经修饰的聚阳离子载体在基因治疗中的应用进展

合成的多聚物载体作为癌细胞靶向治疗的载体在基因治疗中发展迅猛,拥有广阔的研究前景。随着人类基因组测序工作的完成,基因靶序列的不断更新,基因病的存在促使人们发明一种以基因为基础的治疗方案。以往所采取的非病毒载体治疗手段存在着转染效率低、表达含量低等缺陷。在众多合成的多聚物载体中,聚烯酰亚胺、聚酰胺-胺型树状高分子材料在基因治疗中应用广泛。本文综述了以聚乙烯亚胺、聚酰胺-胺型树状高分子材料作为基本结构进行修饰的基因载体的研究进展,并讨论了该领域未来可能的发展方向。     

叶酸-聚酰胺-胺聚合物靶向给药系统的研究进展

简要介绍了聚酰胺-胺树状大分子(PAMAM-D)的特点,叶酸修饰的PAMAM-D复合物的主动靶向性机制,及其在抗肿瘤、基因治疗等领域的研究进展.     

聚酰胺树脂在分离纯化黄酮中的应用研究

对聚酰胺的结构性质和层析分离的机理,以及聚酰胺树脂在分离纯化黄酮类化合物中的应用现状作了综述。聚酰胺是一种应用广泛的吸附剂,在分离纯化黄酮类化合物,蒽醌,多酚类以及金属离子起到了很大的作用;随着研究的深入,相信聚酰胺的层析分离机理研究会有更大的进展,而且也会在更多地领域发挥其作用。     

寡聚酰胺化学与生物学研究进展

含有吡咯和咪唑的寡聚酰胺是一类人工合成的小分子化合物，其分子形状与DNA小沟的曲率相吻合，能高亲合力和特异性地与DNA 小沟中特定序列进行识别并结合，从而起到调控基因表达的作用。寡聚酰胺易透过细胞膜、无细胞毒性、结构稳定和不易降解，是一类很有前途的基因调控工具。本文从寡聚酰胺配对规则、对DNA序列的选择性与结合力、合成方法、透膜吸收、基因调控和动物体内实验等方面的研究进展进行综述，并讨论其在研究中面临的问题，为寡聚酰胺的研究提供参考。     

甲氨蝶呤-G4.0聚酰胺胺树状大分子体外释放初探

目的探讨新型纳米级生物材料G4 0聚酰胺胺(polyamidoamine ,PAMAM )树状大分子对抗癌药物甲氨蝶呤(methotrexate ,MTX)体外释放的影响及释药机理。方法用紫外分光光度法测定G4 0聚酰胺胺树状大分子与甲氨蝶呤复合物在不同浓度和离子强度缓冲溶液( 37℃、pH =7 4 )中甲氨蝶呤的释放行为。结果确定了一个G4 0聚酰胺胺树状大分子可复合14个甲氨蝶呤,在37℃、pH为7 4的10mmol·L-1Tris HCl缓冲溶液中释放甲氨蝶呤80 %需2 0 0h ,但这种缓释作用随离子强度的增加而减弱。结论G4 0聚酰胺胺树状大分子对甲氨蝶呤具有明显的缓释作用,二者的复合作用是聚酰胺胺树状大分子的氨基阳离子与甲氨蝶呤的羧基阴离子之间形成的静电作用导致的。     

PAMAM作为抗肿瘤药物载体研究进展

树枝状大分子是一类新型的高分子材料,具有高度枝化、结构可控、单分散等特点,其中聚酰胺-胺型大分子(Poly-amidoamine,PAMAM)由于能提高药物的水溶性、靶向性和生物利用度,已在抗肿瘤药物载体领域有着广泛的应用。本文将就PAMAM作为一种新型的抗肿瘤药物载体的研究进展进行综述。     

