基因疫苗导入技术研究进展

基因疫苗积极的临床结果证明了,基因免疫是一种有效的临床免疫模式。虽然,喷射注射法的精确作用机制还不太清楚,但临床前研究表明,在皮肤内直接打靶抗原呈递细胞可有效地增强免疫反应。另外,局部给药法和树突细胞体外加载抗原的实验结果显示,直接打靶抗原呈递细胞可放大、控制和调节预防及治疗性基因疫苗的免疫结果。尽管基因枪有许多令人鼓舞的优点,但由于价格和便利性的障碍,它是否能商业化还不能确定。利用基因法治疗和预防疾病所涉及的安全性对基因疫苗要求更严格。这要有可控的质粒导入系统和组织特异性表达系统。     

Dendritic cell delivery of plasmid DNA

Positive human clinical data using biolistic-mediated gene transfer (i.e., gene gun) to administer a nucleic acid-based Hepatitis B vaccine has validated genetic immunization as an effective clinical vaccine modality. Although the precise mechanism of action has yet to be determined, preclinical studies using jet injection have indicated that direct targeting of resident antigen presenting cells (Langerhan's cells) in the skin as the primary immunological driving force for the potent and long-lived immune response. Moreover, positive results with topical delivery of genetic vaccines and ex vivo loading of dendritic cells with antigen has strengthened the movement toward directly targeting antigen presenting cells as a means to amplify, control, and mediate the immunological consequences of prophylactic and/or therapeutic genetic vaccines. Despite these encouraging results with the gene gun, it is unclear whether this technology will translate into commercially available vaccines due to potential product development barriers such as cost and convenience. It is clear that safety concerns in using genetic approaches to treat and prevent disease have highlighted the need for strict product requirements for genetic vaccines. A plausible strategy to meet these requirements is to combine controlled plasmid delivery systems with tissue-specific gene expression systems.     

Skin vaccination against cervical cancer associated human papillomavirus with a novel micro-projection array in a mouse model

Background

Better delivery systems are needed for routinely used vaccines, to improve vaccine uptake. Many vaccines contain alum or alum based adjuvants. Here we investigate a novel dry-coated densely-packed micro-projection array skin patch (Nanopatch™) as an alternate delivery system to intramuscular injection for delivering an alum adjuvanted human papillomavirus (HPV) vaccine (Gardasil®) commonly used as a prophylactic vaccine against cervical cancer.

Methodology/Principal Findings

Micro-projection arrays dry-coated with vaccine material (Gardasil®) delivered to C57BL/6 mouse ear skin released vaccine within 5 minutes. To assess vaccine immunogenicity, doses of corresponding to HPV-16 component of the vaccine between 0.43±0.084 ng and 300±120 ng (mean ± SD) were administered to mice at day 0 and day 14. A dose of 55±6.0 ng delivered intracutaneously by micro-projection array was sufficient to produce a maximal virus neutralizing serum antibody response at day 28 post vaccination. Neutralizing antibody titres were sustained out to 16 weeks post vaccination, and, for comparable doses of vaccine, somewhat higher titres were observed with intracutaneous patch delivery than with intramuscular delivery with the needle and syringe at this time point.

Conclusions/Significance

Use of dry micro-projection arrays (Nanopatch™) has the potential to overcome the need for a vaccine cold chain for common vaccines currently delivered by needle and syringe, and to reduce risk of needle-stick injury and vaccine avoidance due to the fear of the needle especially among children.     

Skin permeability prediction with MD simulation sampling spatial and alchemical reaction coordinates

A molecular-level understanding of skin permeation may rationalize and streamline product development, and improve quality and control, of transdermal and topical drug delivery systems. It may also facilitate toxicity and safety assessment of cosmetics and skin care products. Here, we present new molecular dynamics simulation approaches that make it possible to efficiently sample the free energy and local diffusion coefficient across the skin’s barrier structure to predict skin permeability and the effects of chemical penetration enhancers. In particular, we introduce a new approach to use two-dimensional reaction coordinates in the accelerated weight histogram method, where we combine sampling along spatial coordinates with an alchemical perturbation virtual coordinate. We present predicted properties for 20 permeants, and demonstrate how our approach improves correlation with ex vivo/in vitro skin permeation data. For the compounds included in this study, the obtained log K_Pexp-calc mean square difference was 0.9 cm² h^?2.     

