甘油三酯介质中制备壳聚糖纳米粒工艺研究

用乳化溶剂扩散法结合离子沉淀交联法从甘油三酯介质中制备壳聚糖纳米粒,用L9(34)正交设计优选纳米粒制备的处方工艺条件,用显微镜测定纳米粒的粒径,用透射电镜观察纳米粒的形态。结果:正交设计确定纳米粒制备的最优处方工艺条件为:搅拌速度150 r.min-1,壳聚糖质量分数0.10%,壳聚糖分子量9.1万,甘油三酯与壳聚糖酸溶液体积之比200:1,制备的纳米粒平均粒径为(150±50)nm。甘油三酯介质中制备壳聚糖纳米粒工艺简便,制剂具有广泛应用前景。     

壳聚糖-有机累托石/海藻酸钠复合纳米粒的制备

目的:以BSA作为模型药物,制备壳聚糖季铵盐-OREC复合物纳米微粒,建立一种安全有效的药物控释传递系统。方法:超声条件下,制备不同质量比的具有壳聚糖硅酸盐插层结构的复合物纳米微粒,观察其形态学特征、进行红外光谱分析。同时,测定OREC对BSA包封率和载药量的影响。结果:成功制备了不同质量比的OREC-HTCC纳米粒子。电镜结果显示纳米粒呈圆球形,均匀,平均粒径约为30nm。红外图谱分析证实,HTCC插入了OREC插层中,BSA成功地包裹入HTCC-ALG/OREC混合材料制备的纳米微粒。加入OREC后,纳米粒子的包封率及载药量均明显提高,但随着加入量的增加,包封率及载药量逐渐减少。结论:OREC-HTCC纳米粒子是良好的蛋白药物载体,具有粒径小、包封率高、缓释效果好等优点,为CS-OREC作为潜在的药物给药系统的进一步应用提供科学依据。     

载基因壳聚糖纳米粒的制备及免疫增强作用的初步研究

摘 要 目的: 制备壳聚糖载基因纳米粒,并对其体外转染效率及其在小鼠体内的免疫增强效果进行初步研究。方法: 以本课题组构建的口蹄疫DNA疫苗为模型药物,采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;凝胶阻滞分析测定基因在纳米粒中的位置;用体外基因转染实验评价纳米粒的转染活性。用载基因壳聚糖纳米粒免疫雌性Balb/c小鼠,检测免疫小鼠的细胞免疫和体液免疫水平。结果: 所制备的载基因纳米粒形态规则、大多成球形,平均粒径约为150nm,多分散度＜0.26,zeta电位约为21mV;凝胶分析结果表明质粒DNA与壳聚糖分子间可以通过电性结合作用而完全结合,基因几乎全部被包裹在纳米粒内部;体外基因转染实验表明壳聚糖作为一种新型的非病毒基因递送载体能够高效传递DNA进入BHK-21细胞,基因能够在该细胞中高效表达;小鼠免疫实验表明纳米粒不仅能诱导机体产生较高的细胞免疫水平,而且体液免疫水平也显著提高。结论: 壳聚糖纳米粒能将基因递送到细胞内并且能够表达,小鼠免疫实验显示其具有良好的免疫增强效果。     

抗菌脂肽制备羧甲基壳聚糖纳米粒稳定混悬液及其抑菌活性

利用抗菌脂肽解决羧甲基壳聚糖纳米粒制备过程中出现的团聚问题,并考察其对羧甲基壳聚糖纳米粒的抑菌效果影响.通过抗菌脂肽乳化分散离子交联法制备羧甲基壳聚糖纳米粒,静置观察其稳定性,乌氏粘度计测定其粘度变化,扫描电镜考察其形态改变,比浊法测定不同浓度纳米粒溶液对金黄色葡萄球菌和大肠埃希菌的抑制作用.实验发现,加入抗菌脂肽制备的纳米粒稳定性好,粘度降低,形态均一,并能显著增强对金黄色葡萄球菌和大肠埃希菌的抑制作用.结果表明,抗菌脂肽的加入能很好的解决纳米粒制备过程中出现的团聚现象,而且其抑菌效果也有很大改善.     

