新型多肽聚酰胺-胺型靶向药物载体的载药性能及细胞吸收和毒性

背景:前期研究通过噬菌体展示体内筛选方法获得了一条NCI-H460非小细胞肺癌特异结合的多肽(Lung cancer targetingpe ptide,LCTP),将该多肽与修饰的聚酰胺-胺型(Polyamidoamine,PAMAM)树枝状高分子材料连接制备了纳米靶向药物载体PAMAM-Ac-FITC-LCTP,该载体在体内外对非小细胞肺癌NCI-H460具有很好的靶向性。目的:在前期研究基础上,进一步研究PAMAM-Ac-FITC-LCTP靶向载体对阿霉素的包埋、释放及其细胞吸收和毒性性能。方法:以筛选到的多肽LCTP为靶向剂,构建了PAMAM-Ac-FITC-LCTP靶向载体。采用物理包埋法将PAMAM-Ac-FITC-LCTP与阿霉素连接,通过体外透析实验观察载体对药物的缓释功能,共聚焦显微镜观察细胞对药物的吸收。以游离阿霉素作为对照,MTT法观察载体载药后对NCI-H460细胞的作用。结果与结论:PAMAM-Ac-FITC-LCTP对阿霉素的最大包埋率为7.46%。载体对药物具有明显的缓释作用,离子浓度、pH和温度对药物的释放具有影响,说明PAMAM-Ac-FITC-LCTP主要是通过静电相互作用与阿霉素结合。PAMAM-Ac-FITC-LCTP/阿霉素短时间内较单独药物更高效进入NCI-H460细胞,而复合物24h的细胞毒性与阿霉素对细胞的毒性基本一致。以上结果说明PAMAM-Ac-FITC-LCTP可能是一个肿瘤治疗和诊断中很有用的药物靶向传输载体。     

聚酰胺-胺树状聚合物靶向修饰及放射性核素标记研究进展

纳米分子在体内具有药物载体的特性,与生物靶向分子结合,可实现药物的靶向传递;与放射性核素的结合,可实现在体内靶向分布与靶向聚集的可视化,从而达到对特定病变的显像或治疗作用。本文对近年来新型树状纳米分子聚乙酰胺-胺（PAMAM）的靶向分子修饰及核素标记的研究进展进行综述。     

聚酰胺-胺型树枝状聚合物介导的基因治疗

聚酰胺-胺型(polyamidoamine,PAMAM)树枝状聚合物是一种新兴起的非病毒类纳米材料载体,可有效地转染外源基因进入细胞。本文对其结构特征及转染机理作一综述。     

乙酰化可降低聚酰胺-胺的细胞毒性

背景：聚酰胺-胺型树枝状高分子纳米材料已被广泛应用于药物载体的研究,但由于整代聚酰胺-胺表面有大量带正电荷的氨基,具有一定的细胞毒性.目的：观察乙酰化对聚酰胺-胺细胞毒性的影响.方法：①细胞增殖检测：采用 MTT 法检测在含0,0.125,0.25,0.5,1,2,4μmol/L 乙酰化聚酰胺-胺的培养液中人胚肾293T 细胞的增殖.②细胞形态：倒置荧光显微镜观察在含4μmol/L 乙酰化聚酰胺-胺的培养液中人胚肾293T 细胞的形态.③细胞周期：流式细胞术检测在含0,5,10,15,20 mg/L 乙酰化聚酰胺-胺的培养液中人胚肾293T 细胞的细胞周期.结果与结论：聚酰胺-胺对293T 细胞具有一定的细胞毒性,在4μmol/L 浓度下48 h 的细胞存活率仅为52%,而乙酰化可显著降低聚酰胺-胺的细胞毒性（P 〈0.01）;聚酰胺-胺孵育的细胞发生团缩,伸展性变差,而乙酰化聚酰胺-胺孵育的293T 细胞与正常培养细胞基本一致,具有良好的伸展性;乙酰化聚酰胺-胺对细胞周期无影响,而聚酰胺-胺在20 mg/L 较高质量浓度时可使细胞 S 期产生阻滞.表明乙酰化可以降低聚酰胺-胺的细胞毒性.     

