微针条件下秦艽复杂成分体系的透皮给药研究

本文探讨了秦艽复杂成分体系在微针条件下透过不同部位皮肤的给药特点。采用双室扩散池,分别以离体小鼠不同部位皮肤和微针预处理离体小鼠不同部位的皮肤作为透皮屏障,用高效液相色谱(HPLC)相似度评价接收池中秦艽复杂成分体系的透皮特点及小鼠不同部位皮肤的龙胆苦苷的透过速率和透过量。被动给药和微针条件下给药24 h,接受池中秦艽复杂成分体系与原液相似度基本在83.0%～98.9%之间;微针预处理小鼠不同部位皮肤后,通过腹部皮肤给药接收液中秦艽复杂成分体系与原液相似度达到90%的时间为4 h;而背部皮肤和颈部皮肤给药时,则接收液中秦艽复杂成分体系与原液相似度达到90%的时间分别为18 h和12 h。微针能较理想的用于中药复杂成分的透皮给药,极大地缩短透皮吸收的时滞,提高其生物利用度;相对颈部和背部皮肤,微针通过腹部皮肤更能显著提高中药多成分透皮给药的速率及与原液的相似度。     

微针透皮给药系统应用研究进展

微针透皮给药系统是近年来透皮给药系统研究的热点。微针透皮给药系统具有注射给药和透皮给药的双重优势,具有快速、方便、无痛等众多优点,研究表明可以显著提高药物透皮速率和吸收量,特别是在蛋白质、多肽、DNA和RNA等大分子物质的透皮制剂研究领域表现出良好的效果和应用前景,本文对微针透皮给药系统应用研究的最新进展进行了综述。随着微针加工技术、载药技术和应用研究的不断深入,微针透皮技术在临床领域将会有更广泛的应用。     

Minimally invasive cutaneous delivery of macromolecules and plasmid DNA via microneedles

The stratum corneum (SC) represents a significant barrier to the delivery of gene therapy formulations. In order to realise the potential of therapeutic cutaneous gene transfer, delivery strategies are required to overcome this exclusion effect. This study investigates the ability of microfabricated silicon microneedle arrays to create micron-sized channels through the SC of ex vivo human skin and the resulting ability of the conduits to facilitate localised delivery of charged macromolecules and plasmid DNA (pDNA). Microscopic studies of microneedle-treated human epidermal membrane revealed the presence of microconduits (10-20 microm diameter). The delivery of a macromolecule, beta-galactosidase, and of a 'non-viral gene vector mimicking' charged fluorescent nanoparticle to the viable epidermis of microneedle-treated tissue was demonstrated using light and fluorescent microscopy. Track etched permeation profiles, generated using 'Franz-type' diffusion cell methodology and a model synthetic membrane showed that >50% of a colloidal particle suspension permeated through membrane pores in approximately 2 hours. On the basis of these results, it is probable that microneedle treatment of the skin surface would facilitate the cutaneous delivery of lipid:polycation:pDNA (LPD) gene vectors, and other related vectors, to the viable epidermis. Preliminary gene expression studies confirmed that naked pDNA can be expressed in excised human skin following microneedle disruption of the SC barrier. The presence of a limited number of microchannels, positive for gene expression, indicates that further studies to optimise the microneedle device morphology, its method of application and the pDNA formulation are warranted to facilitate more reproducible cutaneous gene delivery.     

Cutaneous gene expression of plasmid DNA in excised human skin following delivery via microchannels created by radio frequency ablation

The skin is a valuable organ for the development and exploitation of gene medicines. Delivering genes to skin is restricted however by the physico-chemical properties of DNA and the stratum corneum (SC) barrier. In this study, we demonstrate the utility of an innovative technology that creates transient microconduits in human skin, allowing DNA delivery and resultant gene expression within the epidermis and dermis layers. The radio frequency (RF)-generated microchannels were of sufficient morphology and depth to permit the epidermal delivery of 100 nm diameter nanoparticles. Model fluorescent nanoparticles were used to confirm the capacity of the channels for augmenting diffusion of macromolecules through the SC. An ex vivo human organ culture model was used to establish the gene expression efficiency of a beta-galactosidase reporter plasmid DNA applied to ViaDerm treated skin. Skin treated with ViaDerm using 50 microm electrode arrays promoted intense levels of gene expression in the viable epidermis. The intensity and extent of gene expression was superior when ViaDerm was used following a prior surface application of the DNA formulation. In conclusion, the RF-microchannel generator (ViaDerm) creates microchannels amenable for delivery of nanoparticles and gene therapy vectors to the viable region of skin.     