壳聚糖和帕米膦酸双修饰的固体脂质纳米粒的制备

目的:制备壳聚糖和帕米膦酸双修饰的固体脂质纳米粒。方法:首先利用课题组发表的专利合成帕米膦酸修饰Brij78的新型非离子表面活性剂(Pa-Brij78),然后以壳聚糖(CS)溶液为水相,Pa-Brij78为乳化剂,E-Wax为油相,采用微乳法,利用修饰的帕米膦酸基团与壳聚糖分子链中质子化的氨基交联反应原理,通过一系列实验条件的探索,确定了最佳实验工艺条件,成功制备了壳聚糖和帕米膦酸双修饰的固体脂质纳米粒。通过动态光散射(DLS)粒径仪测定了纳米粒的粒径大小和Zeta电位;透射电子显微镜(TEM)对CS-Pa-Brij78-SLNs的形貌结构进行了表征。结果:实验结果显示,制备壳聚糖和帕米膦酸双修饰的固体脂质纳米粒的最佳条件为:p H=6.0,壳聚糖浓度分别为0.1%,0.2%;反应温度65℃,反应时间40 min,在该条件下,制备的壳聚糖和帕米膦酸双修饰的固体脂质纳米粒(CS-Pa-Brij78-SLNs)粒径分别为97.9±6.6 nm和182.4±62.2 nm,表面电位分别为(+5.21±1.4m V);(+7.94±0.80 m V),装载姜黄素时,载药量为10%,包封率在90%以上,透射电镜下观察其形态圆整,清晰可见壳聚糖包裹的电晕。结论:本文以壳聚糖(CS)溶液为水相,合成的新型非离子表面活性剂Pa-Brij78为乳化剂,E-Wax为油相,采用微乳化法,经过最佳实验条件的探索,通过一步法成功制备了稳定的壳聚糖和帕米膦酸双修饰的固体脂质纳米粒(CS-Pa-Brij78-SLNs)。     

壳聚糖纳米粒介导基因转染的影响因素及改性修饰

壳聚糖带正电荷,可与带负电荷的DNA结合形成纳米级的多聚复合物(纳米粒)。作为一种基因载体,壳聚糖对DNA具有很好的结合和保护作用,对生物体无毒、相容性好,被广泛应用于基因转染及基因预防和治疗中。壳聚糖的主要缺点是转染效率较低,但对其进行改性或修饰后,有可能提高其转染效率。     

转铁蛋白修饰磁性纳米粒的制备及其体外抗肿瘤活性研究

目的:本研究旨在构建一种转铁蛋白修饰负载阿霉素(DOX)的磁纳米粒靶向递药系统,以提高阿霉素作用的靶向性。方法:采用化学共沉淀法制备转铁蛋白修饰负载阿霉素的磁性纳米粒(DOX@MNP),采用zeta电位及纳米粒度分析仪测定DOX@MNP的粒径及其zeta电位,透析法评价DOX@MNP的体外释药特征。通过MTT实验,研究DOX@MNP与游离DOX对A549细胞的细胞毒性,通过激光共聚焦显微镜和流式细胞仪观察A549细胞对DOX@MNP与游离DOX的摄取情况。结果:DOX@MNP的释药具有p H依赖性。MTT实验结果显示,DOX@MNP与游离DOX具有相当的细胞毒性;激光共聚焦显微镜和流式细胞仪检测结果显示A549细胞对DOX和DOX@MNP的摄取没有明显差异。结论:本文构建了一种转铁蛋白修饰包载阿霉素的磁纳米粒,体外结果显示其具有与游离DOX相当的细胞毒性,为进一步进行体内实验奠定了基础。     

