Preparation,Characterization and Protein Loading of Hexanoyl-Modified Chitosan Nanoparticles

Hexanoyl chitosan was synthesized through a coupling reaction between chitosan and hexanoic anhydride. Proton nuclear magnetic resonance (¹HNMR) and fourier-transform infrared (FTIR) spectroscopy studies showed the formation of hexanoyl chitosan. The nanoparticles of hexanoyl chitosan were prepared through ionotropic gelation with tripolyphosphate (TPP) followed by sonication. The hexanoyl chitosan-TPP nanoparticles exhibited uniform spherical shape with smooth surface as observed by atomic force microscopy and transmission electron microscopy. The particle size of nanoparticles was between 54.1 to 724 nm with a mean diameter of 324 nm. At 0.2, 0.4, and 0.6 mg/mL bovine serum albumin initial concentration, the encapsulation efficiency and loading capacity of hexanoyl-chitosan-TPP nanoparticles were 58.2, 44.5, and 28.1%, and 14.1, 23.4, and 30.3%, respectively.     

载基因壳聚糖纳米粒的制备及其特性的研究

目的制备壳聚糖纳米粒,并连接上质粒,研究壳聚糖纳米粒的特性及其对DNA的结合及保护能力。方法采用离子交联法制备壳聚糖纳米粒,并用喷金扫描电子显微镜检测,了解粒径的分布与形态;通过静电吸附作用连接上pGenesil-1质粒(报告基因);经琼脂糖凝胶电泳分析壳聚糖纳米载体与质粒DNA的结合能力,及不同pH值的壳聚糖纳米粒对质粒DNA的结合能力;并通过DnaseⅠ消化壳聚糖纳米-质粒结合物以观察壳聚糖纳米载体对质粒的保护作用。结果喷金扫描电镜检测证实壳聚糖纳米粒呈均匀分散的球形颗粒,平均直径为5nm;琼脂糖凝胶电泳的结果显示壳聚糖纳米粒能有效地结合载体pGenesil-1质粒;不同pH值的壳聚糖纳米粒对质粒的保护作用不同,当pH值<7时壳聚糖纳米载体能100%结合质粒;DnaseⅠ消化试验证实壳聚糖纳米载体对质粒DNA有保护作用。结论采用离子交联法制备出粒径较小、均匀的壳聚糖纳米粒,并且壳聚糖纳米粒能有效地连接上质粒并对其有保护作用。     

Preparation and in vitro evaluation of bFGF-loaded chitosan nanoparticles

The objective of our study was to prepare and characterize basic fibroblast growth factor (bFGF)-loaded nanoparticles. Protein-loaded chitosan nanoparticles were obtained by ionotropic gelation process based on the interaction between chitosan and tripolyphosphate (TPP). The protein-loading capacity and encapsulation efficiency were 0.021% and 27.388%, respectively. The bFGF-loaded nanoparticles have a mean diameter of 424 nm, a narrow size distribution, spherical shape and positive surface charges. In vitro release showed that the extent of release was 68% at 24 hr. The protein integrity was investigated by SDS-PAGE analysis that confirmed protein integrity was not affected by the encapsulation procedure and release conditions.     

曲尼司特的壳聚糖纳米粒的制备和体外释放研究

目的：制备载有曲尼司特的壳聚糖纳米粒,考察它的性质和体外释放。方法：用多聚磷酸钠（TPP—Na）做交联剂,采用离子交联法制备的曲尼司特壳聚糖纳米粒。测定纳米粒的大小和电位,并考察纳米粒溶液的稳定性和纳米粒的体外释放。结果：制备了粒径为285．5nm的米粒,而且多分散指数为0．04,药物的包封率为82.4％。结论：壳聚糖可用作制备曲尼司特纳米粒的载体,制备的载药纳米粒有可能开发成注射剂。     

壳聚糖纳米粒Zeta电位与载药量关系初探

目的以壳聚糖作为载体材料、冬凌草甲素为模型药物,制备载药纳米粒,研究载药纳米粒Zeta电位与载药量的关系。方法采用离子交联法在系列pH下制备出不同Zeta电位的冬凌草甲素-壳聚糖纳米粒(Ori-CS-NPs)。测量粒径分布、多分散性和Zeta电位,用HPLC测定载药量,对数据进行回归分析。结果初步得出了ORI-CS-NPs(粒径242.01±11.45nm,PDI<0.3)的Zeta电位随pH升高而降低,载药量随Zeta电位的增高而降低。结论采用离子交联法在不同pH下可制备出粒径分布均匀、Zeta电位及载药量呈一定规律变化的载药纳米粒;纳米粒的Zeta电位与载药量呈线性关系。     

