Cell Penetrating Peptides: Intracellular Pathways and Pharmaceutical Perspectives

Cell penetrating peptides, generally categorized as amphipathic or cationic depending on their sequence, are increasingly drawing attention as a non-invasive delivery technology for macromolecules. Delivery of a diverse set of cargo in terms of size and nature ranging from small molecules to particulate cargo has been attempted using different types of cell penetrating peptides (CPPs) in vitro and in vivo. However, the internalization mechanism of CPPs is an unresolved issue to date, with dramatic changes in view regarding the involvement of endocytosis as a pathway of internalization. A key reason for the lack of consensus on the mechanism can be attributed to the methodology in deciphering the internalization mechanism. In this review, we highlight some of the methodology concerns, focus more on the internalization pathway and also provide a novel perspective about the intracellular processing of CPPs, which is a crucial aspect to consider when selecting a cell penetrating peptide as a drug delivery system. In addition, recent applications of cell penetrating peptides for the delivery of small molecules, peptides, proteins, oligonucleotides, nanoparticles and liposomes have been reviewed.     

Differentiation Restricted Endocytosis of Cell Penetrating Peptides in MDCK Cells Corresponds with Activities of Rho-GTPases

Purpose Cellular entry of biomacromolecules is restricted by the barrier function of cell membranes. Tethering such molecules to cell penetrating peptides (CPPs) that can translocate cell membranes has opened new horizons in biomedical research. Here, we investigate the cellular internalization of hCT(9-32)-br, a human calcitonin derived branched CPP, and SAP, a γ-zein related sequence. Methods Internalization of fluorescence labelled CPPs was performed with both proliferating and confluent MDCK cells by means of confocal laser scanning microscopy (CLSM) and fluorescence activated cell sorting (FACS) using appropriate controls. Internalization was further elaborated in an inflammatory, IFN-γ/TNF-αa induced confluent MDCK model mimicking inflammatory epithelial pathologies. Activities of active form Rho-GTPases (Rho-A and Rac-1) in proliferating and confluent MDCK cells were monitored by pull-down assay and Western blot analysis. Results We observed marked endocytic uptake of the peptides into proliferating MDCK by a process suggesting both lipid rafts and clathrin-coated pits. In confluent MDCK, however, we noted a massive but compound-unspecific slow-down of endocytosis. This corresponded with a down-regulation of endocytosis by Rho-GTPases, previously identified to be intimately involved in endocytic traffic. In fact, we found endocytic internalization to relate with active Rho-A; vice versa, MDCK cell density, degree of cellular differentiation and endocytic slow-down were found to relate with active Rac-1. To our knowledge, this is the first study to cast light on the previously observed differentiation restricted internalization of CPPs into epithelial cell models. In the inflammatory IFN-γ/TNF-αa induced confluent MDCK model mimicking inflammatory epithelial pathologies, CPP internalization was enhanced in a cytokine concentration-dependent way resulting in maximum enhancement rates of up to 90%. We suggest a cytokine induced redistribution of lipid rafts in confluent MDCK to cause this enhancement. Conclusion Our findings emphasize the significance of differentiated cell models in the study of CPP internalization and point towards inflammatory epithelial pathologies as potential niche for the application of CPPs for cellular delivery. This work was supported by the Commission of the European Union (EU project on Quality of Life and Management of Living Resources, Project No. QLK2-CT-2001-01451.     

蛋白质转导结构域的跨血脑屏障药物递送

药物递送入脑的主要障碍是血脑屏障,为克服血脑屏障,目前主要采取神经外科手术、增加分子脂溶性、通过内源性血脑屏障转运载体的递送策略。PTD介导跨血脑屏障药物递送是近来出现的新技术,可以方便、高效地使各种分子通过外周血、腹腔等途径,跨越血脑屏障进入脊髓、脑组织细胞。PTD递送快捷、简单、安全,为中枢神经系统疾病的治疗提供了新的研究思路。     

