Transdermal drug delivery systems for fighting common viral infectious diseases.

Transdermal drug delivery systems (TDDS) have many advantages and represent an excellent alternative to oral delivery and hypodermic injections. TDDS are more convenient and less invasive tools for disease and viral infection treatment, prevention, detection, and surveillance. The emerging development of microneedles for TDDS has facilitated improved skin barrier penetration for the delivery of macromolecules or hydrophilic drugs. Microneedle TDDS patches can be fabricated to deliver virus vaccines and potentially provide a viable alternative vaccine modality that offers improved immunogenicity, thermostability, simplicity, safety, and compliance as well as sharp-waste reduction, increased cost-effectiveness, and the capacity for self-administration, which could improve vaccine distribution. These advantages make TDDS-based vaccine delivery an especially well-suited option for treatment of widespread viral infectious diseases including pandemics. Because microneedle-based bioassays employ transdermal extraction of interstitial fluid or blood, they can be used as a minimally invasive approach for surveying disease markers and providing point-of-care (POC) diagnostics. For cutaneous viral infections, TDDS can provide localized treatment with high specificity and less systemic toxicity. In summary, TDDS, especially those that employ microneedles, possess special attributes that can be leveraged to reduce morbidity and mortality from viral infectious diseases. In this regard, they may have considerable positive impact as a modality for improving global health. In this article, we introduce the possible role and summarize the current literature regarding TDDS applications for fighting common cutaneous or systemic viral infectious diseases, including herpes simplex, varicella or herpes zoster, warts, influenza, measles, and COVID-19.

Microneedle Mediated Transdermal Delivery of Protein, Peptide and Antibody Based Therapeutics: Current Status and Future Considerations.

The success of protein, peptide and antibody based therapies is evident - the biopharmaceuticals market is predicted to reach $388 billion by 2024 [1], and more than half of the current top 20 blockbuster drugs are biopharmaceuticals. However, the intrinsic properties of biopharmaceuticals has restricted the routes available for successful drug delivery. While providing 100% bioavailability, the intravenous route is often associated with pain and needle phobia from a patient perspective, which may translate as a reluctance to receive necessary treatment. Several non-invasive strategies have since emerged to overcome these limitations. One such strategy involves the use of microneedles (MNs), which are able to painlessly penetrate the stratum corneum barrier to dramatically increase transdermal drug delivery of numerous drugs. This review reports the wealth of studies that aim to enhance transdermal delivery of biopharmaceutics using MNs. The true potential of MNs as a drug delivery device for biopharmaceuticals will not only rely on acceptance from prescribers, patients and the regulatory authorities, but the ability to upscale MN manufacture in a cost-effective manner and the long term safety of MN application. Thus, the current barriers to clinical translation of MNs, and how these barriers may be overcome are also discussed.

Techniques for analyzing gene expression using BAC-based reporter constructs.

To characterize the complex regulatory control of gene expression using fluorescent protein reporters, it is often necessary to analyze large genomic regions. Bacteria artificial chromosome (BAC) vectors, which are able to support DNA fragments of up to 300kb, provide stable platforms for experimental manipulation. Using phage-based systems of homologous recombination, BACs can be efficiently engineered for a variety of aims. These include expressing fluorescent proteins to delineate gene expression boundaries using high-resolution, in vivo microscopy, tracing cell lineages using stable fluorescent proteins, perturbing endogenous protein function by expressing dominant negative forms, interfering with development by mis-expressing transcription factors, and identifying regulatory regions through deletion analysis. Here, we present a series of protocols for identifying BAC clones that contain genes of interest, modifying BACs for use as reporter constructs, and preparing BAC DNA for microinjection into fertilized eggs. Although the protocols here are tailored for use in echinoderm embryonic and larval stages, these methods are easily adaptable for use in other transgenic systems. As fluorescent protein technology continues to expand, so do the potential applications for recombinant BACs.

Recent advances in microneedle-based drug delivery: Special emphasis on its use in paediatric population.