基于PAMAM复合脂质载体的分级肿瘤主动靶向给药系统的构建及其性质考察

目的采用薄膜分散法制备了具有肿瘤分级靶向和pH敏感释药功能的载阿霉素聚酰胺-胺复合脂质载体c[RGDyk]-PEG2000-PSL-[PAMAM G5.0-AC80-FITC5-FA5/DOX]并对其相关性质进行了考察。方法通过微柱离心法和荧光分光光度法(spectrofluorometry,SPF)测定了聚酰胺-胺复合脂质载体的载药量及包封率;分别利用透射电镜(transmission electron microscope,TEM)、动态光散射法(dynamic light scattering,DLS)和ξ电位分析仪对聚酰胺-胺复合脂质载体的形态、粒径和表面电位进行了表征;采用透析法分别考察了聚酰胺-胺复合脂质载体在pH=7.4和pH=6.5释放介质中的体外释放行为,以及血浆对其体外释放行为的影响。结果聚酰胺-胺复合脂质载体的DOX包封率为35.17%(w),载药量为0.41%(w);其粒径分布很窄,平均粒径为106.1 nm;在透射电镜下,可见聚酰胺-胺复合脂质载体呈囊泡状,粒径大小均匀(100¹50 nm);其表面ξ电位值为-6.00 mV;聚酰胺-胺复合脂质载体的体外释药行为体现出pH敏感性和对血浆的稳定性。结论以c[RGDyk]-PEG修饰的pH敏感长循环脂质体包载PAMAM G5.0-AC80-FITC5-FA5/DOX复合物构建具有肿瘤分级靶向和pH敏感释药功能的载阿霉素聚酰胺-胺复合脂质载体有可行性及良好的应用前景。     

聚乳酸/乙醇酸共聚物纳米基因载体的制备及转染效率考察

目的：为了制备转染效率高的新型基因载体，本实验制备由聚酰胺一胺型树状大分子（PAMAM）与聚乳酸／乙醇酸共聚物（PLGA）复合的新型基因载体，并评价了其转染效率。方法：采用溶剂挥发法制备了PAMAM-PLGA新型基因载体，并且通过考察纳米基因载体粒径，粒子形态，稳定性，zeta电位、pH值等，从而确定了最佳的制备工艺。用电泳实验和绿色荧光蛋白标记方法研究了它的转染效率。结果：新型基因载体能够很好的携带DNA进入细胞。结论：在适合的制备工艺条件下可以制备出粒径大约100nm，分散系数为0.088的新型纳米基因载体，其转染效率要高于单纯的PAMAM。     

应用HPLC-ED同时测定脑匀浆8种生物胺及其代谢产物含量

高效液相色谱-电化学检测器(HPLC-ED)是测定儿茶酚胺和吲哚胺等生物胺和相关化合物公认的方法,具有简便性、多功能性、精确性和特异性[1].本文报道同时测定脑组织中8种生物胺及其相关化合物含量的方法.     

叠氮化合物还原为胺或酰胺

烷基叠氮、芳基叠氮、芳酰基叠氮、芳基磺酰叠氮化合物在室温用廉价的 Fe Cl3/Zn还原 ,高收率地得到胺或酰胺。 18例收率 80 %～98% ,分子中双键和硝基等不被还原叠氮化合物还原为胺或酰胺@李荣坡     

The use of PAMAM dendrimers in the efficient transfer of genetic material into cells

Polyamidoamine (PAMAM) dendrimers have steadily grown in popularity in the past decade in a variety of disciplines, ranging from materials science to biomedicine. This can be attributed in part to their use in applications that range from computer toners to medical diagnostics. PAMAM dendrimers are safe and nonimmunogenic, and can function as highly efficient cationic polymer vectors for delivering genetic material into cells. They have been shown to be as efficient or more efficient than either cationic liposomes or other cationic polymers (e.g. polyethylenimine, polylysine) for in vitro gene transfer. This article will focus on the application of PAMAM dendrimers as a nonviral gene delivery vector from the initial discovery of this capacity to the most recent experimental findings.     

Permeability of surface-modified polyamidoamine (PAMAM) dendrimers across Caco-2 cell monolayers

Aim of this study was to prepare polyamine-conjugated PAMAM dendrimers and study their permeability across Caco-2 cell monolayers. Polyamines, namely, arginine and ornithine were conjugated to the amine terminals of the G4 PAMAM dendrimers by Fmoc synthesis. The apical-to-basolateral (AB) and basolateral-to-apical (BA) apparent permeability coefficients (P_app) for the PAMAM dendrimers increased by conjugating the dendrimers with both of the polyamines. The enhancement in permeability was dependent on the dendrimer concentration and duration of incubation. The correlation between monolayer permeability and the decrease in transepithelial electrical resistance (TEER) with both the PAMAM dendrimers and the polyamine-conjugated dendrimers suggests that paracellular transport is one of the mechanisms of transport across the epithelial cells. Cytotoxicity of the polyamine-conjugated dendrimers was evaluated in Caco-2 cells by MTT (methylthiazoletetrazolium) assay. Arginine-conjugated dendrimers were slightly more toxic than PAMAM dendrimer as well as ornithine-conjugated dendrimers. Though investigations on the possible involvement of other transport mechanisms are in progress, results of the present study suggest the potential of dendrimer–polyamine conjugates as drug carriers to increase the oral absorption of drugs.     