壳聚糖及其衍生物纳米粒的制备以及在疫苗中的应用

壳聚糖是一种高分子线性阳离子多糖。由壳聚糖及其化学改性衍生物制备的纳米粒具有生物相容性好、细胞毒性低以及可降解等特点,人们对其作为佐剂或递送系统在疫苗中的应用已开展了广泛研究。此文对壳聚糖及其衍生物纳米粒的制备方法以及在疫苗中的应用进行综述。     

Preparation,characterization, and antibacterial activity of diclofenac-loaded chitosan nanoparticles

Emerging antibiotic resistance necessitates the development of new therapeutic approaches. Many studies have reported the antimicrobial activity of diclofenac sodium (DIC) and chitosan nanoparticles (CNPs). Hence, this study aimed to prepare non-antibiotic DIC-loaded CNPs (DIC.CNPs) and characterize their in vitro antibacterial activity. DIC.CNPs were prepared from low and high molecular weight (LMW and HMW, respectively) chitosan using an ionic gelation method. Prepared NPs were characterized, and their antibacterial activity against gram-positive Staphylococcus aureus and Bacillus subtilis was evaluated using the agar diffusion and broth dilution methods. The particle size, polydispersity index (PDI), and encapsulation efficiency of the formulated DIC.CNPs increased with increasing MW of chitosan. The prepared NPs showed a narrow size distribution with low PDI values (0.18 and 0.24) and encapsulation efficiency (29.3% and 31.1%) for LMW.DIC.CNPs and HMW.DIC.CNPs, respectively. The in vitro release profile of DIC from the DIC.CNPs was biphasic with a burst release followed by slow release and was influenced by the MW of chitosan. DIC.CNPs exhibited significantly higher antibacterial activity against S. aureus (minimum inhibitory concentration [MIC₉₀] _LMW.DIC.CNPs?=?35?µg/mL and MIC_{90 HMW.DIC.CNPs}?=?18?µg/mL) and B. subtilis (MIC_{90 LMW.DIC.CNPs}?=?17.5?µg/mL and MIC_{90 HMW.DIC.CNPs}?=?9?µg/mL) than DIC alone did (MIC_{90 DIC}?=?250 and 50?µg/mL against S. aureus and B. subtilis, respectively). The antibacterial activity was influenced by pH and the MW of chitosan. Collectively, these results may suggest the potential usefulness of DIC.CNPs as non-antibiotic antibacterial agent necessitating further future studies to asses the stability of DIC.CNPs prepared.     

壳聚糖-岩藻聚糖硫酸酯纳米粒的制备及性质研究

目的：本文以低分子量岩藻聚糖硫酸酯为交联剂,研究了聚电解质凝聚法制备壳聚糖-岩藻聚糖硫酸酯纳米微粒(Chitosan-fucoidan nanoparticles, CS-Fuc NPs)的制备工艺,并对纳米粒的胃肠道和贮藏稳定性进行研究。方法：采用聚电解质凝聚法制备壳聚糖-岩藻聚糖硫酸酯纳米微粒,采用体外模拟胃液和模拟肠液消化体系,检测纳米粒的胃肠稳定性,常温贮藏实验测定纳米粒的短期贮藏稳定性。结果：壳聚糖-岩藻聚糖硫酸酯纳米微粒的最优制备条件是：壳聚糖(1mg/ml)与岩藻聚糖硫酸酯(1mg/ml)体积比为1.1/1,pH 4.5,温度30℃。所得纳米粒粒径为227.8 nm,Zeta电位为38.4 mV,PDI为0.231,岩藻聚糖硫酸酯复合率(Fuc%)为94.92%。所制备的纳米颗粒在10周内没有显著变化,在模拟胃环境中稳定性良好,在模拟肠道环境中解聚性良好。结论： 壳聚糖-岩藻聚糖硫酸酯纳米粒制备工艺简单,性能良好,有望成为新型的口服药物运送载体。     

阿昔洛韦眼用壳聚糖纳米粒的制备及家兔生物利用度研究

目的:应用离子交联法制备阿昔洛韦壳聚糖纳米粒,考察其体外性质及其经家兔眼部给药后的生物利用度.方法:壳聚糖与三聚磷酸钠通过离子交联作用制备纳米粒,考察了纳米粒的粒径、Zeta电位、包封率以及体外释放性质,通过家兔眼部结膜囊内给药,考察眼房水中药物浓度的变化,并与市售阿昔洛韦滴眼液相比较.结果:阿昔洛韦壳聚糖纳米粒的平均粒径为235 nm,多分散系数为0.256,Zeta电位为43.9 mV;平均包封率为15.6%,平均载药量为1.9%;家兔眼部给药后,AUC0→6 h达到3.69μg·h-1·mL-1,是市售制剂的2.4倍.结论:实验初步证实制备的壳聚糖纳米粒可以促进阿昔洛韦的眼部吸收.     

