保护性特异性抗肿瘤免疫核糖核酸纳米粒制备及质量评价

目的：研究一种具有保护性的特异性肿瘤免疫核糖核酸（iRNA）纳米粒的制备方法。方法：从接种了H22细胞的小鼠腹水中分离肿瘤细胞，裂解后加弗氏完全佐剂充分乳化制备成免疫原，接种于小鼠，1个月后取小鼠的免疫器官提取核糖核酸，复凝聚方法制备壳聚糖-免疫核糖核酸纳米粒，并检测理化特性。结果：提取的肝、脾iRNA为RNA纯品，iRNA白细胞黏附抑制率大于30％，壳聚糖与iRNA形成表面带正电荷的纳米粒，iRNA得到保护不易被破坏。结论：该方法提取并制备的iRNA纳米粒携带供体肿瘤特异性免疫信息，并得到良好保护，能提高抗肿瘤疗效。     

重组水蛭素-2鼻腔黏附纳米粒的体外评价

目的:对制备的重组水蛭素-2(rHV2)壳聚糖鼻腔黏附纳米粒进行体外评价。方法:测定纳米粒的形态、粒径大小分布及表面电位;以超速离心测定纳米粒在不同介质中的释放度;采用在体蟾蜍上腭纤毛运动试验法考察纳米粒的纤毛毒性。结果:制备的rHV2壳聚糖纳米粒呈较为均匀分散的颗粒状,平均粒径为213.2 nm,纳米粒带正电荷,Zeta电位值为+30.61 mV,体系相对稳定性较高;rHV2纳米粒在乙酸缓冲液中的累积释放百分数明显高于在磷酸缓冲液中的累积释放百分数;rHV2纳米粒溶液的纤毛毒性较小(与生理盐水组相比,P>0.05)。结论:壳聚糖鼻腔黏附纳米粒有望成为rHV2鼻腔给药的递释系统。     

抗乙肝免疫核糖核酸前体脂质体的研究

目的制备、研究抗乙肝iRNA前体脂质体。方法采用白细胞黏附抑制试验检测抗乙肝iRNA前体脂质体体外和小鼠口服后体内生物活性。结果抗乙肝iRNA前体脂质体口服有效 ,而iRNA和正常肝RNA前体脂质体均无作用。抗乙肝iRNA前体脂质体体外活性检测未黏附抑制指数在 5 3 .8%～ 76.2 % ;多批次供试品活性检测结果显示其稳定性和重复性较好。结论将抗乙肝iRNA制备为前体脂质体可成为有效的口服剂型     

壳聚糖纳米粒用作基因递送载体的初步研究

目的初步研究基因壳聚糖纳米粒的性质和转染活性。方法用复凝聚法制备纳米粒;用透射电镜观察形态;用纳米粒度分析仪测定粒径、多分散度和zeta电位;用荧光分光光度法测定基因包封率;用凝胶阻滞分析和荧光扫描测定基因在纳米粒中的位置;用体外基因转染实验定性评价纳米粒的转染活性。结果纳米粒形态多呈球形,平均粒径为218.9 nm,多分散度为0.276,zeta电位为+21.2 mV;基因包封率为99.6%;凝胶阻滞分析和荧光扫描表明基因几乎全部被包裹在纳米粒内部,表面吸附很少;体外基因转染实验表明基因壳聚糖纳米粒能够转染人胚胎肾细胞(HEK293)和肝癌细胞(HepG2),基因能够在这两种细胞中表达。结论壳聚糖纳米粒能将基因递送到细胞内并且基因能够表达,因此可以用作基因药物载体。     

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。     

负载胰岛素壳聚糖接枝醋酸乙烯酯纳米粒的制备和性能

目的:制备负载胰岛素壳聚糖-醋酸乙烯酯共聚物纳米粒并研究其性能。方法:用自由基聚合法合成壳聚糖-醋酸乙烯酯共聚物,该聚合物在水中形成具有疏水核心、亲水表面的纳米粒。采用正交设计试验研究了投料比、引发剂浓度和反应时间对纳米粒粒径的影响。结果:对纳米粒进行了热重分析和红外表征。测定纳米粒的形态、粒径和表面电位(Zeta电位),以胰岛素为模型药物,研究纳米粒的包封和释药性能。结论:纳米粒呈球形,粒径均匀,表面荷正电。胰岛素的包封率可达90%以上。pH6.8的磷酸盐缓冲液中胰岛素释放较慢。结论:该纳米制剂具有较好的物理性能和体外缓释特性。     