Polymer Microneedles for Controlled-Release Drug Delivery

Purpose As an alternative to hypodermic injection or implantation of controlled-release systems, this study designed and evaluated biodegradable polymer microneedles that encapsulate drug for controlled release in skin and are suitable for self-administration by patients. Methods Arrays of microneedles were fabricated out of poly-lactide-co-glycolide using a mold-based technique to encapsulate model drugs—calcein and bovine serum albumin (BSA)—either as a single encapsulation within the needle matrix or as a double encapsulation, by first encapsulating the drug within carboxymethylcellulose or poly-l-lactide microparticles and then encapsulating drug-loaded microparticles within needles. Results By measuring failure force over a range of conditions, poly-lactide-co-glycolide microneedles were shown to exhibit sufficient mechanical strength to insert into human skin. Microneedles were also shown to encapsulate drug at mass fractions up to 10% and to release encapsulated compounds within human cadaver skin. In vitro release of calcein and BSA from three different encapsulation formulations was measured over time and was shown to be controlled by the encapsulation method to achieve release kinetics ranging from hours to months. Release was modeled using the Higuchi equation with good agreement (r² ≥ 0.90). After microneedle fabrication at elevated temperature, up to 90% of encapsulated BSA remained in its native state, as determined by measuring effects on primary, secondary, and tertiary protein structure. Conclusions Biodegradable polymer microneedles can encapsulate drug to provide controlled-release delivery in skin for hours to months.     

Drug Delivery by Phonophoresis

Phonophoresis is defined as the migration of drug molecules, contained in a contact agent, through the skin under the influence of ultrasound. Several drugs have been introduced into the body by this technique. The design of a phonophoretic drug delivery system is in developmental stages in various research laboratories. Parameters affecting the delivery of drugs by this technique and devices available for drug delivery purposes are discussed in this review.     

细胞膜修饰纳米粒药物递送系统的研究进展

纳米粒是药物递送系统研究的热点之一,但仍存在体内循环时间短,易被网状内皮系统识别和清除等缺点,限制了其临床应用。近年来,天然细胞膜成分和纳米技术的结合为解决这些问题提供了新的方案。一种由纳米粒核和细胞膜壳组成的新型仿生系统极大地改善了纳米粒的性能。用细胞膜修饰的纳米粒具有独特的功能,如延长血液循环时间,提高主动靶向和增强细胞内化等功能。本文综述了细胞膜修饰纳米粒药物递送系统的最新进展及其在癌症治疗方面的应用前景。     

Cellular Uptake But Low Permeation of Human Calcitonin-Derived Cell Penetrating Peptides and Tat(47-57) Through Well-Differentiated Epithelial Models

PURPOSE: To investigate whether cell penetrating peptides (CPP) derived from human calcitonin (hCT) possess, in addition to cellular uptake, the capacity to deliver their cargo through epithelial barriers. METHODS: Cellular uptake of hCT(9-32) and permeation of six hCT-derived peptides, namely, hCT(9-32), hCT(12-32), hCT(15-32), hCT(18-32), hCT(21-32), and a random sequence of hCT(9-32) were evaluated in fully organized confluent Madin-Darby canine kidney (MDCK), Calu-3, and TR146 cell culture models. For comparison, Tat(47-57) and penetratin(43-58) were investigated. The peptides were N-terminally labeled with carboxyfluorescein (CF). Uptake in the well-differentiated epithelial models was observed by confocal laser scanning microscopy (CLSM), whereas permeation through the models was analyzed by reversed-phase (RP)-HPLC. RESULTS: In MDCK epithelium hCT(9-32), Tat(47-57) and penetratin(43-58) demonstrated punctuated cytoplasmic distribution. In Calu-3, Tat(47-57) and penetratin(43-58) were simultaneously localized in a punctuated cytoplasmic and paracellular distribution, whereas hCT(9-32) showed strict paracellular distribution. By contrast, in TR146 cells, Tat(47-57) was located strictly paracellularily, whereas penetratin(43-58) showed a punctuated cytoplasmic pattern and hCT(9-32) both. The transepithelial permeability of all tested peptides and their cargo was lower than that of paracellular markers. CONCLUSIONS: The CPP uptake pattern depends on both the type of peptide and the cell culture model. In general, the investigated CPP have no apparent potential for systemic drug delivery across epithelia. Nevertheless, distinct patterns of cellular distribution may offer a potential for localized epithelial delivery.     