半乳糖基壳聚糖/5-氟尿嘧啶纳米粒抑制小鼠原位肝癌移植模型的实验研究

目的:观察半乳糖基壳聚糖(GC)/5-氟尿嘧啶(5-FU)纳米粒抑制小鼠原位肝癌移植模型的疗效及机制.方法:合成GC/5-FU纳米粒,建立小鼠原位肝癌移植模型,通过动物实验观察GC/5-FU纳米粒治疗小鼠原位肝癌移植模型的疗效,通过原位末端标记法(TUNEL法)检测肝癌的凋亡.结果:GC/5-FU纳米粒成功合成,呈规则的球形,表面光滑,大小均匀,纳米粒间无粘连.实验结束时,小鼠的肝癌组织称重,进行方差分析发现GC/5-Fu的瘤重为(0.4361±0.1153)g,5-Fu组为(0.7932±0.1283)g,GC为(1.3989±0.2125)g,和对照组为(1.5801±0.2821)g,4组瘤重之间差异有显著的统计学意义(P＜0.01).GC/5-FU组和5-FU组的瘤重明显小于GC组及对照组,差异有统学意义(P＜0.01);GC/5-FU的瘤重又显著小于5-Fu组(P＜0.01).观察各组小鼠的生存期发现对照组的中位生存时间为12d,GC组为13d,5-FU为17d,而GC/5-FU组最高为30d.GC/5-FU组的生存时间最长.通过TUNEL法观察肝癌细胞的凋亡发现GC/5-FU组的平均凋亡指数(AI)为21.34％较5-FU组的14.74％明显增高(P＜0.05),均较GC组和对照组明显增高,而GC组和对照组间的AI无明显差异(P＞0.05).结论:GC/5-FU纳米粒在体内对小鼠原位肝癌具有明显抑制作用,较5-FU明显增强.其机理可能与GC能通过细胞膜将5-FU从细胞外转移到细胞内,增强5-FU对肝癌细胞的凋亡.     

叶酸-壳聚糖Prdx6 shRNA纳米粒的构建及其对胃癌细胞增殖的影响

目的:探讨叶酸-壳聚糖Prdx6 shRNA纳米粒对胃癌细胞生长的影响。方法:制备靶向性叶酸-壳聚糖Prdx6 shRNA纳米粒,原子力显微镜观察其形态,激光粒度分析仪测定纳米粒的粒径;倒置荧光显微镜观察叶酸-壳聚糖Prdx6 shRNA纳米粒的转染效率;采用蛋白质印迹法检测胃癌细胞Prdx6蛋白的表达变化;CCK8细胞增殖实验检测胃癌细胞的存活率。结果:1制备成功叶酸-壳聚糖Prdx6 shRNA向纳米粒。2荧光显微镜下观察靶向性叶酸-壳聚糖Prdx6 shRNA纳米粒转染胃癌细胞的效率明显高于非靶向纳米粒;胃癌细胞转染靶向组纳米粒后Prdx6蛋白的表达显著低于非靶向组。3与对照组相比,叶酸-壳聚糖Prdx6 shRNA纳米粒能够明显抑制胃癌细胞的增殖(P0.01)。结论:1叶酸-壳聚糖Prdx6 shRNA纳米粒可高效转染胃癌细胞。2转染叶酸-壳聚糖Prdx6 shRNA纳米粒后胃癌细胞的生长明显受抑制。     

酸敏阿霉素脂质纳米粒的制备及其体外抗肿瘤活性研究

目的:本研究旨在制备具有被动靶向和酸敏特性的脂质混合纳米粒,以期提高阿霉素(doxorubicin,DOX)的靶向递药效率,降低DOX的毒副作用,提高抗肿瘤活性。方法:采用微乳法制备磷酸钙纳米粒核,薄膜分散法制备脂质混合纳米粒,硫酸铵梯度法包封DOX。采用透射电镜观察外观形态,用zeta电位及纳米粒度分析仪测定纳米粒的粒径及zeta电位,透析法评价阿霉素脂质纳米粒体外释药特征。用MTT方法研究阿霉素脂质混合纳米粒对A549细胞的细胞毒性。采用流式细胞仪和激光共聚焦显微镜观察A549细胞对阿霉素脂质纳米粒的摄取。结果:体外释药结果显示阿霉素脂质纳米粒具有酸敏特性。流式结果说明A549细胞对阿霉素脂质纳米粒的摄取具有明显的时间依赖性,激光共聚焦显示阿霉素脂质纳米粒能将阿霉素递送至细胞核中。结论:阿霉素脂质体对A549细胞有明显的细胞毒性,为进一步进行体内实验提供了基础。     