Transdermal drug delivery offers several attractive advantages over the traditional oral and parenteral routes. Particularly, in case of paediatric patients, it helps to overcome the issues specific to this population, such as difficulty in swallowing and palatability of oral medicines as well as fear and pain associated with needles. However, due to the formidable barrier characteristic of the stratum corneum, it fails in the effective systemic delivery of broad range of therapeutic molecules, especially macromolecules and genetic material. Over the last two decades, microneedle technology has been portrayed as a strategy to infringe the stratum corneum, in a minimally invasive manner, and enable the successful passage of molecules by creating transient channels across the skin. There has been an exponential surge in the number of studies exploring the design, development and fabrication of microneedles. This article reviews the evolution of microneedle technology and provides a comprehensive summary of microneedle research to date. It provides a detailed overview of the microneedle types, advanced fabrication strategies including the biodegradability and compatibility of the new materials used in fabrication. Research on microneedle-mediated paediatric drug delivery as well as insights on the application of this novel technology has been discussed. The up-to-date progress in clinical translation of microneedles and the regulatory requirements for their commercialization are highlighted along with a brief perspective on the future prospects of microneedle-mediated paediatric drug delivery. This review proposes that advanced research can further contribute to the improved therapeutic efficiency of microneedle-based delivery of numerous molecules, which are otherwise difficult to administer via the conventional transdermal delivery mechanisms.

Microinjection and Micromanipulation: A Historical Perspective.

Microinjection/micromanipulation is more than 100 years old. It is a technique that is instrumental in biomedical research and healthcare. Its longevity lies in its preciseness in mechanical retrieval, or delivery of biological materials, which in some cases is simply necessary or more effective than other retrieval/delivery means. Microinjection is favored for its straightforwardness in transferring contents from micromolecules to macromolecules and from organelles to cells. Microinjection/micromanipulation has been practiced over the century like an art form. Variations in handlings and instruments can be tolerated to a surprising degree with satisfactory outcomes. Throughout the century, microinjection developed as an indispensable tool along with the evolution of biomedical fields: from transgenics to gene targeting, from animal cloning to human infertility treatment, from nuclease-guided genetic engineering to RNA-guided genome editing (Fig. 1). The birth of the CRISPRology rejuvenated microinjection. For microinjection/micromanipulation, the second century has already begun with the early arrival of computerized instrumentation and lately of the high-throughput nanomanipulators potentially operable by artificial intelligence. As we yin-yang both systemic and precision approaches in research and medicine, microinjection will no doubt continue to find its unique place in the future.

The ICSI procedure from past to future: a systematic review of the more controversial aspects.

BACKGROUND: ICSI is currently the most commonly used assisted reproductive technology, accounting for 70-80% of the cycles performed. This extensive use, even excessive, is partly due to the high level of standardization reached by the procedure. There are, however, some aspects that deserve attention and can still be ameliorated. The aim of this systematic review was to evaluate the results of available publications dealing with the management of specific situations during ICSI in order to support embryologists in trying to offer the best laboratory individualized treatment. METHODS: This systematic review is based on material obtained by searching PUBMED between January 1996 and March 2015. We included peer-reviewed, English-language journal articles that have evaluated ICSI outcomes in the case of (i) immature oocytes, (ii) oocyte degeneration, (iii) timing of the various phases, (iv) polar body position during injection, (v) zona-free oocytes, (vi) fertilization deficiency, (vii) round-headed sperm, (viii) immotile sperm and (ix) semen samples with high DNA fragmentation. RESULTS: More than 1770 articles were obtained, from which only 90 were specifically related to the issues developed for female gametes and 55 for the issues developed for male gametes. The studies selected for this review were organized in order to provide a guide to overcome roadblocks. According to these studies, the injection of rescue metaphase I oocytes should be discouraged due to poor clinical outcomes and a high aneuploidy rates; laser-assisted ICSI represents an efficient method to solve the high oocyte degeneration rate; the optimal ICSI timing and the best polar body position during the injection have not been clarified; injected zona-free oocytes, if handled carefully, can develop up to blastocyst stage and implant; efficient options can be offered to patients who suffered fertilization failure in previous conventional ICSI cycles. Most controversial and inconclusive are data on the best method to select a viable spermatozoa when only immotile spermatozoa are available for ICSI and, to date, there is no reliable approach to completely filter out spermatozoa with fragmented DNA from an ejaculate. However, most of the studies do not report essential clinical outcomes, such as live birth, miscarriage and fetal abnormality rate, which are essential to establish the safety of a procedure. CONCLUSIONS: This review provides the current knowledge on some controversial technical aspects of the ICSI procedures in order to improve its efficacy in specific contexts. Notwithstanding that embryologists might benefit from the approaches presented herein in order to improve ICSI outcomes, this area of expertise still demands a greater number of well-designed studies, especially in order to solve open issues about the safety of these procedures.

Microneedles for drug delivery: trends and progress.