Transcutaneous antigen delivery system

Transcutaneous immunization refers to the topical application of antigens onto the epidermis. Transcutaneous immunization targeting the Langerhans cells of the skin has received much attention due to its safe, needle-free, and noninvasive antigen delivery. The skin has important immunological functions with unique roles for antigen-presenting cells such as epidermal Langerhans cells and dermal dendritic cells. In recent years, novel vaccine delivery strategies have continually been developed; however, transcutaneous immunization has not yet been fully exploited due to the penetration barrier represented by the stratum corneum, which inhibits the transport of antigens and adjuvants. Herein we review recent achievements in transcutaneous immunization, focusing on the various strategies for the enhancement of antigen delivery and vaccination efficacy. [BMB Reports 2013; 46(1): 17-24]     

Topical Gene Electrotransfer to the Epidermis of Hairless Guinea Pig by Non-Invasive Multielectrode Array

Topical gene delivery to the epidermis has the potential to be an effective therapy for skin disorders, cutaneous cancers, vaccinations and systemic metabolic diseases. Previously, we reported on a non-invasive multielectrode array (MEA) that efficiently delivered plasmid DNA and enhanced expression to the skin of several animal models by in vivo gene electrotransfer. Here, we characterized plasmid DNA delivery with the MEA in a hairless guinea pig model, which has a similar histology and structure to human skin. Significant elevation of gene expression up to 4 logs was achieved with intradermal DNA administration followed by topical non-invasive skin gene electrotransfer. This delivery produced gene expression in the skin of hairless guinea pig up to 12 to 15 days. Gene expression was observed exclusively in the epidermis. Skin gene electrotransfer with the MEA resulted in only minimal and mild skin changes. A low level of human Factor IX was detected in the plasma of hairless guinea pig after gene electrotransfer with the MEA, although a significant increase of Factor IX was obtained in the skin of animals. These results suggest gene electrotransfer with the MEA can be a safe, efficient, non-invasive skin delivery method for skin disorders, vaccinations and potential systemic diseases where low levels of gene products are sufficient.     

基于壳聚糖的可控缓释型微针制备与性能评价

在临床应用上,一种能够持续递送药物的微针(microneedle,MN)系统对于一些疫苗、激素类药物的递送具有重要价值。本文研究设计了一种基于壳聚糖的可控缓释型微针阵列(PVA/CS-MN),将微针贴片与药物相结合用于药物的可控长效缓释。重点研究了PVA/CS-MN的制备优化工艺,并对MN阵列外观形貌、力学性能、溶解与溶胀性能以及体外刺入性能进行了表征。实验结果表明,使用最优工艺制备的PVA/CS-MN具有良好的形貌以及力学性能,可以顺利在皮肤上打开微通道,并实现可控的溶解与溶胀功能。同时,体外透皮扩散实验表明,以抗坏血酸(l-ascorbic acid)为模型药物制备的Vc-PVA/CS-MN在1 h内即释放了约57%的药物,随后12 h内缓慢释放了约66.7%的药物,7 d后最终释放了92%的药物。PVA/CS-MN具备可控的缓释特性以及优良的药物递送效率,为药物的持续透皮递送提供了一个新选择。     

Hollow microneedle-based sensor for multiplexed transdermal electrochemical sensing

The development of a minimally invasive multiplexed monitoring system for rapid analysis of biologically-relevant molecules could offer individuals suffering from chronic medical conditions facile assessment of their immediate physiological state. Furthermore, it could serve as a research tool for analysis of complex, multifactorial medical conditions. In order for such a multianalyte sensor to be realized, it must be minimally invasive, sampling of interstitial fluid must occur without pain or harm to the user, and analysis must be rapid as well as selective. Initially developed for pain-free drug delivery, microneedles have been used to deliver vaccines and pharmacologic agents (e.g., insulin) through the skin. Since these devices access the interstitial space, microneedles that are integrated with microelectrodes can be used as transdermal electrochemical sensors. Selective detection of glucose, glutamate, lactate, hydrogen peroxide, and ascorbic acid has been demonstrated using integrated microneedle-electrode devices with carbon fibers, modified carbon pastes, and platinum-coated polymer microneedles serving as transducing elements. This microneedle sensor technology has enabled a novel and sophisticated analytical approach for in situ and simultaneous detection of multiple analytes. Multiplexing offers the possibility of monitoring complex microenvironments, which are otherwise difficult to characterize in a rapid and minimally invasive manner. For example, this technology could be utilized for simultaneous monitoring of extracellular levels of, glucose, lactate and pH, which are important metabolic indicators of disease states (e.g., cancer proliferation) and exercise-induced acidosis.     