抗乙肝免疫核糖核酸活性的测定

目的研究抗乙肝免疫核糖核酸免疫活性的测定方法。方法采用巨噬细胞吞噬法和白细胞黏附抑制法测定其细胞免疫活性 ,酶联免疫法和间接血凝法测定其体液免疫活性。结果实验组与对照组相比 ,巨噬细胞吞噬率显著增加 ,白细胞黏附率显著下降 ,血清中可检测到HBsAb的存在。结论用以上方法可测定免疫核糖核酸的活性     

叶酸偶联壳聚糖纳米粒的制备

目的制备叶酸偶联的壳聚糖纳米粒。方法根据叶酸与壳聚糖的偶联比选择最佳工艺条件,通过叶酸活性酯与壳聚糖上的氨基反应,制得叶酸偶联的壳聚糖,再通过离子交联法制得叶酸偶联壳聚糖纳米粒,并测定纳米粒的粒径和表面电位。结果正交实验结果显示叶酸活性酯用量和反应温度是影响偶联比的主要因素,在叶酸活性酯与壳聚糖用量比为2∶1,反应温度50℃,反应时间2 h的条件下可得到偶联比大致为每个壳聚糖分子上偶联3个叶酸分子的叶酸偶联壳聚糖。所制得的纳米粒粒径316 nm,表面电位为(24.85±1.14)mV,透射电镜下观察其形态圆整。结论该方法可成功制备叶酸偶联壳聚糖纳米粒。     

胰岛素纳米粒温敏凝胶的制备及体外释药性能

目的:制备胰岛素壳聚糖温度敏感型原位凝胶(INS-CS-NP-TISG)并进行体外释药动学考察。方法:采用离子凝胶化法制备胰岛素壳聚糖纳米粒;均匀设计法优化其处方及制备工艺,观察形态,测定粒径、表面电位、包封率和载药量;冷法配液的方法制备温度敏感型原位凝胶,改进透析袋-恒温水浴法研究胰岛素壳聚糖纳米粒温度敏感型原位凝胶溶液的体外释药动学。结果:优化制得的纳米粒呈类球形,均匀圆整,分散性好;平均粒径为(255.3±143.5)nm,在175.2~349.6nm范围内的纳米粒子达99.4%,大小均匀,分布较窄;高效液相色谱法(HPLC)测定胰岛素壳聚糖纳米粒平均包封率和载药量分别为75.84%与58.52%;表面电位(ζ)为+32.67;在人工鼻黏液中,胰岛素壳聚糖纳米粒温度敏感型原位凝胶的体外释药符合双相动力学方程,且持续释药24h。结论:选用合适的处方制备胰岛素壳聚糖纳米粒温度敏感型原位凝胶,方法简便,药物载药量高,具有较好的生物黏附性,并有一定的缓释作用。     

壳聚糖-甲基丙烯酸甲酯共聚物纳米粒的制备和表征

目的制备壳聚糖-甲基丙烯酸甲酯共聚物纳米粒。方法用自由基聚合法合成壳聚糖-甲基丙烯酸甲酯共聚物,该聚合物在水中形成具有疏水核心、亲水表面的纳米粒。测定纳米粒的形态、粒径和表面电位(Zeta电位),并研究了壳聚糖含量、聚合物总浓度和引发剂浓度对纳米粒粒径的影响。以胰岛素为模型药物,研究纳米粒的包封和释药性能。结果纳米粒呈球形,粒径均匀,表面荷正电。胰岛素的包封率可达90%以上。pH 5.8的磷酸盐缓冲液中胰岛素的释放较慢,16 h释放量为66.8%。结论该纳米制剂具有较好的物理性能和体外缓释特性。     

TMC–MCC (N-trimethyl chitosan–mono-N-carboxymethyl chitosan) nanocomplexes for mucosal delivery of vaccines

In this study, for the first time, TMC/MCC complex nanoparticles as a delivery system and as an adjuvant were developed and evaluated to obtain systemic and mucosal immune responses against nasally administered tetanus toxoid (TT). Nanoparticles were developed by complexation between the oppositely charged chitosan derivatives, N-trimethyl chitosan (TMC, polycationic) and mono-N-carboxymethyl chitosan (MCC, polyampholytic) without using any crosslinker for mucosal vaccination. The cellular viability was found to be higher with TMC/MCC complex compared to that of MCC and TMC alone. Size, zeta potential and morphology of the nanoparticles were investigated as a function of preparation method. Nanoparticles with high loading efficacy (95%) and positively charged surface were obtained with an average particle size of 283 ± 2.5 nm. The structural integrity of the TT in the nanoparticles was confirmed by SDS–PAGE electrophoresis analysis. Cellular uptake studies indicated that FITC-BSA loaded nanoparticles were effectively taken up into the mouse Balb/c monocyte macrophages. Mice were nasally immunized with TT loaded TMC/MCC complex nanoparticles and compared to that of TMC and MCC nanoparticles. TMC/MCC complex nanoparticles were shown to induce both the mucosal and systemic immune response indicating that this newly developed system has potential for mucosal administration of vaccines.     

