvantages and

8、improve chemotherapeutic activity, researchers have focused on the development of nano-sized drug carriers 810. Nanoparticles, with highly con-trolled shapes, sizes, have been studied extensively as drug carrierswhich can improve the bioavailability of drug with poor absorption characteristics 11. N

9、anoparticles also can be able to overcome biological barriers, accumulate preferentially in tumors and specifically recognise single cancer cells for detection and treatment. Therefore, nanoparticles also could be as an effective delivery system for improving tea polyphenols bioavailability and anti

10、cancer effect.Chitosan is a favorable type of drug deliver system. Chitosan is a biodegradable polysaccharide derived by partial deacetylation of chitin, which is a copolymer of glucosamine and N-acetyl-d-glucosamine linked together by (1,4) glycosidic bonds 12. Chitosan has been widely used in phar

11、maceutical and medical areas, due to its favorable biological properties such as biodegradability, biocompatibility, low toxicity, hemostatic, bacteriostatic, fungistatic, anticancerogen, and anticholesteremic properties 13. Because of its chemical structure, chitosan and its derivative have been in

12、vestigated in the development of controlled release drug delivery systems, since chitosans mucoadhesive property can enhance drug transmucosal absorption and promote sustained release of drug 14,15. Carboxymethyl chitosan and chitosan hydrochloride are two different water-soluble chitosan with anion

13、ic and cationic respectively. They can form nanoparticles through ionic gelation between the carboxyl groups of carboxymethyl chitosan and the amine groups of chitosan hydrochloride in aqueous solution. The encapsulant prepared as a novel nano-scale carrier has biocompatible and biodegradable charac

14、teristics, and also can limit the release of encapsulated materials more effectively 16. It is also thought to contribute to longer in vivo circulation times and allow encapsulation of water-soluble biomolecules 17.In this study, we prepared one kind of novel chitosan nanoparticles complexation usin

15、g carboxymethyl chitosan and chitosan hydrochloride as encapsulant materials for entrapment of teapolyphenols. The morphology, structure and characteristic of the CS-TP NPs were studied by DLS, TEM and FTIR. The application of chitosan nanoparticles as carriers of tea polyphenols was evaluated by me

16、asuring its drug content, encapsulation rate and cytotoxicity in vitro.2. Materials and methods2.1. MaterialsTea polyphenols with 93% purity were obtained from the green tea by extraction with 50% ethanol and then purification with H1020 resins (Nankai University chemical plant, Tianjin, China). Nca

17、rboxymethyl chitosan (Mv = 61 kDa, degree of deacetylation 83%) and chitosan hydrochloride (Mv = 90 kDa, degree of deacetylation 85%) were purchased from Haidebei Marine Bioengineering Company (Jinan, Shandong, China). HepG2 cells were obtained from Nanjing University (Nanjing, Jiangsu, China). Othe

18、r reagents were of analytical grade.2.2. Preparation of CS-TP NPsCS-TP NPs were prepared by an ionic interaction method, performed according to the following procedure 18: Carboxymethyl chitosan and chitosan hydrochloride were dissolved in distilled water with sonication until the solution was trans

19、parent. The aqueous solution of carboxymethyl chitosan and chitosan hydrochloride were obtained at a concentration of 3.0mg/mL and 1.2 mg/mL, and solution pH about 8.5 and 3.3, respectively. A certain amount of tea polyphenols was added into chitosan hydrochloride solution. As a consequence of the a

20、ddition of carboxymethyl chitosan solution (12 mL) was dropped slowly into tea polyphenols and chitosan hydrochloride mixture solution (30 mL) with stirring at room temperature, and continuous stirring for 30 min. The formation of nanoparticles started spontaneously via the ionic gelation mechanism.