Peptide delivery to tissues via reversibly linked protein transduction sequences

The development of peptide-based therapeutics has suffered from challenges associated with delivery to intact tissue. In skin, an array of protein targets resides only tens of micrometers below the surface; however, because of difficulties in traversing the cutaneous barrier, the potentialfor peptide-based therapeutics remains unrealized. We have developed a general approach for topical peptide delivery into skin using releasable protein transduction sequences to enable peptide transport across tissue boundaries. Upon entry into the cell, the disulfide linkage between the peptide transduction sequences and peptide cargo is cleaved, permitting the dissociation of the highly charged peptide transduction sequences from the active peptide. A protype cargo peptide, the hemagglutinin (HA) epitope, was conjugated to a hepta-arginine protein transduction sequence via a releasable disulfide linkage. This construct penetrated the skin to deep dermis within 1 h after topical application. Consistent with the dissociation of the protein transduction and cargo sequences, absorbed protein transduction sequences and HA peptides displayed differential intracellular localization. Reversible protein transduction sequence linkage thus represents a noninvasive platform for tissue delivery of intact peptides with no requirement for viral vectors or parenteral injection and may be of broad utility in molecular therapy.     

Transdermal protein delivery by a coadministered peptide identified via phage display

Efficient transdermal drug delivery of large hydrophilic drugs is challenging. Here we report that the short synthetic peptide, ACSSSPSKHCG, identified by in vivo phage display, facilitated efficient transdermal protein drug delivery through intact skin. Coadministration of the peptide and insulin to the abdominal skin of diabetic rats resulted in elevated systemic levels of insulin and suppressed serum glucose levels for at least 11 h. Significant systemic bioavailability of human growth hormone was also achieved when topically coadministered with the peptide. The transdermal-enhancing activity of the peptide was sequence specific and dose dependent, did not involve direct interaction with insulin and enabled penetration of insulin into hair follicles beyond a depth of 600 microm. Time-lapse studies suggested that the peptide creates a transient opening in the skin barrier to enable macromolecular drugs to reach systemic circulation.     

Glycan arrays: biological and medical applications

Carbohydrates and their conjugates are involved in various biological events, including viral and bacterial infection, the immune response, differentiation and development, and the progression of tumor cell metastasis. Glycan arrays are a new technology that has enabled the high-sensitivity and rapid analysis carbohydrate-protein interaction and contribute to significant advances in glycomics. Glycan arrays use a minute amount of materials and can be used for high-throughput profiling and quantitative analysis and provide information for the development of carbohydrate-based vaccines and new drug discovery.     

Lecithin organogels as a potential phospholipid-structured system for topical drug delivery: A review

The purpose of this review is to give an insight into the considerable potential of lecithin organogels (LOs) in the applications meant for topical drug delivery. LOs are clear, thermodynamically stable, viscoelastic, and biocompatible jelly-like phases, chiefly composed of hydrated phospholipids and appropriate organic liquid. These systems are currently of interest to the pharmaceutical scientist because of their structural and functional benefits. Several therapeutic agents have been formulated as LOs for their facilitated transport through topical route (for dermal or transdermal effect), with some very encouraging results. The improved topical drug delivery has mainly been attributed to the biphasic drug solubility, the desired drug partitioning, and the modification of skin barrier function by the organogel components. Being thermodynamically stable, LOs are prepared by spontaneous emulsification and therefore posses prolonged shelf life. The utility of this novel matrix as a topical vehicle has further increased owing to its very low skin irritancy potential. Varied aspects of LOs viz formation, composition, phase behavior, and characterization have been elaborated, including a general discussion on the developmental background. Besides a comprehensive update on the topical applications of lecithin organogels, the review also includes a detailed account on the mechanistics of organogelling. Published: October 6, 2005     

Effective induction of anti-melanoma immunity following genetic vaccination with synthetic mRNA coding for the fusion protein EGFP.TRP2

RNA-based genetic immunization represents an alternative novel strategy for antigen-specific cancer vaccines. In the present paper we investigate the use of synthetic messenger RNA in an experimental melanoma model. We show that gene gun-based immunization using synthetic RNA mediates gene expression in the epidermis and effectively induces antigen-specific cellular and humoral immunity in mice in vivo. Importantly, bombardment of the skin with RNA coding for the melanocytic self-antigen TRP2 linked to the immunogenic protein EGFP was associated with protection against experimentally induced B16 melanoma lung metastases and vitiligo-like fur depigmentation. Our results provide a scientific basis for clinical trials using synthetic mRNA encoding melanocytic antigens linked to immunogenic helper proteins for vaccination of patients with melanoma. Julia Steitz and Cedrik M. Britten contributed equally to this work.     

Efficient delivery of transgenes to human hair follicle progenitor cells using topical lipoplex

The topical delivery of transgenes to hair follicles has potential for treating disorders of the skin and hair. Here we show that the topical administration of liposome-DNA mixtures (lipoplex) to mouse skin and to human skin xenografts resulted in efficient in vivo transfection of hair follicle cells. Transfection depended on liposome composition, and occurred only at the onset of a new growing stage of the hair cycle. Manipulating the hair follicle cycle with depilation and retinoic acid treatment resulted in nearly 50% transfection efficiency-defined as the proportion of transfected, newly growing follicles within the xenograft. Transgenes administered in this fashion are selectively expressed in hair progenitor cells and therefore have the potential to affect the characteristics of the follicle. These findings form a foundation for the future use of topical lipoplex applications to alter hair follicle phenotype and treat diseases of the hair and skin.     