Plasmid DNA loaded chitosan nanoparticles for nasal mucosal immunization against hepatitis B

This work investigates the preparation and in vivo efficacy of plasmid DNA loaded chitosan nanoparticles for nasal mucosal immunization against hepatitis B. Chitosan pDNA nanoparticles were prepared using a complex coacervation process. Prepared nanoparticles were characterized for size, shape, surface charge, plasmid loading and ability of nanoparticles to protect DNA against nuclease digestion and for their transfection efficacy. Nasal administration of nanoparticles resulted in serum anti-HBsAg titre that was less compared to that elicited by naked DNA and alum adsorbed HBsAg, but the mice were seroprotective within 2 weeks and the immunoglobulin level was above the clinically protective level. However, intramuscular administration of naked DNA and alum adsorbed HBsAg did not elicit sIgA titre in mucosal secretions that was induced by nasal immunization with chitosan nanoparticles. Similarly, cellular responses (cytokine levels) were poor in case of alum adsorbed HBsAg. Chitosan nanoparticles thus produced humoral (both systemic and mucosal) and cellular immune responses upon nasal administration. The study signifies the potential of chitosan nanoparticles as DNA vaccine carrier and adjuvant for effective immunization through non-invasive nasal route.     

Chitosan nanoparticles as a novel delivery system for ammonium glycyrrhizinate

The ammonium glycyrrhizinate-loaded chitosan nanoparticles were prepared by ionic gelation of chitosan with tripolyphosphate anions (TPP). The particle size and zeta potential of nanoparticles were determined, respectively, by dynamic light scattering (DLS) and a zeta potential analyzer. The effects, including chitosan molecular weight, chitosan concentration, ammonium glycyrrhizinate concentration and polyethylene glycol (PEG) on the physicochemical properties of the nanoparticles were studied. These nanoparticles have ammonium glycyrrhizinate loading efficiency. The encapsulation efficiency decreased with the increase of ammonium glycyrrhizinate concentration and chitosan concentration. The introduction of PEG can decrease significantly the positive charge of particle surface. These studies showed that chitosan can complex TPP to form stable cationic nanoparticles for subsequent ammonium glycyrrhizinate loading.     

Immune response by nasal delivery of hepatitis B surface antigen and codelivery of a CpG ODN in alginate coated chitosan nanoparticles

Alginate coated chitosan nanoparticles were previously developed with the aim of protecting the antigen, adsorbed on the surface of those chitosan nanoparticles, from enzymatic degradation at mucosal surfaces. In this work, this new delivery system was loaded with the recombinant hepatitis B surface antigen (HBsAg) and applied to mice by the intranasal route. Adjuvant effect of the delivery system was studied by measuring anti-HBsAg IgG in serum, anti-HBsAg sIgA in faeces extracts or nasal and vaginal secretions and interferon-gamma production in supernatants of the spleen cells. The mice were primed with 10 microg of the vaccine associated or not with nanoparticles and associated or not with 10 microg CpG oligodeoxynucleotide (ODN) followed by two sequential boosts at three week intervals. The association of HBsAg with the alginate coated chitosan nanoparticles, administered intranasally to the mice, gave rise to the humoral mucosal immune response. Humoral systemic immune response was not induced by the HBsAg loaded nanoparticles alone. The generation of Th1-biased antigen-specific systemic antibodies, however, was observed when HBsAg loaded nanoparticles were applied together with a second adjuvant, the immunopotentiator, CpG ODN. Moreover, all intranasally vaccinated groups showed higher interferon-gamma production when compared to na?ve mice.     