Immobilization and characterization of horseradish peroxidase into chitosan and chitosan/PEG nanoparticles: A comparative study

Chitosan (CS) is considered a suitable biomaterial for enzyme immobilization. CS combination with polyethylene glycol (PEG) can improve the biocompatibility and the properties of the immobilized system. Thus, the present work investigated the effect of the PEG in the horseradish peroxidase (HRP) immobilization into chitosan nanoparticles from the morphological, physicochemical, and biochemical perspectives. CS and CS/PEG nanoparticles were obtained by ionotropic gelation and provided immobilization efficiencies (IE) of 65.8 % and 51.7 % and activity recovery (AR) of 76.4 % and 60.4 %, respectively. The particles were characterized by DLS, ZP, SEM, FTIR, TGA and DSC analysis. Chitosan nanoparticles showed size around 135 nm and increased to 229 nm after PEG addition and HRP immobilization. All particles showed positive surface charges (20−28 mV). Characterizations suggest nanoparticles formation and effective immobilization process. Similar values for optimum temperature and pH for immobilized HRP into both nanoparticles were found (45 °C, 7.0). V_max value decreased by 5.07 to 3.82 and 4.11 mM/min and K_M increased by 17.78 to 18.28 and 19.92 mM for free and immobilized HRP into chitosan and chitosan/PEG nanoparticles, respectively. Another biochemical parameters (K_cat, K_e, and K_α) evaluated showed a slight reduction for the immobilized enzyme in both nanoparticles compared to the free enzyme.     

Chitosan nanoparticle as gene therapy vector via gastrointestinal mucosa administration: results of an in vitro and in vivo study

This study was designed to investigate the in vitro and in vivo transfection efficiency of chitosan nanoparticles used as vectors for gene therapy. Three types of chitosan nanoparticles [quaternized chitosan -60% trimethylated chitosan oligomer (TMCO-60%), C(43-45 KDa, 87%), and C(230 KDa, 90%)] were used to encapsulate plasmid DNA (pDNA) encoding green fluorescent protein (GFP) using the complex coacervation technique. The morphology, optimal chitosan-pDNA binding ratio and conditions for maximal in vitro transfection were studied. The in vivo transfection was conducted by feeding the chitosan/pDNA nanoparticles to 12 BALB/C-nu/nu nude mice. Both conventional and TMCO-60% could form stable nanoparticles with pDNA. The in vitro study showed the transfection efficiency to be in the following descending order: TMCO-60%>C(43-45 KDa, 87%)>C(230 KDa, 90%). TMCO-60% proved to be the most efficient and the optimal chitosan/pDNA ratio being 3.2:1. In vivo study showed most prominent GPF expression in the gastric and upper intestinal mucosa. GFP expression in the mucosa of the stomach and duodenum, jejunum, ileum, and large intestine were found, respectively, in 100%, 88.9%, 77.8% and 66.7% of the nude mice examined. TMCO-60%/pDNA nanoparticles had better in vitro and in vivo transfection activity than the other two, and with minimal toxicity, which made it a desirable non-viral vector for gene therapy via oral administration.     

The study of renin–angiotensin–aldosterone in experimental diabetes mellitus

It is generally accepted that hypertension and other vascular pathologies increase in diabetes mellitus (DM) patients as a result of the renin–angiotensin–aldosterone (RAA) system. In this study, changes in the renin‐angiotensin‐aldosterone (RAA) system level was determined in Streptozotocin (STZ)‐injected rats. A total of 46 female Wistar albino rats (180–220 g body weight) was utilized in these experiments. STZ was given intraperitoneally to induce diabetes in rats. Streptozotocin (60 mg kg⁻¹ body weight) was dissolved in 0·1 m citrate–‐phosphate buffer (pH 4–5). The non‐diabetic rats were injected with sterilized buffer alone to act as a control group. Blood glucose levels were 398±8·2 mg dl⁻¹, 488±11·75 mg dl⁻¹ and 658±29·6 mg dl⁻¹ at days 3, 12 and 30 respectively. The level of plasma renin activity (PRA) was measured as 7·69±1·07 ng ml⁻¹ h⁻¹; 1·82±0·22 ng ml⁻¹ h⁻¹ and 0·67±0·12 ng ml⁻¹ h⁻¹ at days 3, 12 and 30, respectively. These values showed that the PRA levels are decreased with increased time period. Serum angiotensin converting enzyme (ACE, E.C. 3.4.15.1) levels were increased at days 12 and 30 (p<0·05 and p<0·005), whereas serum aldosterone levels were increased at days 3 and 12 (p<0·05). The level of urea and creatinine increased at days 12 and 30 (p<0·05 and p<0·005, respectively) when compared to the control group. The data from these experiments indicate that the PRA level decreased whereas ACE activity level increased in diabetic rats compared with the control. Aldosterone levels increased at the first stage of the experiment, but then decreased by the end of the experiment as a result of changes in renin and ACE levels. Copyright © 1999 John Wiley & Sons, Ltd.     