Gene Delivery to the Epidermal Cells of Human Skin Explants Using Microfabricated Microneedles and Hydrogel Formulations

Purpose Microneedles disrupt the stratum corneum barrier layer of skin creating transient pathways for the enhanced permeation of therapeutics into viable skin regions without stimulating pain receptors or causing vascular damage. The cutaneous delivery of nucleic acids has a number of therapeutic applications; most notably genetic vaccination. Unfortunately non-viral gene expression in skin is generally inefficient and transient. This study investigated the potential for improved delivery of plasmid DNA (pDNA) in skin by combining the microneedle delivery system with sustained release pDNA hydrogel formulations. Materials and Methods Microneedles were fabricated by wet etching silicon in potassium hydroxide. Hydrogels based on Carbopol polymers and thermosensitive PLGA-PEG-PLGA triblock copolymers were prepared. Freshly excised human skin was used to characterise microneedle penetration (microscopy and skin water loss), gel residence in microchannels, pDNA diffusion and reporter gene (β-galactosidase) expression. Results Following microneedle treatment, channels of approximately 150–200 μm depth increased trans-epidermal water loss in skin. pDNA hydrogels were shown to harbour and gradually release pDNA. Following microneedle-assisted delivery of pDNA hydrogels to human skin expression of the pCMVβ reporter gene was demonstrated in the viable epidermis proximal to microchannels. Conclusions pDNA hydrogels can be successfully targeted to the viable epidermis to potentially provide sustained gene expression therein.     

Advances in skin gene therapy

Specific anatomical and biological properties make the skin a very interesting target organ for gene therapy approaches. Different cell types of the epidermis, such as keratinocytes, melanocytes, or dendritic cells, can be genetically modified to treat a broad spectrum of diseases, including genetically inherited skin disorders, tumour diseases, metabolic disorders and infectious diseases. The easy accessibility of skin suggests that different methods for gene delivery can be pursued, depending on the desired application. The approach used to deliver DNA to the skin will influence not only the efficiency of DNA delivery, but also the level and duration of transgene expression. Furthermore, the desired biological effect will also influence the decision of which gene transfer method is the best choice. Among the current challenges of cutaneous gene therapy are: optimising the efficiency of direct in vivo gene delivery; targeting specific epidermal cells, including keratinocyte stem cells; achieving sustained gene expression and regulating gene expression in vivo. This review summarises recent advances in the field of skin gene therapy and evaluates possible strategies to overcome obstacles and achieve successful clinical applications of skin gene therapy.     

Advances in skin gene therapy

Specific anatomical and biological properties make the skin a very interesting target organ for gene therapy approaches. Different cell types of the epidermis, such as keratinocytes, melanocytes, or dendritic cells, can be genetically modified to treat a broad spectrum of diseases, including genetically inherited skin disorders, tumour diseases, metabolic disorders and infectious diseases. The easy accessibility of skin suggests that different methods for gene delivery can be pursued, depending on the desired application. The approach used to deliver DNA to the skin will influence not only the efficiency of DNA delivery, but also the level and duration of transgene expression. Furthermore, the desired biological effect will also influence the decision of which gene transfer method is the best choice. Among the current challenges of cutaneous gene therapy are: optimising the efficiency of direct in vivo gene delivery; targeting specific epidermal cells, including keratinocyte stem cells; achieving sustained gene expression and regulating gene expression in vivo. This review summarises recent advances in the field of skin gene therapy and evaluates possible strategies to overcome obstacles and achieve successful clinical applications of skin gene therapy.     