聚羟基脂肪酸酯和聚酰胺-胺树状高分子构筑的经皮给药系统

背景：聚羟基脂肪酸化合物具有黏着性及与药物分子混合、分散的特性,可作为体外给药剂型的基质;树状高分子具有独特的物理、化学性质,能够作为药物的载体。目的：将聚羟基脂肪酸化合物与聚酰胺-胺树状高分子混合,构筑能够促进难溶型药物的透皮的经皮给药系统。方法：将恶臭假单胞菌 KT2442在含有NH4Cl的培养基中30℃培养72 h,然后在培养体系中添加5 g/L辛烷酸培养24 h,用氯仿抽提聚羟基脂肪酸化合物。各取10 mg聚羟基脂肪酸化合物基质与含聚酰胺-胺与苯乙胺树状高分子的聚羟基脂肪酸化合物基质,分别与5 mg磺酰胺衍生物Tamsulosin混合,构建经皮给药系统;同时构建体外渗透研究装置,研究基质促进药物渗透的效果。X射线衍射分析Tamsulosin、聚羟基脂肪酸化合物基质、基质聚羟基脂肪酸化合物-Tamsulosin 及含聚酰胺-胺树状高分子聚羟基脂肪酸化合物基质-Tamsulosin的内部原子空间分布状况。结果与结论：聚羟基脂肪酸化合物及聚羟基脂肪酸化合物与聚酰胺-胺树状高分子的混合物都能作为经皮给药的基质,促进难溶药物的透皮作用;并且在相同的条件下,聚羟基脂肪酸化合物与聚酰胺-胺树状高分子的混合物作为基质的经皮给药系统能够满足药物的临床血药浓度。聚羟基脂肪酸化合物与聚酰胺-胺树状高分子的混合物能够使模型药物很好地晶体化并呈现高度有序的方向性。     

抗肿瘤药物载体的主动靶向策略

背景:抗肿瘤药物给药后,在机体内往往非特异性分布,毒副作用大,生物利用度低,具有主动靶向功能的药物载体可成功解决这一问题。目的:综合论述了近年来国内外制备主动靶向药物载体应用于抗肿瘤药物输送的最新研究,归纳总结出药物载体的各类主动靶向策略。方法:应用计算机检索平台ISI Web of Knowledge检索2004/2011各英文数据库,检索词采用与抗肿瘤药物载体靶向给药密切相关的关键词,如"tumor,drug carrier,active targeting"等,共检索到文献58篇,最终纳入符合标注的文献26篇。结果与结论:主动靶向策略是设计抗肿瘤药物载体时首先需要考虑也是最为重要的环节,它决定了药物载体实际的药物输送效果以及药物的生物利用度。具有主动靶向策略的药物载体可携带抗肿瘤药物到达肿瘤部位,显著减少机体内其他部位药物的非特异性分布,从而降低其毒副作用,提高生物利用度。     

肿瘤靶向治疗的新型细胞载体分析

靶向治疗不仅可以直接杀伤肿瘤细胞，还能够用作靶向性病毒载体，因此具有十分广泛的应用领域。现阶段肿瘤靶向治疗多利用具备高杀伤力、低免疫原性的病毒，例如 SPG500、ONYX015以及 CV706等源自腺病毒的低免疫原性病毒，或MTH-68／H、PV701等源自新城疫病毒的低免疫原性病毒等。     

聚酰胺-胺型树枝状聚合物介导人凝血因子Ⅷ的体外高效稳定表达

目的 应用纳米材料聚酰胺-胺型(PAMAM)树枝状聚合物／逆病毒基础的质粒载体复合体，在表达水平、持续时间和细胞毒性三方面进行血友病A基因治疗的体外研究。方法 PAMAM树枝状聚合物与含B区缺失(760aa～1639aa)人FⅧ cDNA(FⅧBD cDNA)的以逆病毒为框架的重组表达质粒载体pLNC-FⅦBD形成复合体后，转染NIH3T3细胞系，于转染后第2，5，10，15，30d留取细胞培养上清，分别采用一期法和ELISA法检测其中人FⅧ的凝血活性(FⅧ：C)和抗原含量(FⅧ：Ag)，并应用RTPCR方法检测细胞中FⅧBD cDNA的转录，以细胞生长抑制率来表示PAMAM的细胞毒性。结果 PAMAM载体介导的人FⅧ的体外表达可持续30d，24h内每ml细胞上清中10^6细胞表达FⅧ：C平均为0．929U，FⅧ：Ag平均为0．188μg，期间FⅧ表达未见降低；PAMAM的细胞生长抑制率为5．32％。结论 PAMAM能够有效介导逆病毒基础的质粒载体pLNC-FⅧBD转染靶细胞，并维持高效稳定表达，且PAMAM的细胞毒性较低。     