21、 The nanoparticles suspensions were immediately subjected to further analysis and applications. The non-loaded nanoparticles without tea polyphenols were also prepared as control.2.3. Characterizations of CS-TP NPsDynamic light scattering (DLS) (Brookhaven Instruments Corporation, Holtsville, NY) wa

22、s used to measure the average particle size. Zeta potentialwasperformed on a Zeta sizer Nano-ZS (Malvern Instruments, England, UK) on the basis of DLS techniques. All measurements were performed in triplicate. The morphology of the CS-TP NPs were observed by TEM (JEOL H-7650, Hitachi High-Technologi

23、es Corporation, Tokyo, Japan). The sample for TEM analysis was obtained by placing a drop of the CS-TP NPs dispersed aqueous solution onto a copper micro-grid and evaporated in air at room temperature. FTIR spectroscopy of nanoparticles was obtained by using a FTIR spectrophotometer (FT-IR200, Nicol

24、et,USA).2.4. Determination of drug content and entrapment efficiencyCS-TP NPs suspensions were centrifuged at 15,000rpm at 4 C for 30 min. The free tea polyphenols in the clear supernatant was determined in triplicate by the tea polyphenols colorimetric assay method described below 19.The clear supe

25、rnatant was transferred into 50mL volumetric flask and achieved to the fixed scale with distilled water. To determine tea polyphenols concentration of the sample, tea polyphenols aqueous solution 5mL was taken from the sample and added into a 25mL volumetric flask, then 5mL ferrous tartrate tetrahyd

26、rate solution (containing 1 g ferrous sulfate and 5 g potassium sodium tartrate tetrahydrate dissolved in 1000mL water) was also added into the volumetric flask and made up to 25mL with potassium phosphate buffer solution (pH 7.5). Several minutes were required for the color to develop. With a blank

27、 solution (without tea polyphenols) as reference solution, the absorbance at 540nm in a 10mm quartz cell was used to calculate the amount of free tea polyphenols according to the prepared standard equation of tea polyphenols solution (y = 0.0322x0.0156, R2 = 0.9993). The drug content and entrapment

28、efficiency of CS-TP NPs was calculated by the following formulas:A = total amount of tea polyphenols in added solution; B = total amount of tea polyphenols in supernatant after ultrafiltration; and C =weight of the nanoparticles measured after freeze-drying.2.5. In vitro drug release experimentRelea

29、se rate measurements in vitro were carried out as follows20: 5mL of CS-TP NPs solution (10mg/mL in a dialysis tube) was placed in 20mL of phosphate-buffered saline solution (PBS) at pH 7.4 and incubated shaker operated at 120rpm at 37 C. At predetermined time intervals, 2mL medium was removed and re

30、placed by 2mL fresh PBS to maintain sink conditions. The amount of tea polyphenols in the solution was determined by UV spectrometry at 540 nm. Calibration curves were made with the incubation medium. All measurements were performed in triplicate.2.6. Cell cultureHuman hepatoma HepG2 cells were cult

31、ured in Dulbeccos modified Eagles medium (DMEM) equilibrated with 5% CO2 and 95% air at 37 C. The medium was supplemented with 10% fetal calf serum (FCS), 50 mg/L streptomycin and 75 mg/L penicillin sulfate.2.7. Assay of in vitro cytotoxicityTheMTT3-(4,5-dimethylthazol-2-yl)-2,5-diphenyltetrazolium

32、bromide blue-indicator dye-based assay is a simple nonradioactive colorimetric assay to measure cell cytotoxicity, proliferation or viability. HepG2 cells were used for the analysis of cytotoxicity in vitro. The cells (1105 cells/mL) were placed into 96-well tissue-culture plates and incubated at 37

33、 C. After 24 h, cells were treated with three different concentrations of CS-TP NPs (1 mg/mL, 0.5 mg/mL, 0.25 mg/mL, respectively) and CS-NPs (1 mg/mL), respectively. Untreated cells were used as controls. Plates were incubated in a humidified 5% CO2 balanced-air incubator at 37 C for 24 h, 48 h and

34、 72 h, respectively. Then 10L of 5mg/mL MTT solution was added to each well and the plates were incubated for another 4 h, and then the medium was discarded. Dimethyl sulfoxide (100L) was added to each well, and the solution was vigorously mixed to dissolve tetrazolium dye. The absorbance of each we

35、ll was measured by enzyme-linked immunosorbent assay reader (BioTek; Austria) at a test wavelength of 570nm and the cell apoptosis rate was calculated by the following equation:where C is the number of viable cells after 24 h, 48 h and 72 h of incubation without nanoparticles and T is the number of