DNA delivery for vaccination and therapeutics through the skin

Cutaneous gene therapy and DNA vaccination are potential applications of plasmid delivery methods where a gene for an antigen or a therapeutic protein is inserted in the plasmid and applied to the skin. However, the delivery of the DNA plasmid is a major challenge due to the unusual physicochemical properties of the DNA, the tissue and cellular barriers and expression difficulties. Even though the skin is the most accessible organ of the body and it is an ideal target for gene therapy, the delivery of plasmid DNA across the skin is very difficult due to the specific barrier function of the stratum corneum and the inconsistent transfection rate of keratinocytes and other epidermal cells. To date there is no gene delivery system that was shown to be optimal for cutaneous gene therapy. In order to develop an efficient non-viral delivery vehicle we need to design a system that provides the combined properties of effective DNA condensation, cutaneous permeation, cellular transfection and sufficiently sustained expression. This paper reviews the formulation approaches and delivery methods for DNA through the skin in the context of the barriers both at the tissue and cellular levels for both vaccine and gene therapy applications.     

Topical Gene Transfer into Rat Skin Using Electroporation

Purpose. To investigate whether electroporation can be used for topical gene delivery and for DNA expression in rat keratinocytes.Methods. The localization of a fluorescent-labelled plasmid and the expression of a reporter gene (pEGFP-N1) coding for Green Fluorescent Protein (GFP) in stripped skin were assessed by Confocal Laser Scanning Microscopy (CLSM).Results. The plasmid penetrated into the epidermis within minutes after electroporation and entered the keratinocyte cytoplasm within hours. A localized expression of GFP was observed for at least 7 days in the epidermis. Skin viability was not compromised by electroporation.Conclusions. Electroporation enhances the delivery, and hence the expression, of topically applied plasmid DNA on the skin. It could be a promising alternative method to administer DNA, particularly for DNA vaccines, in the skin in vivo.     

Microneedles and other physical methods for overcoming the stratum corneum barrier for cutaneous gene therapy

The outermost layer of skin, the epidermis, has developed formidable physical and immunological barrier properties that prevent infiltration of deleterious chemicals and pathogens. Consequently, transdermal delivery of medicaments is currently restricted to a limited number of low molecular weight drugs. As a corollary, there has been significant recent interest in providing strategies that disrupt or circumvent the principal physical barrier, the stratum corneum, for the efficient cutaneous delivery of macromolecular and nucleic acid based therapeutics. These strategies include: electrical methods, intradermal injection, follicular delivery, particle acceleration, laser ablation, radiofrequency ablation, microscission, and microneedles. The application of microfabricated microneedle arrays to skin creates transient pathways to enable transcutaneous delivery of drugs and macromolecules. Microneedle use is simple, pain-free, and causes no bleeding, with further advantages of convenient manufacture, distribution, and disposal. To date, microneedles have been shown to deliver drug, peptide, antigen, and DNA efficiently through skin. Robust and efficient microneedle designs and compositions can be inserted into the skin without fracture. Further progress in microneedle array design, microneedle application apparatus, and integrated formulation will confirm this methodology as a realistic clinical strategy for delivering a range of medicaments, including DNA, to and through skin.     

Epidermal delivery of protein and DNA vaccines

Targeting vaccines to the skin epidermis results in the activation of an immune inductive site that is rich in antigen-presenting cells. The superficial location of the skin makes it accessible to vaccine delivery. However, it is difficult to access the epidermis using needle and syringe delivery, and vaccine antigens are too large to be effectively delivered using standard topical formulations. Needle-free vaccine delivery systems have been developed for efficient delivery of particulate vaccines into the epidermal tissue. Particle-mediated epidermal delivery of DNA vaccines is based on the delivery of DNA-coated gold particles directly into the cytoplasm and nuclei of living cells of the epidermis, facilitating DNA delivery and gene expression. Alternatively, protein vaccines can be formulated into a dense powder, which can be propelled into the skin epidermis by epidermal powder immunisation using similar delivery devices and principles, but in this instance the protein is delivered to the extracellular space. Preclinical and clinical data will be reviewed, demonstrating applications of epidermal vaccine delivery to a wide range of experimental infectious disease vaccines.     