肿瘤靶向药物静脉给药流程优化的方法与效果

目的：探讨应用优化流程在肿瘤靶向药物静脉给药过程中的管理效果。方法依据《静脉输液护理实践指南》对传统的经验式靶向药物静脉给药流程进行优化,为靶向药物治疗的患者提供个体化、标准化、全程监控的静脉给药流程。比较流程优化前后给药错误发生率、患者满意度。结果流程优化前静脉给药的错误发生率为3．6％,优化流程后无一例给药错误,差异有统计学意义（χ2＝4．903, P＝0．027）;患者对护理工作的满意度从94．4％升高至97．8％,差异有统计学意义（χ2＝4．745, P＝0.029）。结论优化靶向药物静脉给药的护理流程,进一步确保了护理用药安全,增加了患者的满意度,有效提高了护士专业水平。     

聚酰胺-胺型树枝状高聚合物介导Survivin反义寡核苷酸抑制人肝癌裸鼠移植瘤生长的作用

背景：采用反义寡核苷酸等治疗方法可以抑制凋亡抑制因子Survivin的表达,从而诱导肿瘤细胞凋亡。目前使用Survivin治疗恶性肿瘤仍存在基因载体转染效率低下、不能长期稳定表达、易被酶降解等难题。目的：观察聚酰胺-胺型树枝状高聚合物（polyamidoamine,PAMAM）脂质体介导Survivin-反义寡核苷酸对人肝癌裸鼠移植瘤的抑制作用。方法：以人肝癌细胞SMMC-7721裸鼠皮下注射建立人肝癌裸鼠皮下移植瘤模型,将PAMAM脂质体和PAMAM分别与Survivin-反义寡核苷酸混合得到载反义基因转染复合物。测定复合物的zeta电位及包封率。将两种载基因复合物注射道裸鼠移植瘤体内,观察两组移植瘤大小,检测移植瘤组织中survivin基因的表达。结果与结论：PAMAM脂质体-survivin-反义寡核苷酸复合物的zeta电位高于PAMAM-survivin-反义寡核苷酸复合物（P〈0.05）,基因包封率两组比较差异无显著性意义（P〉0.05）。PAMAM脂质体-survivin-反义寡核苷酸复合物治疗组裸鼠移植瘤质量及survivin蛋白表达低于PAMAM-survivin-反义寡核苷酸复合物组（P〈0.05）。提示PAMAM脂质体能将Survivin-反义寡核苷酸高效递送到人肝癌移植瘤细胞,降低survivin蛋白的表达,诱导移植瘤细胞凋亡。     

药物载体系统中肝靶向药物的研究与进展

学术背景:靶向制剂是利用药物载体系统将治疗药物最大限度地运送到靶区,使治疗药物在靶区浓集,提高治疗效果。目的:总结近10年来靶向制剂在肝脏疾病中的研究进展。检索策略:应用计算机检索PUBMED 1995-01/2007-06相关文章,检索词为"liver target,drug"并限定语言种类为"English";同时检索万方数据库1995-01/2007-06相关文章,检索词为"肝,靶向制剂,药物载体",限定语言种类为中文。选择内容与肝靶向给药系统相关的文章,排除重复及陈旧文献。文献评价:初检得到92篇文献,阅读标题和摘要后筛除28篇,查找全文后保留29篇符合标准者进行综述,其中8篇涉及靶向药物机制研究,10篇涉及肝靶向药物制剂载体研究,11篇涉及不同载体的肝靶向制剂在治疗各种肝脏疾病中的应用。资料综合:肝靶向给药系统主要包括被动靶向制剂、主动靶向制剂和物理靶向制剂。被动靶向制剂主要介绍了脂质体、前体药物、纳米粒、药质体和胆酸,主动靶向制剂介绍了半乳糖受体及三硝基苯基-抗生物素蛋白链菌素介导的肝主动靶向制剂的应用,物理靶向制剂主要指磁性靶向制剂。结论:近年来各种肝靶向药物治疗肝脏疾病均取得很大进展,载体选择、运输途径、释放途径日趋多样化,但距临床应用还有一段距离。     

Bacterial ghosts as drug carrier and targeting vehicles.