氧化石墨烯生物材料在经皮给药系统中的研究进展

氧化石墨烯含有丰富的含氧活性基团、巨大的比表面积和独特的离域π电子结构,是负载生物活性分子的理想平台。利用氧化石墨烯的电热效应可为提高活性分子的生物利用度提供安全有效的促透皮吸收途径;同时,它还具有生物相容性、抗菌抗炎活性、促伤口愈合活性以及远红外共振效应等生物活性,因此氧化石墨烯生物材料在经皮给药系统中发挥重要作用。文章介绍了氧化石墨烯的结构、理化性质和生物活性,概述了其在药物载体、抗菌材料和伤口敷料等经皮给药系统中的应用研究,并展望其在临床医学的应用前景,希望为氧化石墨烯的生物医用研究提供新的思路。     

脉冲释药系统的新进展

综述近年来脉冲释药系统的新进展。脉冲释药系统是一种按照生理节律设计、定时定量脉冲式释放有效治疗量药物的剂型。根据剂型设计原理和药物释药机制,将脉冲释药系统分为预设药物传递系统、闭环反馈型传递系统和开环传递系统三大类。     

脑靶向给药系统的研究进展

介绍了近年来脑靶向给药的研究进展，为研制可透过血－脑屏蔽的治疗中枢神经系统疾病的脑内给药系统提供参考。     

Respiratory Drug Delivery X

There were ～ 700 delegates who attended Respiratory Drug Delivery X (RDD-X) at the Boca Raton Resort and Club in Boca Raton, Florida, between the 24th and 27th April 2006. Participants from North America, Europe and many other parts of the world came together to hear a series of invited podium presentations covering the latest scientific developments in pulmonary and nasal drug delivery, along with regulatory and quality control issues. A total of 150 proffered posters were also presented, and a Technology Exhibition involved the products of 78 companies. The conference also provided unparalleled networking opportunities. The proceedings of RDD-X will prove to be an invaluable resource for years to come.     

Colonic Drug Delivery: Prodrug Approach

The colon is largely being investigated as a site for administration of protein and peptides, which are degraded by digestive enzymes in the upper GIT. Also for local diseases of the colon, drug administration to the site of action can not only reduce the dose to be administered, but also decrease the side effects. One of the approaches used for colon specific drug delivery is the formation of a prodrug which optimizes drug delivery and improves drug efficacy. Many prodrugs have been evaluated for colon drug delivery. These prodrugs are designed to pass intact and unabsorbed from the upper GIT and undergo biotransformation in the colon releasing the active drug molecule. This biotransformation is carried out by a variety of enzymes, mainly of bacterial origin present in the colon (e.g. azoreductase, glucuronidase, glycosidase, dextranase, esterase, nitroreductase, cyclodextranase, etc.). The present review includes varius prodrug approaches investigated for colon drug delivery and their site specificity.     

微乳作为皮肤局部用药载体的研究进展

微乳是目前药剂学研究的热点,通过皮肤给药以达到局部或全身治疗目的的1种给药载体。综述近年来国内外对微乳作为皮肤局部用药载体的作用机制、影响因素、评价方法以及临床应用等方面的研究进展。     

自乳化药物传递系统的研究概况

目的:总结自乳化药物传递系统的研究概况.方法:对近期自乳化药物传递系统的形成机制、处方组成、体外质量评价及其在药剂学方面的应用进行介绍.结果与结论:自乳化药物传递系统对亲脂性和水难溶性的药物是一个非常有前景的新型载体系统.     

Recent Perspectives in Ocular Drug Delivery

Anatomy and physiology of the eye makes it a highly protected organ. Designing an effective therapy for ocular diseases, especially for the posterior segment, has been considered as a formidable task. Limitations of topical and intravitreal route of administration have challenged scientists to find alternative mode of administration like periocular routes. Transporter targeted drug delivery has generated a great deal of interest in the field because of its potential to overcome many barriers associated with current therapy. Application of nanotechnology has been very promising in the treatment of a gamut of diseases. In this review, we have briefly discussed several ocular drug delivery systems such as microemulsions, nanosuspensions, nanoparticles, liposomes, niosomes, dendrimers, implants, and hydrogels. Potential for ocular gene therapy has also been described in this article. In near future, a great deal of attention will be paid to develop non-invasive sustained drug release for both anterior and posterior segment eye disorders. A better understanding of nature of ocular diseases, barriers and factors affecting in vivo performance, would greatly drive the development of new delivery systems. Current momentum in the invention of new drug delivery systems hold a promise towards much improved therapies for the treatment of vision threatening disorders. All the authors contributed equally to this work.     