Epidermal delivery of protein and DNA vaccines

Targeting vaccines to the skin epidermis results in the activation of an immune inductive site that is rich in antigen-presenting cells. The superficial location of the skin makes it accessible to vaccine delivery. However, it is difficult to access the epidermis using needle and syringe delivery, and vaccine antigens are too large to be effectively delivered using standard topical formulations. Needle-free vaccine delivery systems have been developed for efficient delivery of particulate vaccines into the epidermal tissue. Particle-mediated epidermal delivery of DNA vaccines is based on the delivery of DNA-coated gold particles directly into the cytoplasm and nuclei of living cells of the epidermis, facilitating DNA delivery and gene expression. Alternatively, protein vaccines can be formulated into a dense powder, which can be propelled into the skin epidermis by epidermal powder immunisation using similar delivery devices and principles, but in this instance the protein is delivered to the extracellular space. Preclinical and clinical data will be reviewed, demonstrating applications of epidermal vaccine delivery to a wide range of experimental infectious disease vaccines.     

Skin targeted DNA vaccine delivery using electroporation in rabbits. I: efficacy

Genetic immunization through skin is highly desirable as skin has plenty of antigen presenting cells (APCs) and is easily accessible. The purpose of this study was to investigate the effects of electroporation pulse amplitude, pulse length and number of pulses on cutaneous plasmid DNA vaccine delivery and immune responses, following intradermal injection in vivo in rabbits. Expression of the delivered plasmid was studied using a reporter plasmid, coding for beta-galactosidase. The efficiency of DNA vaccine delivery was investigated using a DNA vaccine against Hepatitis B, coding for Hepatitis B surface antigen (HBsAg). Serum samples and peripheral blood mononuclear cells (PBMC) were analyzed for humoral and cellular immunity, respectively, following immunization. The expression of transgene in the skin was transient and reached its peak in 2 days post-delivery with 200 and 300 V pulses. The expression levels with 200 and 300 V pulses were 48- and 129-fold higher, respectively, compared with the passive on day 2. In situ histochemical staining of skin with X-gal demonstrated the localized expression of beta-galactosidase with electroporation pulses of 200 and 300 V. Electroporation mediated cutaneous DNA vaccine delivery significantly enhanced both humoral and cellular immune responses (p<0.05) to Hepatitis B compared to passive delivery. The present study demonstrates the enhanced DNA vaccine delivery to skin and immune responses by topical electroporation. Hence, electroporation mediated cutaneous DNA vaccine delivery could be developed as a potential alternative for DNA vaccine delivery.     

Skin targeted DNA vaccine delivery using electroporation in rabbits II. Safety

The Achilles heel of gene-based therapy is gene delivery into the target cells efficiently with minimal toxic effects. Viral vectors for gene/DNA vaccine delivery are limited by the safety and immunological problems. Recently, nonviral gene delivery mediated by electroporation has been shown to be efficient in different tissues including skin. There are no detailed reports about the effects of electroporation on skin tissue, when used for gene/DNA vaccine delivery. In a previous study we demonstrated the efficacy of skin targeted DNA vaccine delivery using electroporation in rabbits [Medi, B.M., Hoselton, S., Marepalli, B.R., Singh, J., 2005. Skin targeted DNA vaccine delivery using electroporation in rabbits. I. Efficacy. Int. J. Pharm. 294, 53-63]. In the present study, we investigated the safety aspects of the electroporation technique in vivo in rabbits. Different electroporation parameters (100-300 V) were tested for their effects on skin viability, macroscopic barrier property, irritation and microscopic changes in the skin. Skin viability was not affected by the electroporation protocols tested. The electroporation pulses induced skin barrier perturbation and irritation as indicated by elevated transepidermal water loss (TEWL) and erythema/edema, respectively. Microscopic studies revealed inflammatory responses in the epidermis following electroporation using 200 and 300 V pulses. However, these changes due to electroporation were reversible within a week. The results suggest that the electroporation does not induce any irreversible changes in the skin and can be a useful technique for skin targeted DNA vaccine delivery.     