A novel system for the packaging of drugs as well as vaccines is presented. Bacterial ghosts are intact, non-denatured bacterial envelopes that are created by lysis of bacteria through the expression of cloned phage PhiX174 gene E. Inhibition of induced E-mediated lysis by MgSO(4), harvesting of cells by centrifugation, and resuspension in low-ionic-strength buffers leads to rapid, violent lysis and results in empty bacterial envelopes with large (approximately 1 microm in diameter) openings. The construction of plasmid pAV1, which encodes a streptavidin fusion protein with an N-terminal membrane anchor sequence, allows the loading of the inner side of the cytoplasmic membrane with streptavidin. The functionality and efficacy of binding of even large biotinylated compounds in such streptavidin ghosts (SA-ghosts) was assessed using the enzyme alkaline phosphatase. The successful binding of biotinylated fluorescent dextran, as well as fluorescent DNA complexed with biotinylated polylysine, was demonstrated microscopically. The display by bacterial ghosts of morphological and antigenic surface structures of their living counterparts permits their attachment to target tissues such as the mucosal surfaces of the gastrointestinal and respiratory tract, and their uptake by phagocytes and M cells. In consequence, SA-ghosts are proposed as drug carriers for site-specific drug delivery.     

Combination of a new generation of PNAs with a peptide-based carrier enables efficient targeting of cell cycle progression

The design of potent systems for the delivery of charged and noncharged molecules that target genes of interest remains a challenge. We describe a novel technology that combines a new generation of peptide nucleic acids (PNAs), or HypNA-pPNAs, with a new noncovalent peptide-based delivery system, Pep-2, which promotes efficient delivery of PNAs into several cell lines. We have validated the potential of this technology by showing that Pep2-mediated delivery of an antisense HypNA-pPNA chimera directed specifically against cyclin B1 induces rapid and robust downregulation of its protein levels and efficiently blocks cell cycle progression of several cell lines, as well as proliferation of cells derived from a breast cancer. Pep-2-based delivery system was shown to be 100-fold more efficient in delivering HypNA-pPNAs than classical cationic lipid-based methods. Whereas Pep-2 is essential for improving the bioavailability of PNAs and HypNA-pPNAs, the latter contribute significantly to the efficiency and specificity of the biological response. We have found that Pep-2/HypNA-pPNA strategy promotes potent antisense effects, which are approximately 25-fold greater than with classical antisense oligonucleotide directed specifically against the same cyclin B1 target. Taken together, these data demonstrate that peptide-mediated delivery of HypNA-pPNAs constitutes a very promising technology for therapeutic applications.     

Bioevaluation of magnetic mesoporous silica rods: cytotoxicity,cell uptake and biodistribution in zebrafish and rodents