Crystal structure of a phosphonotripeptide K-26 in complex with angiotensin converting enzyme homologue (AnCE) from Drosophila melanogaster

Angiotensin-I converting enzyme (ACE, a zinc dependent dipeptidyl carboxypeptidase) is a major target of drugs due to its role in the modulation of blood pressure and cardiovascular disorders. Here we present a crystal structure of AnCE (an ACE homologue from Drosophila melanogaster with a single enzymatic domain) in complex with a natural product-phosphonotripeptide, K-26 at 1.96 Å resolution. The inhibitor binds exclusively in the S₁ and S₂ binding pockets of AnCE (coordinating the zinc ion) through ionic and hydrogen bond interactions. A detailed structural comparison of AnCE·K-26 complex with individual domains of human somatic ACE provides useful information for further exploration of ACE inhibitor pharmacophores involving phosphonic acids.     

ACE activity during the hypotension produced by standardized aqueous extract of Cecropia glaziovii Sneth: A comparative study to captopril effects in rats

To evaluate the effect of the standardized aqueous extract (AE) of Cecropia glaziovii Sneth on the plasma angiotensin I converting enzyme (ACE-EC 3.4.15.1) activity, rats were treated with a single dose of AE (1 g/kg, p.o.) or repeatedly (0.5 g/kg/bid, p.o.) for 60 days. Captopril (50 mg/kg, p.o.) was used as positive control on the same animals. The effects on the blood pressure were recorded directly from the femoral artery (single dose), or indirectly by the tail cuff method (repeated doses) in conscious rats. The plasma ACE activity was determined spectrofluorimetrically using Hypuril-Hystidine-Leucine as substrate. The arterial blood pressure, heart rate and plasma ACE activity were not significantly modified within 24 h after a single dose administration of AE. Comparatively, blood pressure in captopril treated rats was reduced by 7-16% and heart rate was increased by 10-20% from 30 min to 24 h after drug administration. ACE activity after captopril presented a dual response: an immediate inhibition peaking at 30 min and a slow reversal to 32% up-regulation after 24 h. To correlate the drug effects upon repeated administration of either compound, normotensive rats were separated in three groups: animals with high ACE (48.8+/-2.6 nmol/min/ml), intermediate ACE (39.4+/-1.4 nmol/min/ml) and low ACE (23.5+/-0.6 nmol/min/ml) activity, significantly different among them. Repeated treatment with AE reduced the mean systolic blood pressure (121.7+/-0.5 mm Hg) by 20 mm Hg after 14 days. The hypotension was reversed upon washout 60 days afterwards. Likely, repeated captopril administration decreased blood pressure by 20 mm Hg throughout treatment in all groups. After 30 days treatment with AE (0.5 g/kg/bid, p.o.) the plasma ACE activity was unchanged in any experimental group. After captopril (50 mg/kg/bid, p.o.) administration the plasma ACE activity was inhibited by 50% within 1 h treatment but it was up-regulated by 120% after 12 h in all groups. It is concluded that the hypotension produced by prolonged treatment with AE of C. glaziovii is unrelated to ACE inhibition.     