Transport of surface engineered polyamidoamine (PAMAM) dendrimers across IPEC-J2 cell monolayers

The aim of our study was to prepare arginine-and ornithine-conjugated Polyamidoamine (PAMAM) dendrimers and study their permeability across IPEC-J2 cell monolayers, a new intestinal cell line model for drug absorption studies. Arginine and ornithine were conjugated to the amine terminals of the PAMAM(G4) dendrimers by Fmoc synthesis. The apical-to-basolateral (AB) and basolateral-to-apical (BA) apparent permeability coefficients (P(app)) for the PAMAM dendrimers increased by conjugating the dendrimers with both of these polyamines. The enhancement in permeability was dependent on the dendrimer concentration and duration of incubation. Correlation between monolayer permeability and the decrease in transepithelial electrical resistance (TEER) with the PAMAM dendrimers and the polyamine-conjugated dendrimers suggests that paracellular transport is one of the mechanisms of transport across the epithelial cells. Cytotoxicity of these surface-modified dendrimers was evaluated in IPEC-J2 cells by MTT (methylthiazoletetrazolium) assay. Arginine-conjugated dendrimers were insignificantly more toxic than PAMAM dendrimer as well as ornithine-conjugated dendrimers. Though investigations on the possible involvement of other transport mechanisms are in progress, results of the present study suggest the potential of dendrimer-polyamine conjugates as the carriers for antigen/drug delivery through the oral mucosa.     

PEG-conjugated PAMAM Dendrimers Mediate Efficient Intramuscular Gene Expression

Generations 5 and 6 (G5 and G6) poly(amidoamine) (PAMAM) dendrimers have been shown to be highly efficient nonviral carriers in in vitro gene delivery. However, their high toxicity and unsatisfied in vivo efficacy limit their applications. In this study, to improve their characteristics as gene delivery carriers, polyethylene glycol (PEG, molecular weight 5,000) was conjugated to G5 and G6 PAMAM dendrimers (PEG-PAMAM) at three different molar ratios of 4%, 8%, and 15% (PEG to surface amine per PAMAM dendrimer molecular). Compared with unconjugated PAMAM dendrimers, PEG conjugation significantly decreased the in vitro and in vivo cytotoxicities and hemolysis of G5 and G6 dendrimers, especially at higher PEG molar ratios. Among all of the PEG-PAMAM dendrimers, 8% PEG-conjugated G5 and G6 dendrimers (G5-8% PEG, G6-8% PEG) resulted in the most efficient muscular gene expression when polyplexes were injected intramuscularly to the quadriceps of neonatal mice. Consistent with the in vivo results, these two 8% PEG-conjugated PAMAM dendrimers could also mediate the highest in vitro transfection in 293A cells. Therefore, G5-8% PEG and G6-8% PEG possess a great potential for gene delivery both in vivo and in vitro.     

Developmental toxicity of low generation PAMAM dendrimers in zebrafish

Biological molecules and intracellular structures operate at the nanoscale; therefore, development of nanomedicines shows great promise for the treatment of disease by using targeted drug delivery and gene therapies. PAMAM dendrimers, which are highly branched polymers with low polydispersity and high functionality, provide an ideal architecture for construction of effective drug carriers, gene transfer devices and imaging of biological systems. For example, dendrimers bioconjugated with selective ligands such as Arg-Gly-Asp (RGD) would theoretically target cells that contain integrin receptors and show potential for use as drug delivery devices. While RGD-conjugated dendrimers are generally considered not to be cytotoxic, there currently exists little information on the risks that such materials pose to human health. In an effort to compliment and extend the knowledge gleaned from cell culture assays, we have used the zebrafish embryo as a rapid, medium throughput, cost-effective whole-animal model to provide a more comprehensive and predictive developmental toxicity screen for nanomaterials such as PAMAM dendrimers. Using the zebrafish embryo, we have assessed the developmental toxicity of low generation (G3.5 and G4) PAMAM dendrimers, as well as RGD-conjugated forms for comparison. Our results demonstrate that G4 dendrimers, which have amino functional groups, are toxic and attenuate growth and development of zebrafish embryos at sublethal concentrations; however, G3.5 dendrimers, with carboxylic acid terminal functional groups, are not toxic to zebrafish embryos. Furthermore, RGD-conjugated G4 dendrimers are less potent in causing embryo toxicity than G4 dendrimers. RGD-conjugated G3.5 dendrimers do not elicit toxicity at the highest concentrations tested and warrant further study for use as a drug delivery device.     