Mesoporous silica nanoparticles (MSN) characterized by large surface area, pore volume, tunable chemistry, and biocompatibility have been widely studied in nanomedicine as imaging and therapeutic carriers. Most of these studies focused on spherical particles. In contrast, mesoporous silica rods (MSR) that are more challenging to prepare have been less investigated in terms of toxicity, cellular uptake, or biodistribution. Interestingly, previous studies showed that silica rods penetrate fibrous tissues or mucus layers more efficiently than their spherical counterparts. Recently, we reported the synthesis of MSR with distinct aspect ratios and validated their use in multiple imaging modalities by loading the pores with maghemite nanocrystals and functionalizing the silica surface with green and red fluorophores. Herein, based on an initial hypothesis of high liver accumulation of the MSR and a future vision that they could be used for early diagnosis or therapy in fibrotic liver diseases; the cytotoxicity and cellular uptake of MSR were assessed in zebrafish liver (ZFL) cells and the in vivo safety and biodistribution was investigated via fluorescence molecular imaging (FMI) and magnetic resonance imaging (MRI) employing zebrafish larvae and rodents. The selection of these animal models was prompted by the well-established fatty diet protocols inducing fibrotic liver in zebrafish or rodents that serve to investigate highly prevalent liver conditions such as non-alcoholic fatty liver disease (NAFLD). Our study demonstrated that magnetic MSR do not cause cytotoxicity in ZFL cells regardless of the rods'' length and surface charge (for concentrations up to 50 μg ml⁻¹, 6 h) and that MSR are taken up by the ZFL cells in large amounts despite their length of ∼1 μm. In zebrafish larvae, it was observed that they could be safely exposed to high MSR concentrations (up to 1 mg ml⁻¹ for 96 h) and that the rods pass through the liver without causing toxicity. The high accumulation of MSR in rodents'' livers at short post-injection times (20% of the administered dose) was confirmed by both FMI and MRI, highlighting the utility of the MSR for liver imaging by both techniques. Our results could open new avenues for the use of rod-shaped silica particles in the diagnosis of pathological liver conditions.

MSR as multimodal imaging agents could be safely exposed to zebrafish larvae in high concentrations. Their high accumulation in rodents'' livers could open new avenues of rod-shaped particles in pathological fibrotic liver conditions.     

Structure-property relationship in cytotoxicity and cell uptake of poly(2-oxazoline) amphiphiles

The family of poly(2-oxazoline)s (POx) is being increasingly investigated in the context of biomedical applications. We tested the relative cytotoxicity of POx and were able to confirm that these polymers are typically not cytotoxic even at high concentrations. Furthermore, we report structure-uptake relationships of a series of amphiphilic POx block copolymers that have different architectures, molar mass and chain termini. The rate of endocytosis can be fine-tuned over a broad range by changing the polymer structure. The cellular uptake increases with the hydrophobic character of the polymers and is observed even at nanomolar concentrations. Considering the structural versatility of this class of polymers, the relative ease of preparation and their stability underlines the potential of POx as a promising platform candidate for the preparation of next-generation polymer therapeutics.     

SMA-doxorubicin, a new polymeric micellar drug for effective targeting to solid tumours.

Copolymer of styrene-maleic acid (SMA) was used to construct micelles containing doxorubicin by means of a hydrophobic interaction between the styrene moiety of SMA and doxorubicin (Dox). The micelles obtained (SMA-Dox) showed a high solubility in water and a constant doxorubicin release rate of about 3-4%/day in vitro. The SMA-Dox micelle preparation was less (36-70%) cytotoxic to the SW480 human colon cancer cell line in vitro compared with free doxorubicin. In vivo assay of SMA-Dox in ddY mice bearing S-180 tumor revealed a potent anticancer effect with no remarkable toxicity up to a dose of 100 mg/kg of free doxorubicin equivalent. The drug concentration in tumor after administration of SMA-Dox was 13 times higher than that after the free drug. This result can be attributed to the enhanced permeability and retention (EPR) effect of macromolecular drugs observed in solid tumors. Complete blood counts and cardiac histology showed no serious side effects for intravenous (i.v.) doses of the micellar formulation as high as 100 mg/kg doxorubicin equivalent in mice. These data indicate that i.v. administration of SMA-Dox micellar formulation can enhance the therapeutic effect of doxorubicin while reducing greatly cardiac and bone marrow toxicity, which should allow safe use of high doses of this agent.     

Therapeutic potential of a peptide targeting BCL-2 cell guardians in cancer

A promising approach to cancer therapy is to elicit apoptosis with "BH3 mimetic" drugs, which target proteins of the BCL-2 family. As of yet, however, such drugs can target only certain BCL-2 family proteins. Hence, in this issue of the JCI, LaBelle et al. assess instead the therapeutic potential of a "stapled" BH3 peptide from the BIM protein, which inactivates all its prosurvival relatives. The peptide killed cultured hematologic tumor cells and abated growth of a leukemia xenograft, without perturbing the hematopoietic compartment. Hence, such peptides might eventually provide a new way to treat refractory leukemias.