Cholic acid modified N-(2-hydroxy)-propyl-3-trimethylammonium chitosan chloride for superoxide dismutase delivery

A series of novel amphiphilic chitosan derivatives, cholic acid modified N-(2-hydroxy)-propyl-3-trimethylammonium chitosan chloride (HTCC-CA) with different quaternization degrees and cholic acid substitutions were synthesized in this study. HTCC-CA is biocompatible and forms particles in aqueous solution. The binding with superoxide dismutase (SOD) at pH 6.8 destroys the original aggregates of HTCC-CA and produces smaller SOD/HTCC-CA complex nanoparticles via electrostatic and hydrophobic interactions. The SOD loading efficiency and loading capacity of HTCC-CA can reach to more than 90% and 45%, respectively. Confocal laser scanning microscopy observation and flow cytometry analysis reveal that SOD/HTCC-CA complex nanoparticles greatly enhance the cellular internalization of the loaded SOD. The SOD activities and malonaldehyde concentrations in the serum and organs of the rats, administrated intravenously with free SOD, free HTCC-CA, and SOD/HTCC-CA nanoparticles, were assayed to evaluate the antioxidant efficiency in vivo. The results demonstrate that free HTCC-CA is effective to scavenge superoxide radicals in the blood circulation and SOD/HTCC-CA nanoparticles have better antioxidant efficiency than free SOD as well as free HTCC-CA.     

Evaluation and modification of N-trimethyl chitosan chloride nanoparticles as protein carriers

N-Trimethyl chitosan chloride (TMC) nanoparticles were prepared by ionic crosslinking of TMC with tripolyphosphate (TPP). Two model proteins with different pI values, bovine serum albumin (BSA, pI=4.8) and bovine hemoglobin (BHb, pI=6.8), were used to investigate the loading and release features of the TMC nanoparticles. TMC samples with different degrees of quaternization were synthesized to evaluate its influence on the physicochemical properties and release profiles of the nanoparticles. Sodium alginate was used to modify the TMC nanoparticles to reduce burst release. The results indicated that the TMC nanoparticles had a high loading efficiency (95%) for BSA but a low one (30%) for BHb. The particle size and zeta potential were significantly affected by the BSA concentration but not by the BHb concentration. Nanoparticles of TMC with a lower degree of quaternization showed an increase in particle size, a decrease in zeta potential and a slower drug-release profile. As for the alginate-modified nanoparticles, a smaller size and lower zeta potential were observed and the burst release of BSA was reduced. These studies demonstrated that TMC nanoparticles are potential protein carriers, and that their physicochemical properties and release profile could be optimized by means of various modifications.     

Mannose receptor targeted bioadhesive chitosan nanoparticles of clofazimine for effective therapy of tuberculosis

Drug-resistant tuberculosis (TB) is one of the most lethal diseases, and it is imperative to exploit an advanced drug formulation for its effective treatment. This work aims to develop a mannose receptor-targeted bioadhesive chitosan nanoparticles for effective drug-resistant tuberculosis treatment. The clofazimine loaded chitosan nanoparticles were formulated; their size, charge, polydispersity (PDI), surface morphology, entrapment efficiency (EE) and in-vitro release pattern were established. Also, cellular uptake study on C2C12 cell lines and anti-mycobacterial activity against H37Rv (a standard strain of Mycobacterium tuberculosis) were evaluated. The particle sizes of formulated chitosan nanoparticles were in the range of 132–184 nm and EE was also found to be between 73 and 95%. The functionalization of bioadhesive chitosan nanoparticles with mannose was confirmed by infrared spectroscopy (FTIR). The uptake studies on the C2C12 cell lines showed that mannosylated nanoparticles were more efficiently internalized when compared to non-targeted nanoparticles. Further, luciferase reporter phage (LRP) assay against H37Rv strain showed that clofazimine nanoparticles were found to be 49.5 times superior in terms of inhibition and anti-mycobacterial activity than free clofazimine. This excellent activity might be attributed to enhanced drug delivery with a promising bioadhesion property of chitosan-based nanoparticles.     