Hydrogel-Forming Microneedles Prepared from “Super Swelling” Polymers Combined with Lyophilised Wafers for Transdermal Drug Delivery

We describe, for the first time, hydrogel-forming microneedle arrays prepared from “super swelling” polymeric compositions. We produced a microneedle formulation with enhanced swelling capabilities from aqueous blends containing 20% w/w Gantrez S-97, 7.5% w/w PEG 10,000 and 3% w/w Na₂CO₃ and utilised a drug reservoir of a lyophilised wafer-like design. These microneedle-lyophilised wafer compositions were robust and effectively penetrated skin, swelling extensively, but being removed intact. In in vitro delivery experiments across excised neonatal porcine skin, approximately 44 mg of the model high dose small molecule drug ibuprofen sodium was delivered in 24 h, equating to 37% of the loading in the lyophilised reservoir. The super swelling microneedles delivered approximately 1.24 mg of the model protein ovalbumin over 24 h, equivalent to a delivery efficiency of approximately 49%. The integrated microneedle-lyophilised wafer delivery system produced a progressive increase in plasma concentrations of ibuprofen sodium in rats over 6 h, with a maximal concentration of approximately 179 µg/ml achieved in this time. The plasma concentration had fallen to 71±6.7 µg/ml by 24 h. Ovalbumin levels peaked in rat plasma after only 1 hour at 42.36±17.01 ng/ml. Ovalbumin plasma levels then remained almost constant up to 6 h, dropping somewhat at 24 h, when 23.61±4.84 ng/ml was detected. This work represents a significant advancement on conventional microneedle systems, which are presently only suitable for bolus delivery of very potent drugs and vaccines. Once fully developed, such technology may greatly expand the range of drugs that can be delivered transdermally, to the benefit of patients and industry. Accordingly, we are currently progressing towards clinical evaluations with a range of candidate molecules.     

Vitamin E in human skin: organ-specific physiology and considerations for its use in dermatology

Vitamin E has been used for more than 50 years in experimental and clinical dermatology. While a large number of case reports were published in this time, there is still a lack of controlled clinical studies providing a rationale for well defined dosages and clinical indications. In contrast, advances in basic research on the physiology, mechanism of action, penetration, bioconversion and photoprotection of vitamin E in human skin has led to the development of numerous new formulations for use in cosmetics and skin care products. This article reviews basic mechanisms and possible cosmetic as well as clinical implications of the recent advances in cutaneous vitamin E research. Experimental evidence suggests that topical and oral vitamin E has antitumorigenic, photoprotective, and skin barrier stabilizing properties. While the current use of vitamin E is largely limited to cosmetics, controlled clinical studies for indications such as atopic dermatitis or preventions of photocarcinogenesis are needed to evaluate the clinical benefit of vitamin E.     

In Vivo Electroporation of Minicircle DNA as a Novel Method of Vaccine Delivery To Enhance HIV-1-Specific Immune Responses

DNA vaccines offer advantage over conventional vaccines, as they are safer to use, easier to produce, and able to induce humoral as well cellular immune responses. Unfortunately, no DNA vaccines have been licensed for human use for the difficulties in developing an efficient and safe in vivo gene delivery system. In vivo electroporation (EP)-based DNA delivery has attracted great attention for its potency to enhance cellular uptake of DNA vaccines and function as an adjuvant. Minicircle DNA (a new form of DNA containing only a gene expression cassette and lacking a backbone of bacterial plasmid DNA) is a powerful candidate of gene delivery in terms of improving the levels and the duration of transgene expression in vivo. In this study, as a novel vaccine delivery system, we combined in vivo EP and the minicircle DNA carrying a codon-optimized HIV-1 gag gene (minicircle-gag) to evaluate the immunogenicity of this system. We found that minicircle-gag conferred persistent and high levels of gag expression in vitro and in vivo. The use of EP delivery further increased minicircle-based gene expression. Moreover, when delivered by EP, minicircle-gag vaccination elicited a 2- to 3-fold increase in cellular immune response and a 1.5- to 3-fold augmentation of humoral immune responses compared with those elicited by a pVAX1-gag positive control. Increased immunogenicity of EP-assisted minicircle-gag may benefit from increasing local antigen expression, upregulating inflammatory genes, and recruiting immune cells. Collectively, in vivo EP of minicircle DNA functions as a novel vaccine platform that can enhance efficacy and immunogenicity of DNA vaccines.