The role of particle-mediated DNA vaccines in biodefense preparedness

Particle-mediated epidermal delivery (PMED) of DNA vaccines is based on the acceleration of DNA-coated gold directly into the cytoplasm and nuclei of living cells of the epidermis, facilitating DNA delivery and gene expression. Professional antigen-presenting cells and keratinocytes in the skin are both targeted, resulting in antigen presentation via direct transfection and cross-priming mechanisms. Only a small number of cells need to be transfected to elicit humoral, cellular and memory responses, requiring only a low DNA dose. In recent years, data have accumulated on the utility of PMED for delivery of DNA vaccines against a number of viral pathogens, including filoviruses, flaviviruses, poxviruses, togaviruses and bunyaviruses. PMED DNA immunization of rodents and nonhuman primates results in the generation of neutralizing antibody, cellular immunity, and protective efficacy against a broad range of viruses of public health concern.     

Skin hydration and possible shunt route penetration in controlled estradiol delivery from ultradeformable and standard liposomes.

Human skin delivery of estradiol from ultradeformable and traditional liposomes was explored, comparing occlusive and open application, with the aim of examining the role of skin hydration. Partially hydrated epidermis was used for open hydration, but fully hydrated membranes were used for occluded studies. In addition, we developed a novel technique to investigate the role of shunt route penetration in skin delivery of liposomal estradiol. This compared delivery through epidermis with that through a stratum corneum (SC)/epidermis sandwich from the same skin with the additional SC forming the top layer of the sandwich. This design was based on the fact that orifices of shunts only occupy 0.1% of skin surface area and thus for SC/epidermis sandwiches there will be a negligible chance for shunts to superimpose. The top SC thus blocks most shunts available on the bottom membrane. If shunts play a major role then the delivery through sandwiches should be much reduced compared with that through epidermis, taking into consideration the expected reduction owing to increased membrane thickness. After open application, both ultradeformable and traditional liposomes improved estradiol skin delivery, with the ultradeformable liposomes being superior. Occlusion reduced the delivering efficiency of both vesicle types, supporting the theory that a hydration gradient provides the driving force. Shunt route penetration was found to play only a very minor role in liposomal delivery. In conclusion, full hydration of skin reduces estradiol delivery from liposomes and the shunt route is not the main pathway for this delivery.     

Epidermal cell culture model with tight stratum corneum as a tool for dermal gene delivery studies

The purpose of this study was to evaluate the feasibility of organotypic cultures of rat epidermal cells as a tool to study non-invasive dermal gene delivery. Also, a novel transfection method employing liposomal pre-treatment of stratum corneum (SC) was evaluated. Rat epidermal cells were cultured on Transwell tissue culture inserts and formation of stratum corneum barrier was evaluated in permeability studies with two model compounds. Transfections were performed with naked pCMV-SEAP2 plasmid and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP)/dioleyl-phosphatidylethanolamine (DOPE)/DNA lipoplexes. Naked DNA was administered on the stratum corneum of the cell culture model with or without prior treatment of the stratum corneum with DOTAP/DOPE liposomes. Transfection was evaluated non-invasively by monitoring concentrations of secreted alkaline phosphatase (SEAP) in the culture medium of the basolateral compartment at 24-h intervals. Transfection with lipoplexes produced significant gene expression in rat epidermal keratinocyte (REK) epidermal culture model. Likewise, delivery of naked DNA on stratum corneum after DOTAP/DOPE liposome pre-treatment produced gene expression. Naked DNA alone did not result in detectable gene expression. In dermal gene delivery studies REK epidermal culture model is a suitable tool that includes tight stratum corneum and allows transgene expression in viable epidermis and non-invasive sampling of secreted gene product in the basolateral compartment. Liposomal pre-treatment of the stratum corneum augments transfection of viable epidermis.     