Preparation,characterization and biodistribution of ultrafine chitosan nanoparticles

Chitosan nanoparticles cross-linked with glutaraldehyde have been prepared in AOT/n hexane reverse micellar system. The cross-linking in the polymeric network has been confirmed from FTIR data. Because of the adhesive nature of these particles, their sizes, as measured by QELS, have been found dependent on the particle density in aqueous buffer. The particle size has also been found to vary with the amount of cross-linking. The actual particle size of these chitosan nanoparticles with a particular degree of cross-linking has been determined at infinite dilution of particles in water. The particle size at infinite dilution is approximately 30 nm diameter, when 10% of the amine groups in the polymeric chains have been cross-linked and it shoots up to 110 nm diameter when all the amine groups are cross-linked (100% cross-linked). TEM pictures show that these particles are spherical in shape and remain in the form of aggregation. The biodistribution of these particles after intravenous injections in mice showed that these particles readily evade the RES system and remain in the blood for a considerable amount of time. The gamma image of the rabbit after administration of (99m)Technetium (99mTc) tagged chitosan nanoparticles also confirms the above observation, as the blood pool is readily visible even after 2 h. The gamma picture shows distribution of particles in the heart, liver, kidneys, bladder and the vertebral column. Interestingly, the biodistribution studies of the chitosan nanoparticles have indicated that these particles are distributed in the bone marrow also, implying the possibility of using these nanoparticles for bone imaging and targeting purpose.     

Overcoming the protein corona in chitosan-based nanoparticles

Numerous properties of chitosan have led to its extensive use in the formulation of nanomaterials for drug delivery. However, the cationic surface of chitosan-based nanoparticles adsorbs proteins upon exposure to biological fluids, forming a phenomenon known as ‘protein corona’. This causes several effects such as decreased bioavailability and limited in vivo clinical applications of chitosan nanoparticles. Understanding and overcoming the effects of protein adsorption on chitosan nanoparticles is key for drug delivery purposes. This review focuses on the strategies implemented to increase the stability of chitosan nanoparticles in the systemic circulation by averting the formation of protein corona and the limitations of PEGylation.     

Preparation of N,O-carboxymethyl chitosan nanoparticles as an insulin carrier

The aim of this research was to develop pH-sensitive insulin-loaded NOCC (N,O-carboxymethyl chitosan) nanoparticles for the controlled release of insulin via the oral route. Thus, in this study, insulin-loaded NOCC nanoparticles were prepared by ionic gelation of NOCC with TPP (tripolyphosphate). NOCC nanoparticles were formed at conditions of 2?mg/ml of NOCC and 1?mg/ml of TPP. It was found that the encapsulation efficiency and process yield decreased with increasing NOCC to TPP weight ratio. Furthermore, the cumulative release of insulin from insulin-loaded NOCC nanoparticles decreased with decreasing NOCC-to-TPP weight ratio, but it increased with decreasing the initial concentration of insulin. The higher the pH of the phosphate buffered saline, the greater the amount of cumulative release of insulin-loaded NOCC nanoparticles, and thus they could protect insulin from acid.     

生物素化壳聚糖纳米粒的制备及其相关性质

制备了生物素化的壳聚糖纳米粒(biotinylated chitosan nanoparticles，Bio-CS-NP)并测定其相关性质，以此作为抗癌药物的载体。制备过程为：先用磺酸琥珀酰亚胺生物素与壳聚糖反应生成生物素化的壳聚糖(biotinylated chitosan，Bio-CS)，再采用氯化钠沉淀法制备Bio-CS-NP。采用试剂盒测定Bio-CS-NP表面配体连接密度，用透射电镜和激光粒度分析仪分别检测纳米粒的形态和粒径，并比较了人肝癌HepG2细胞对Bio-CS-NP和未经生物素修饰的壳聚糖纳米粒(chitosan nanoparticles，CS-NP)的摄取情况。结果显示，Bio-CS-NP表面配体连接密度为2.2 biotin CS；纳米粒形态为圆球形，表面光滑，平均粒径为296.8 nm，多分散指数为0.155；HepG2细胞对Bio-CS-NP的摄取能力显著高于CS-NP(P<0.05)。以上研究结果表明Bio-CS-NP有望成为一种新型的药物载体，用于抗癌药物对癌细胞的主动靶向。生物素的检测方法简便、可行。     

Melatonin-loaded lecithin/chitosan nanoparticles: Physicochemical characterisation and permeability through Caco-2 cell monolayers