微针经皮给药技术

微针是介于皮下注射和透皮贴剂之间的一种给药方式,利用在皮肤角质层产生的微小孔道来显著增加药物的经皮吸收。综述微针经皮给药技术的研究进展,介绍制造微针的材料和方法、微针的给药方式及其在经皮给药系统中的应用。     

Transferrin-Oligomers as Potential Carriers in Anticancer Drug Delivery

PURPOSE: To investigate if the cross-linking of transferrin receptor (TfR) induced by Tf-oligomers alters the endocytosis of receptor-ligand complexes in cultured tumor cells and hence increases intracellular drug release. METHODS: An average of 3.5 Tf molecules per aggregate were cross-linked either by using homobifunctional linker (1, 11-bis-maleimidotetraethyleneglycol) [Tf(3.5-BM(PEO)4)] or heterobifunction linker [succinimidyl 4-(-p-maleimidophenyl)-butyrate] (Tf(3.5-SMPB)). Cell surface binding and competition experiments with 125I-Tf for TfR binding were studied to demonstrate that Tf-oligomers maintain specificity of the TfR-binding. To determine the degradation of Tf-oligomers in TfR-mediated endocytosis, cultured tumor cells were pulsed for 15 min with 125I-Tf-oligomers and chased for 2 h at 37 degrees C in the presence of excess unlabeled Tf. The chase medium was subjected to TCA precipitation to separate the intact and degraded Tf. To investigate if the alteration of TfR-trafficking facilitates the intracellular release of the drug from the Tf-conjugated form, methotrexate (MTX) was conjugated to Tf-oligomer (Agg-Tf-MTX) and its antiproliferative activity was compared with monomeric-Tf-MTX (Mono-Tf-MTX) in human colon carcinoma (Caco-2) cells, human breast adenocarcinoma (MCF-7) cells, wild-type Chinese hamster ovary (CHO) cells, and MTX-resistant CHO (CHO-MTX-RII) cells. RESULTS: TfR-mediated degradation of Tf-oligomers was higher than that of monomeric Tf in both Caco-2 and MCF-7 cells. The IC50 of Agg-Tf-MTX was lower than that of Mono-Tf-MTX in both tumor cell lines. The IC50 of MTX and Mono-Tf-MTX in CHO-MTX-RII cells was higher than that in wild-type CHO cells, whereas the Agg-Tf-MTX was almost identical in both the resistant and wild-type cells. CONCLUSIONS: Cross-linking of TfR induced by oligomeric Tf binding alters the intracellular trafficking of Tf-TfR complexes, redirects them out of the recycling pathway, and targets them to intracellular degradation in cultured tumor cells. The alteration of TfR-trafficking facilitates the intracellular release of the drug from the Tf-conjugated form. Consequently, Agg-Tf-MTX is more effective than Mono-Tf-MTX as a TfR-mediated antiproliferative agent in tumor cells, as well as in MTX-resistant transport deficient cells. Therefore, Tf-oligomers are potentially effective TfR-targeting carriers for intracellular delivery of anticancer drugs.     

穿膜肽提高纳米银穿膜活性的研究

目的合成纳米银,在其表面修饰穿膜肽(TAT),并检测修饰后纳米银粒对人乳腺癌耐阿霉素细胞(MCF-7/ADR)的穿膜活性。方法通过化学还原法制备纳米银(AgNP),并通过Ag-S共价键与TAT连接修饰AgNP。通过粒度仪、透射电镜、激光共聚焦显微镜、流式细胞仪以及二喹林甲酸法等仪器及方法对其表征、共价连接及TAT的介导活性进行测定。结果成功制备了10 nm以下的AgNP,且修饰TAT后的AgNP(AgNP-TAT)表现出了比AgNP更强的穿膜活性。结论 TAT修饰AgNP后能显著提高其穿膜活性。     

鼻腔给药新制剂的研究进展

目的介绍国内鼻腔给药新制剂的研究进展。方法系统查阅国内1999-2004年有关鼻腔给药制剂的研究报道及文献，并进行综合分析。结果鼻腔用药具有吸收起效快、生物利用度高、使用方便等优点。结论鼻腔给药治疗全身性疾病具有广阔的前景。     

电致孔技术在透皮给药中的应用

电致孔可显著提高药物的经皮吸收,有望用于多肽和蛋白质类生物大分子药物的透皮给药.本文对电致孔法的透皮促渗机制、影响因素以及安全性进行了讨论,并介绍了有关实验装置及其在透皮给药中的应用.