36、viable cells after 24 h, 48 h and 72 h of incubation with nanoparticles.Fig. 1. (a) Particle size distribution by intensity of CS-TP NPs and (b) Transmissionelectron micrograph of CS-TP NPs at pH 6.13. Results and discussion3.1. Particle size, morphology and zeta potential measurementsNanoparticles

37、of chitosan-coated tea polyphenols were synthesized by ionic cross-linking between the positively charged amine of chitosan hydrochloride and negatively charged carboxyl groups of carboxymethyl chitosan. Using TEM and DLS measurements, the morphology and size distributions for the CS-TP NPs were obt

38、ained. Fig. 1 shows the morphology and size distribution for the nanoparticles. The self-aggregated nanoparticles are non-spherical and roughly irregular shape and themeandiameter is about 407nm determined by the DLS. Zeta potential was an important parameter to reflect the physicochemical and biolo

39、gical stability of nanoparticles in suspension. Surface charge and thereby the stability of the prepared nanoparticles systems was determined by zeta potential measurements. Zeta potential values for the CS-TP NPs were found to be +26.1mV at pH 6.1. It indicated that the nanoparticles systems prepar

40、ed were relatively stable condition. The CS-TP NPs with positive surface charge showed that this delivery system has mucoadhesive potential and absorption enhancement properties.Table 1The loading characterizations of CS-TP NPs.样品物料比（CCS:CSH:TP)载药量（%）包封率（%）3:3:112-1680-832:2:18-1044-50Fig. 2. FT-IR

41、spectra of (a) tea polyphenols, (b) carboxymethyl chitosan, (c) chitosanhydrochloride, and (d) CS-TP NPs.3.2. FT-IR studiesThe FTIR spectra of pure tea polyphenols, carboxymethyl chitosan, chitosan hydrochloride, and CS-TP NPs were presented in Fig. 2. The basic characteristics of tea polyphenols (F

42、ig. 2a) at: 3398cm1 (strong and wide OH stretch), 16001450cm1 (C C stretch), 1241cm1 (CO stretch). However, The FTIR of tea polyphenols showed a sharper peak at 1700cm1, which was not observed in the CS-TP NPs. This peak was found to overlap with the peaks in a broad band ranging from 1700 to 1550cm

43、1. This indicated interaction between the hydroxyl groups of the polyphenols and the amine functionality of the chitosan molecule 20,21. In Fig. 2b, the IR of carboxymethyl chitosan at 1609cm1 and 1416cm1 had two strong peaks, which were observed due to the asymmetrical and symmetrical stretching of

44、 COO group 22. Corresponding, the typical absorption peak of the nanoparticles (Fig. 2d) carboxylic group was still present at 1400cm1. Comparing Fig. 2c and d, the peak at 1077cm1 and 1155cm1 in the spectrum of chitosan hydrochloride were appeared and this can be attributed to the COC and CH stretc

45、hing, where as in the nanoparticle this peak also appeared at 1065cm1 and 1151cm1. In the chitosan hydrochloride spectrum, a peak at 1639cm1 was appeared and this can be attributed to the amine stretching 23, where as in the nanoparticles a weak peak appeared at 1636cm1. The peak at 1545cm1 in the s

46、pectrum of the nanoparticles was indicative of interaction between the carboxylic groups of carboxymethyl chitosan and amino groups of chitosan hydrochloride. It was in agreement with the reported spectra 24,25. The appearing of these peaks was an indication of nanoparticles formation3.3. Drug conte

47、nt and Entrapment efficiency of CS-TP NPsThe drug content and encapsulation efficiency were determined by varying the feed weight ratio of carboxymethyl chitosan, chitosan hydrochloride and tea polyphenols particles. The loading characteristics of two different CS-TP NPs samples were summarized in T

48、able 1. When the feed weight ratio was 3:3:1 (sample I), the drug content was above 12%. However, if the feed ratio was 2:2:1 (sample II), the drug content slightly decreased to 8% and meanwhile the excess of tea polyphenols could not totally be loaded into chitosan (encapsulation rate decreased fro

49、m 80% to about 44%). The reason could be, under the condition of maintaining constant amount of drug, the shielding effect of chitosan skeleton on drug became stronger with the increasing amount of chitosan. Moreover, there was a possibility of polyphenol sorption on chitosan that affected on efficiency of drug loading in CS-TP NPs system. Kosaraju et al. 21 reported that the