In this study, the potential of lecithin/chitosan nanoparticles (NPs) as a mucoadhesive colloidal nanosystem for transmucosal delivery of melatonin was investigated. The size, zeta potential and melatonin loading of the lecithin/chitosan NPs were investigated as a function of lecithin type (Lipoid S45, S75 and S100) and chitosan content in the preparation. The NPs were characterised by mean diameter and zeta potential ranging between 121.6 and 347.5 nm, and 7.5 and 32.7 mV, respectively, and increasing with lecithin-negative charge and chitosan content in the preparation. Melatonin loadings were up to 7.1%. All NPs were characterised by prolonged release profiles with an initial burst (approximately 25%), followed by a slow release phase. Approximately 60–70% of melatonin was released in 4 h. The permeability of melatonin was investigated using Caco-2 cells as an in vitro model of the epithelial barrier. Melatonin permeability from an NP suspension prepared with Lipoid S45 lecithin and a lecithin-to-chitosan weight ratio (L/C) of 20:1 (sample C2) was significantly improved compared to the permeability of melatonin from the solution (P < 0.001) and from all other NPs investigated (P < 0.05). The results obtained by the cell viability studies (MTT and LDH leakage assays) showed that C2 NP suspension did not induce plasma membrane damage or decrease cell viability and could be safely applied to Caco-2 cells in the concentration range tested (<400 μg/ml).     

Design and evaluation of novel pH-sensitive chitosan nanoparticles for oral insulin delivery

Chitosan nanoparticles (CS NPs) have been commonly regarded as potential carriers for the mucosal delivery of therapeutic peptides because of their biocompatibility, bioadhesion and permeation enhancing properties. However, they have limited colloidal stability and readily dissociate and dissolve in the acidic gastric conditions. In the current study, CS NPs were formulated by ionic cross-linking with hydroxypropyl methylcellulose phthalate (HPMCP) as a pH-sensitive polymer and evaluated for the oral delivery of insulin. In vitro results revealed a superior acid stability of CS/HPMCP NPs with a significant control over insulin release and degradation in simulated acidic conditions with or without pepsin. Furthermore, fluorescently-labeled CS/HPMCP NPs showed a 2- to 4-fold improvement in the intestinal mucoadhesion and penetration compared to CS/TPP NPs as evidenced by quantitative fluorescence analysis and confocal microscopy. After s.c. injection to rats, no significant difference in the hypoglycemic effect of insulin solution or insulin-loaded CS/HPMCP NPs was observed, confirming the physico-chemical stability and biological activity of the entrapped peptide. Following peroral administration, CS/HPMCP NPs increased the hypoglycemic effect of insulin by more than 9.8 and 2.8-folds as compared to oral insulin solution and insulin-loaded CS/tripolyphosphate (TPP) NPs, respectively.     

载基因壳聚糖纳米粒的制备及其相关性质的初步研究

目的制备壳聚糖载基因纳米粒,并对其体外相关性质进行初步研究。方法采用复凝聚法制备载基因纳米粒;用纳米粒度仪测量粒度分布、多分散性和Zeta电位;用透射电镜观察粒子的形态;用荧光分光光度法和比色法测定包封率和载药量,并对主要影响因素进行考察;用凝胶阻滞分析和电性结合分析对载药方式进行初步推测。结果所制备的载基因纳米粒形态规则,大多呈球形,平均粒径约150nm,PDI＜0．2,Zeta电位约20mV;包封率大于90％,载药量约30％;凝胶阻滞和电性结合分析结果表明,pDNA与壳聚糖分子间可通过电性结合作用而完全结合。结论采用复凝聚法可制备粒度分布均匀,形态规则,具有较高包封率和载药量的载基因壳聚糖纳米粒;电性结合作用是载基因壳聚糖纳米粒载药的主要方式。     

Preparation and characterization of liposomes encapsulating chitosan nanoparticles

Liposomes are an important colloidal carrier system for controlled drug delivery. However some highly hydrophilic small molecules are difficult to entrap into liposomes and store stably, resulting in poor encapsulation efficiency and fast leakage. In the present work, fluorescein sodium (FS) was used as a model drug that was loaded into chitosan nanoparticles and then encapsulated into liposomes by reverse-phase evaporation (RPV). The encapsulation efficiency, particle size, zeta potential, release in vitro and pharmacokinetics in rats were determined in order to characterize the novel drug delivery system. The entrapment efficiency was above 80% in nanoparticles (Np) and 95% in liposomes encapsulating the nanoparticles (Lip-Np). The Lip-Np was composed of soybean phospholipids, cholesterol and chitosan, which the average diameter was 202.6 nm and zeta potential was -34.8 mV. The release rate of fluorescein sodium from Lip-Np was slower than from Np and liposomes. FS in Lip-Np administered to rats exhibited prolonged circulation and higher bioavailability than FS in Np. The results indicated that liposomal release kinetics can be controlled by encapsulating nanoparticles and thus solid-cored liposomes can be used as a potential drug delivery system.