The expression in E. coli of a polymeric gene coding for an esterase mimic catalyzing the hydrolysis of p-nitrophenyl esters

We have prepared a hybrid protein consisting of seven esterase units, Glu-Ala-His-Ala-Ser-Phe-Phe-Phe, fused to the N-terminal of galactokinase (E. coli). The structural gene for this bifunctional protein was obtained by cloning a polymer made up of three chemically synthesized oligonucleotides to which the galactokinase gene was fused in frame. The hybrid protein was purified to homogeneity with the aid of the galactokinase moiety and showed an M_r of 51 000-53 000. The preparation could catalyze the hydrolysis of p-nitrophenyl esters and, due to the inbuilt hydrophobic spacers, Phe-Phe-Phe, improved catalysis of more hydrophobic substrates was obtained.     

The cytoprotection of chitosan based hydrogels in xenogeneic islet transplantation: An in vivo study in streptozotocin-induced diabetic mouse

Immune rejection and scarcity of donor tissues are the restrictions of islets transplantation. In this study, the cytoprotection of chitosan hydrogels in xenogeneic islet transplantation was demonstrated. Wistar rat islets encapsulated in chitosan hydrogels were performed glucose challenge test and live/dead cell staining in vitro. Islets/chitosan hydrogels were transplanted into the renal subcapsular space of diabetic C57BL/6 mice. Non-fasting blood glucose level (NFBG), body weight, intraperitoneal glucose tolerance test (IPGTT), and glucose disappearance rate were determined perioperatively. The serum insulin level was analyzed, and the kidney transplanted with islets/chitosan hydrogels were retrieved for histological examination after sacrifice. The present results showed that islets encapsulated in chitosan hydrogels secreted insulin in response to the glucose stimulation as naked islets with higher cell survival. The NFBG of diabetic mice transplanted with islets/chitosan hydrogels decreased from 487 ± 46 to 148 ± 32 at one day postoperation and maintained in the range of 201 ± 36 mg/dl for four weeks with an increase in body weight. IPGTT showed the glucose disappearance rate of mice transplanted with islets/chitosan hydrogels was significant faster than that of mice transplanted with naked islets; the serum insulin level increased from 0.29 ± 0.06 to 1.69 ± 0.65 μg/dl postoperatively. Histological examination revealed that the islets successfully engrafted at renal subcapsular space with positive insulin staining. The immunostain was negative for neither the T-cell lineages nor the monocyte/macrophages. This study indicates that the chitosan hydrogels deliver and protect encapsulated islets successfully in xenotransplantation.     

The functional study of human umbilical cord mesenchymal stem cells harbouring angiotensin‐converting enzyme 2 in rat acute lung ischemia‐reperfusion injury model

Acute lung ischemia‐reperfusion injury (ALIRI) is featured as non‐specific alveolar damage, lung edema and hypoxemia, often occurring within 72 h after surgery. It is the leading cause for primary graft failure and mortality after lung surgery and transplantation. Here we aimed to find a more effective therapeutic approach to treat ALIRI. We evaluated the combinational effects of human umbilical cord mesenchymal stem cells (HUMSCs) and angiotensin‐converting enzyme 2 (ACE2) in the rat ALIRI model. HUMSCs were isolated for lentiviral‐ACE2 transfection. Fifty rats were randomly divided into five groups: sham surgery, physiological saline (PS), ACE2, HUMSCs and HUMSCs‐ACE2 group. Several physiological, biochemical and histological indicators were examined and compared among the five groups, such as blood oxygen saturation (Sat O₂%) and right ventricular systolic blood pressure (RVSBP), pulmonary morphology observations, several kinds of cell markers and the abundance of glutathione reductase (GR), glutathione peroxidase (GPX) and NAD(P)H quinone oxidoreductase (NQO1). Compared with HUMSCs and ACE2 groups, HUMSCs‐ACE2 group showed lighter lung injuries, higher CD31 and vWF expression (endothelial cell surface markers), lower γ‐H2AX (DNA damage marker) and CD68 (inflammatory cell marker) and higher anti‐oxidants expression (GR, GPX and NQO1). The results indicated that HUMSCs harbouring ACE2 were more effective than either HUMSCs or ACE2 alone in alleviating the ALIRI damages. The synergistic effects of HUMSCs and ACE2 provide informative clues for mechanism study and therapeutic method development of ALIRI. Copyright © 2014 John Wiley & Sons, Ltd.