Noninvasive delivery of siRNA into the epidermis by iontophoresis using an atopic dermatitis-like model rat

Topical application of siRNA to the skin should be an effective treatment for serious skin disorders, such as atopic dermatitis. However, it is difficult to introduce hydrophilic macromolecules, including siRNA, into the skin by conventional methods. For efficient delivery of siRNA, we examined an iontophoretic technique, since it is suitable for the delivery of charged molecules. Naked siRNA effectively accumulated in the epidermis (and not in the dermis) after iontophoretic delivery. In contrast, siRNA did not penetrate tape-stripped skin by passive diffusion. In a rat model of atopic dermatitis, skin was sensitized with ovalbumin to stimulate IL-10 mRNA expression as observed in skin lesions. Iontophoretic delivery of anti-IL-10 siRNA significantly reduced (73%) the level of IL-10 mRNA. In conclusion, we successfully delivered naked siRNA into the epidermis and concomitantly suppressed the expression of an endogenous immuno-regulatory cytokine.     

Enhancing effect of hydroxypropyl-beta-cyclodextrin on cutaneous penetration and activation of ethyl 4-biphenylyl acetate in hairless mouse skin.

The effect of hydroxypropyl-beta-cyclodextrin (HP-beta-CyD) on the cutaneous penetration and activation of ethyl 4-biphenylyl acetate (EBA), a prodrug of non-steroidal anti-inflammatory drug 4-biphenylylacetic acid (BPAA), from hydrophilic ointment was investigated, using hairless mouse skin in vitro. When the hydrophilic ointment containing a complex of EBA with HP-beta-CyD was applied to the full-thickness skin, HP-beta-CyD facilitated the penetration of EBA into the skin, the conversion of EBA to BPAA in the epidermis and the transfer of BPAA to the receptor phase. Under the present condition, pre- and post-application of the ointment containing HP-beta-CyD onto the skin did not affect the cutaneous penetration of EBA and its activation. When the ointment containing the EBA:HP-beta-CyD complex was applied to the skin, the flux of BPAA through the tape-stripped skin was greater than that through the full-thickness skin, while the activation of the prodrug in the skin was slowed down by the tape-stripping. When propylene glycol was used as a vehicle, HP-beta-CyD no longer enhanced the cutaneous permeation of BPAA through the full-thickness skin. These results suggest that the enhancing effect of HP-beta-CyD on the cutaneous penetration of EBA would be ascribable largely to an increase in effective concentration of EBA in the ointment. Furthermore, the slow diffusion of EBA solubilized in HP-beta-CyD through the stratum corneum, together with the vehicle effect, could make the prodrug more susceptible to the metabolic process that is active in the epidermis, eventually leading to the facilitated activation of the prodrug.     

Effect of lipophilicity on in vivo iontophoretic delivery. II. Beta-blockers.

The objective of this study was to investigate the relationship between drug lipophilicity and the transdermal absorption processes in the iontophoretic delivery in vivo. Anodal iontophoresis of beta-blockers as model drugs having different lipophilicity (atenolol, pindolol, metoprolol, acebutolol, oxprenolol and propranolol) was performed with rats (electrical current, 0.625 mA/cm2; application period, 90 min), and the drug concentrations in skin, cutaneous vein and systemic vein were determined. Increasing the lipophilicity of beta-blockers caused a greater absorption into the skin. Exceptionally, it was found that pindolol had high skin absorption, irrespective of its hydrophilic nature. Further, the drug transfer rate from skin to cutaneous vein (R(SC)) was evaluated from the arterio-venous plasma concentration difference of drug in the skin. Normalized R(SC) by skin concentration showed a negative correlation with the logarithm of n-octanol/buffer partition coefficient (Log P, pH 7.4), suggesting the partitioning between stratum corneum and viable epidermis was a primary process to determine the transfer properties of beta-blockers to local blood circulation. Pindolol exhibited both high skin absorption and high transfer from skin to cutaneous vein. These characteristics of pindolol could be explained by the chemical structure, molecular size and hydrophilicity. These findings for pindolol should be valuable for the optimal design of drug candidates for iontophoretic transdermal delivery.