In recent years, there has been a surge in the research and development of microneedles (MNs), a transdermal delivery system that combines the technology of transdermal patches and hypodermic needles. The needles are in the hundreds of micron length range and therefore allow relatively little or no pain. For example, biodegradable MNs have been researched in the literature and have several advantages compared with solid or hollow MNs, as they produce non-sharp waste and can be designed to allow rapid or slow release of drugs. However, they also pose a disadvantage as successful insertion into the stratum corneum layer of the skin relies on sufficient mechanical strength of the biodegradable material. This review looks at the various technologies developed in MN research and shows the rapidly growing numbers of research papers and patent publications since the first invention of MNs (using time series statistical analysis). This provides the research and industry communities a valuable synopsis of the trends and progress being made in this field.

Optimizing the success of cell transplantation therapy for stroke.

Stem cell transplantation has evolved as a promising experimental treatment approach for stroke. In this review, we address the major hurdles for successful translation from basic research into clinical applications and discuss possible strategies to overcome these issues. We summarize the results from present pre-clinical and clinical studies and focus on specific areas of current controversy and research: (i) the therapeutic time window for cell transplantation; (ii) the selection of patients likely to benefit from such a therapy; (iii) the optimal route of cell delivery to the ischemic brain; (iv) the most suitable cell types and sources; (v) the potential mechanisms of functional recovery after cell transplantation; and (vi) the development of imaging techniques to monitor cell therapy.

Endovascular management of spinal dural arteriovenous fistulas. A review.

OBJECT: Spinal dural arteriovenous fistulas (DAVFs) are the most common spinal vascular malformations and can be a significant cause of myelopathy, yet remain inefficiently diagnosed lesions. Over the last several decades, the treatment of spinal DAVFs has improved tremendously due to improvements in neuroimaging, microsurgical, and endovascular techniques. The aim of this paper was to review the existing literature regarding the clinical characteristics, classification, and endovascular management of spinal DAVFs. METHODS: A search of the PubMed database from the National Library of Medicine and reference lists of all relevant articles was conducted to identify all studies pertaining to spinal DAVFs, spinal dural fistulas, and spinal vascular malformations, with particular attention to endovascular management and outcomes. RESULTS: The ability to definitively treat spinal DAVFs using endovascular embolization has significantly improved over the last several decades. Overall rates of definitive embolization of spinal DAVFs have ranged between 25 and 100%, depending in part on the embolic agent used and the use of variable stiffness microcatheters. The majority of recent studies in which N-butyl cyanoacrylate or other liquid embolic agents were used have reported success rates of 70-90%. Surgical treatment remains the definitive option in cases of failed embolization, repeated recanalization, or lesions not amenable to embolization. Clinical outcomes have been comparable to surgical treatment when the fistula and draining vein remain persistently occluded. Improvements in gait and motor function are more likely following successful treatment, whereas micturition symptoms are less likely to improve. CONCLUSIONS: Endovascular embolization is an increasingly effective therapy in the treatment of spinal DAVFs, and can be used as a definitive intervention in the majority of patients that undergo modern endovascular intervention. A multidisciplinary approach to the treatment of these lesions is required, as surgery is required for refractory cases or those not amenable to embolization. Newer embolic agents, such as Onyx, hold significant promise for future therapy, yet long-term follow-up studies are required.

Microinjection as a tool of mechanical delivery.

Microinjection to single cells has been widely used in the studies of transduction-challenged cells, transgenic animal production, and in vitro fertilization to mechanically transfer DNAs, RNA interferences, sperms, proteins, peptides, and drugs. The advantages of microinjection include the precision of delivery dosage and timing, high efficiency of transduction as well as low cytotoxicity. However, manual microinjection is labor intensive and time consuming, which limits the application of this technique to large number of cells in a sample. New cell culture matrix ensuring all cells grow in a desired position and orientation is needed for application of high throughput automatic injection systems, which will significantly increase injection speed, cell survival, and success rates.

Microneedles for transdermal drug delivery: a minireview.

The stratum corneum is the main barrier for transdermal drug transport. It could be bypassed by microneedles, which have a length of a few tens to a few hundreds of microns. They are usually arranged in arrays and can be used in several ways to enhance transdermal drug transport. Microneedles can be inserted into the skin in order to increase its permeability, after which the drug is applied (poke with patch). Drugs could also be coated onto the microneedles and be inserted into the skin (coat and poke). Hollow microneedles are used to inject drug solutions in to the skin. This review aims to discuss recently published in vivo and in vitro studies on microneedle aided transdermal drug delivery.

Exogenous DNA expression in eukaryotic cells following microinjection.