Transepithelial transport and toxicity of PAMAM dendrimers: implications for oral drug delivery

This article summarizes efforts to evaluate poly(amido amine) (PAMAM) dendrimers as carriers for oral drug delivery. Specifically, the effect of PAMAM generation, surface charge and surface modification on toxicity, cellular uptake and transepithelial transport is discussed. Studies on Caco-2 monolayers, as models of intestinal epithelial barrier, show that by engineering surface chemistry of PAMAM dendrimers, it is possible to minimize toxicity while maximizing transepithelial transport. It has been demonstrated that PAMAM dendrimers are transported by a combination of paracellular and transcellular routes. Depending on surface chemistry, PAMAM dendrimers can open the tight junctions of epithelial barriers. This tight junction opening is in part mediated by internalization of the dendrimers. Transcellular transport of PAMAM dendrimers is mediated by a variety of endocytic mechanisms. Attachment or complexation of cytotoxic agents to PAMAM dendrimers enhances the transport of such drugs across epithelial barriers. A remaining challenge is the design and development of linker chemistries that are stable in the gastrointestinal tract (GIT) and the blood stream, but amenable to cleavage at the target site of action. Recent efforts have focused on the use of PAMAM dendrimers as penetration enhancers. Detailed in vivo oral bioavailability of PAMAM dendrimer-drug conjugates, as a function of physicochemical properties will further need to be assessed.     

Polyamidoamine dendrimers can improve the pulmonary absorption of insulin and calcitonin in rats

The absorption-enhancing effects of polyamidoamine (PAMAM) dendrimers with various generations (G0-G3) and concentrations [0.1%-1.0% (w/v)] on the pulmonary absorption of peptide and protein drugs were studied in rats. Insulin and calcitonin were chosen as models of peptide and protein drugs, and their pulmonary absorption with or without PAMAM dendrimers was examined by in vivo pulmonary absorption studies. PAMAM dendrimers significantly increased the pulmonary absorption of insulin and calcitonin in rats, and their absorption-enhancing effects were generation dependent. The rank order of absorption enhancement effect of these PAMAM dendrimers was G3 > G2 > G1 > G0. For the same generation, the absorption-enhancing effects of PAMAM dendrimers were shown to be concentration dependent. The toxicity of these PAMAM dendrimers in the lung tissues was evaluated by measuring the release of protein and the activities of lactate dehydrogenase (LDH) in bronchoalveolar lavage fluid (BALF). The PAMAM dendrimers with various generations and concentrations did not significantly increase the release of protein and the activities of LDH in BALF, indicating that these dendrimers did not cause any membrane damage to the lung tissues. The zeta potentials of insulin and calcitonin solutions changed to positive by the addition of PAMAM dendrimers, and the degree of positive charge as determined by the zeta potentials was linearly correlated with the absorption-enhancing effects of the PAMAM dendrimers. This positive charge of the PAMAM dendrimers might be related to their absorption-enhancing mechanisms for improving the pulmonary absorption of insulin and calcitonin in rats. In conclusion, the PAMAM dendrimers are suitable absorption enhancers to improve the pulmonary absorption of insulin and calcitonin without any membrane damage to the respiratory tissues.     