Alginate coated chitosan nanoparticles are an effective subcutaneous adjuvant for hepatitis B surface antigen

We recently described a delivery system that is composed of a chitosan core to which the hepatitis B surface antigen (HBsAg) was adsorbed and subsequently coated with sodium alginate. In this present work, alginate coated chitosan nanoparticles were evaluated as a subcutaneous adjuvant for HBsAg. HBsAg loaded, alginate coated or uncoated chitosan nanoparticles, associated or not with CpGODN were subcutaneously administered to mice and several immunological parameters were evaluated. A high anti-HBsAg IgG titer (2271+/-120 mIU/ml), with the majority of antibodies being of Th2 type, was observed within group I, vaccinated with HBsAg loaded onto coated nanoparticles. However, regarding cellular immune response, no significant differences were observed for antigen-specific splenocyte proliferation or for the secretion of IFN-gamma and IL-4, when compared to the control group. The co-delivery of antigen-loaded nanoparticles in the presence of the immunopotentiator, CpG ODN 1826, resulted in an increase of anti-HBsAg IgG titers that was not statistically different from the first group; however, an increase of the IgG2a/IgG1 ratio from 0.1 to 1.0 and an increase (p<0.01) of the IFN-gamma production by the splenocytes stimulated with the HBV antigen was observed. The enhancement of the immune response observed with the antigen-loaded nanoparticles demonstrated that chitosan is a promising platform for parenteral HBsAg delivery and, when co-administered with the CpG ODN, resulted in a mixed Th1/Th2 type immune response.     

Dendritic cell targeted chitosan nanoparticles for nasal DNA immunization against SARS CoV nucleocapsid protein

This work investigates the formulation and in vivo efficacy of dendritic cell (DC) targeted plasmid DNA loaded biotinylated chitosan nanoparticles for nasal immunization against nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus (SARS-CoV) as antigen. The induction of antigen-specific mucosal and systemic immune response at the site of virus entry is a major challenge for vaccine design. Here, we designed a strategy for noninvasive receptor mediated gene delivery to nasal resident DCs. The pDNA loaded biotinylated chitosan nanoparticles were prepared using a complex coacervation process and characterized for size, shape, surface charge, plasmid DNA loading and protection against nuclease digestion. The pDNA loaded biotinylated chitosan nanoparticles were targeted with bifunctional fusion protein (bfFp) vector for achieving DC selective targeting. The bfFp is a recombinant fusion protein consisting of truncated core-streptavidin fused with anti-DEC-205 single chain antibody (scFv). The core-streptavidin arm of fusion protein binds with biotinylated nanoparticles, while anti-DEC-205 scFv imparts targeting specificity to DC DEC-205 receptor. We demonstrate that intranasal administration of bfFp targeted formulations along with anti-CD40 DC maturation stimuli enhanced magnitude of mucosal IgA as well as systemic IgG against N protein. The strategy led to the detection of augmented levels of N protein specific systemic IgG and nasal IgA antibodies. However, following intranasal delivery of naked pDNA no mucosal and systemic immune responses were detected. A parallel comparison of targeted formulations using intramuscular and intranasal routes showed that the intramuscular route is superior for induction of systemic IgG responses compared with the intranasal route. Our results suggest that targeted pDNA delivery through a noninvasive intranasal route can be a strategy for designing low-dose vaccines.     

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.     

The influence of chitosan content in cationic chitosan/PLGA nanoparticles on the delivery efficiency of antisense 2′-O-methyl-RNA directed against telomerase in lung cancer cells

Tailorable cationic chitosan/PLGA nanoparticles (CPNP) were used for the delivery of an antisense 2′-O-methyl-RNA (2OMR) directed against RNA template of human telomerase. Here, we describe the influence of the chitosan content on binding efficiency, complex stability, uptake in different human lung cell types and finally demonstrate the efficacy of this nanoplex system.CPNPs were prepared by the emulsion-solvent evaporation method using different amounts of chitosan and purified by preparative size exclusion chromatography. The characterization by photon correlation spectroscopy and zeta potential measurements showed a small increase in size and an increase of zeta potential with increasing amounts of chitosan. Binding efficiency and complex stability with 2OMR was high in water and correlated well with the chitosan content of particles but was weak in physiologically relevant media (PBS and RPMI cell culture medium). However, flow cytometry analysis showed that the uptake of 2OMR into A549 lung cancer cells was considerably higher in combination with nanoparticles and dependent on the amount of chitosan when compared to 2OMR alone. Confocal laser scanning microscopy revealed that the uptake into A549 cells is mediated via complexes of 2OMR and chitosan/PLGA nanoparticles despite the weak binding in cell culture medium. The nanoparticles were well tolerated and efficient in inhibiting telomerase activity.