Microinjection of nucleic acids, DNA, RNA, proteins, and any soluble material into living eukaryotic cells makes it possible to design experiments focused on single cells. In contrast facilitated transfer protocols requires hundreds of thousands of cells from which the expressed gene or intracellular effect must be detected within the culture. In addition to the immediate observable nature of the expressed product and intracellular reaction, microinjection bypasses the uptake toxicity associated with facilitated transfer of foreign material into cultured cells. The direct injection of material into the nucleus or cytoplasm allows the number of treated cells to be monitored and expression efficiencies to be observed directly. Microinjection of a hundred cells grown on small glass coverslips and subsequently counted for expression of the foreign material determines expression efficiency as a percentage of cells injected. The efficiency is based on detection of the foreign inserted gene product and does not control for relative promoter efficiency between constructs. The purpose is not to compare two constructs to each other but to monitor dual expression. The creation of marker fluorescent proteins, such as the green fluorescent protein (GFP) in the same expression plasmid with a test gene allows the immediate observation of the GFP injected cells and within the same cells the positive or negative expression of the test gene. Expression of a foreign gene, such as SV40 T antigen cloned into an expression vector can be detected four hours after microinjection of the DNA. Fusing GFP into the same expression region of the T coding sequence labels T-GFP as a fusion protein with characteristic T immunological staining nuclear patterns but allows the cells to be studied without fixation through sequential periods of observation. The direct nature of microinjection allows comparison of gene expression in a variety of cells and the determination of the number of cells expressing the exogenous material in relationship to the number of cells injected.

Progress in reproductive biotechnology in swine.

This article summarizes recent progress in reproductive biotechnology in swine with special reference to in vitro production of embryos, generation of identical multiples, and transgenic pigs useful for xenotransplantation. In vitro production (in vitro maturation, in vitro fertilization, and in vitro culture) of viable porcine embryos is possible, although with much lower success rates than in cattle. The main problems are insufficient cytoplasmic maturation of porcine oocytes, a high proportion of polyspermic fertilization and a low proportion of blastocysts that, in addition, are characterized by a low number of cells, hampering their development in vivo upon transfer to recipients. Microsurgical bisection of morula and blastocyst stage embryos leads to a 2 to 3% monozygotic twinning rate of the transferred demiembryos, which is similar to that in rabbits and mice but considerably lower than in ruminants. It was found that with decreasing quality an increasing proportion of demi-embryos did not possess an inner cell mass. Porcine individual blastomeres derived from 4- and 8-cell embryos can be cultured in defined medium to the blastocyst stage. Leukemia inhibitory factor has been shown to be effective at defined embryonic stages and supports the formation of the inner cell mass in cultured isolated blastomeres in a concentration-dependent manner. For maintaining pregnancies with micromanipulated porcine embryos, it is not necessary to transfer extraordinarily high numbers of embryos. Porcine nuclear transfer is still struggling from the inefficiency of producing normally functioning blastocysts. Blastomeres, blastocyst-derived cells, fibroblasts and granulosa cells have been employed as donor cells in porcine nuclear transfer and have yielded blastocysts. Recently, the generation of the first piglets from somatic cell nuclear transfer has been achieved. DNA-microinjection into pronuclei of porcine zygotes has reliably resulted in the generation of transgenic pigs, which have special importance for the production of valuable pharmaceutical proteins in milk and xenotransplantation. It has been demonstrated that by expression of human complement regulatory proteins in transgenic pigs the hyperacute rejection response occurring after xenotransplantation can be overcome in a clinically relevant manner. Although biotechnological procedures in swine have recently undergone tremendous progress, the development is still lagging behind that in cattle and sheep. With regard to genetic engineering, considerable progress will originate from the possibility of employing homologous recombination in somatic cell lines and their subsequent use in nuclear transfer. In combination with the increasing knowledge in gene sequences this will allow in the foreseeable future widespread use in the pig industry either for agricultural or biomedical purposes.

[Future of the treatment of male infertility]. / Avenir du traitement de l'infertilité masculine.

The aim of this paper is to introduce inherent difficulties leading to relative delay in the field of male infecundity treatment. Four theoretical possibilities are exposed: infertility prevention improvement, qualitative or quantitative improvement of spermatogenesis, spermatozoa fecundity ability improvement and micro-fertilization procedures. These methods are exposed in their technical aspects and in their results. Critical analysis is conducted about some impressive reported series. Basic knowledge improvement seems to be necessary to explore new therapeutic ways.