Transport of Surface Engineered Polyamidoamine (PAMAM) Dendrimers Across IPEC-J2 Cell Monolayers

The aim of our study was to prepare arginine-and ornithine-conjugated Polyamidoamine (PAMAM) dendrimers and study their permeability across IPEC-J2 cell monolayers, a new intestinal cell line model for drug absorption studies. Arginine and ornithine were conjugated to the amine terminals of the PAMAM_G4 dendrimers by Fmoc synthesis. The apical-to-basolateral (AB) and basolateral-to-apical (BA) apparent permeability coefficients (P_app) for the PAMAM dendrimers increased by conjugating the dendrimers with both of these polyamines. The enhancement in permeability was dependent on the dendrimer concentration and duration of incubation. Correlation between monolayer permeability and the decrease in transepithelial electrical resistance (TEER) with the PAMAM dendrimers and the polyamine-conjugated dendrimers suggests that paracellular transport is one of the mechanisms of transport across the epithelial cells. Cytotoxicity of these surface-modified dendrimers was evaluated in IPEC-J2 cells by MTT (methylthiazoletetrazolium) assay. Arginine-conjugated dendrimers were insignificantly more toxic than PAMAM dendrimer as well as ornithine-conjugated dendrimers. Though investigations on the possible involvement of other transport mechanisms are in progress, results of the present study suggest the potential of dendrimer-polyamine conjugates as the carriers for antigen/drug delivery through the oral mucosa.     

Efficient in vitro siRNA delivery and intramuscular gene silencing using PEG-modified PAMAM dendrimers

Although siRNA techniques have been broadly applied as a tool for gene knockdown, substantial challenges remain in achieving efficient delivery and in vivo efficacy. In particular, the low efficiency of target gene silencing in vivo is a critical limiting step to the clinical application of siRNA therapies. Poly(amidoamine) (PAMAM) dendrimers are widely used as carriers for drug and gene delivery; however, in vivo siRNA delivery by PAMAM dendrimers remains to be carefully investigated. In this study, the effectiveness of G5 and G6 PAMAM dendrimers with 8% of their surface amines conjugated to MPEG-5000 was studied for siRNA delivery in vitro and for intramuscular in vivo delivery in mice. The results from the PEG-modified dendrimers were compared to the results from the parent dendrimers as well as Lipofectamine 2000 and INTERFERin. Both PEG-modifed dendrimers protect the siRNA from being digested by RNase and gave high transfection efficiency for FITC-labeled siRNA in the primary vascular smooth muscle cells (VSMC) and mouse peritoneal macrophages. The PEG-modified dendrimers achieved knockdown of both plasmid (293A cells) and adenovirus-mediated green fluorescence protein (GFP) expression (Cos7 cells) in vitro with efficiency similar to that shown for Lipofectamine 2000. We further demonstrated in vivo that intramuscular delivery of GFP-siRNA using PEG-modified dendrimer significantly suppressed GFP expression in both transiently adenovirus infected C57BL/6 mice and GFP transgenic mice.     

Transport of Poly(Amidoamine) Dendrimers across Caco-2 Cell Monolayers: Influence of Size, Charge and Fluorescent Labeling

Purpose To investigate the transport of poly(amidoamine) (PAMAM) dendrimers with positive, neutral and negatively charged surface groups across Caco-2 cell monolayers. Methods Cationic PAMAM-NH₂ (G2 and G4), neutral PAMAM-OH (G2), and anionic PAMAM-COOH (G1.5–G3.5) dendrimers were conjugated to fluorescein isothiocyanate (FITC). The permeability of fluorescently labeled PAMAM dendrimers was measured in the apical-to-basolateral direction. ¹⁴C-Mannitol permeability was measured in the presence of unlabeled and FITC labeled PAMAM dendrimers. Caco-2 cells were incubated with the dendrimers followed by mouse anti-occludin or rhodamine phalloidin, and visualized using confocal laser scanning microscopy to examine tight junction integrity. Results The overall rank order of PAMAM permeability was G3.5COOH > G2NH₂ > G2.5COOH > G1.5COOH > G2OH. ¹⁴C-Mannitol permeability significantly increased in the presence of cationic and anionic PAMAM dendrimers with significantly greater permeability in the presence of labeled dendrimers compared to unlabeled. PAMAM dendrimers had a significant influence on tight junction proteins occludin and actin, which was microscopically evidenced by disruption in the occludin and rhodamine phalloidin staining patterns. Conclusions These studies demonstrate that enhanced PAMAM permeability is in part due to opening of tight junctions, and that by appropriate engineering of PAMAM surface chemistry it is possible to increase polymer transepithelial transport for oral drug delivery applications.