Gene Delivery

Non-viral vectors such as cationic lipids are capable of delivering nucleic acids, including genes, siRNA or antisense RNA into cells, thus potentially resulting in their functional expression. These vectors are considered as an attractive alternative for virus-based delivery systems, which may suffer from immunological and mutational hazards. However, the efficiency of cationic-mediated gene delivery, although often sufficient for cell biological purposes, runs seriously short from a therapeutics point of view, as realizing this objective requires a higher level of transfection than attained thus far. To develop strategies for improvement, there is not so much a need for novel delivery systems. Rather, better insight is needed into the mechanism of delivery, including lipoplex–cell surface interaction, route of internalization and concomitant escape of DNA/RNA into the cytosol, and transport into the nucleus. Current work indicates that a major obstacle involves the relative inefficient destabilization of membrane-bounded compartments in which lipoplexes reside after their internalization by the cell. Such an activity requires the capacity of lipoplexes of undergoing polymorphic transitions such as a membrane destabilizing hexagonal phase, while cellular components may aid in this process. A consequence of the latter notion is that for development of a novel generation of delivery devices, entry pathways have to be triggered by specific targeting to select delivery into intracellular compartments which are most susceptible to lipoplex-induced destabilization, thereby allowing the most efficient release of DNA, a minimal requirement for optimizing non-viral vector-mediated transfection.

Solid lipid nanoparticles (SLNs) are a promising system for the delivery of lipophilic and hydrophilic drugs. They consist of a solid lipid core that is stabilized by a layer of surfactants. By the incorporation of cationic lipids in the formulation, positively charged SLNs can be generated, that are suitable carriers for nucleic acids (DNA, siRNA). Considering the beneficial effect of helper lipids on the transfection efficiency with cationic liposomes, the effect of the helper lipid 1,2dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) on transfection with cationic lipid-containing solid lipid nanoparticles was investigated in PC3 prostate cancer cells. The inclusion of DOPE in SLN formulations, instead of promoted, strongly inhibited SLN transfection efficiency, by frustrating the accommodation of DNA by the particles, as was revealed by biochemical analysis. SLNs devoid of DOPE maintained a homogenous size distribution of ∼ 150 nm following lipoplex assembly and cellular delivery, and showed transfection efficiency comparable to that of Lipofectamine 2000 ® (LF2k). Moreover, the SLNs maintain their high transfection efficiency after lyophilization and long-term storage (1-2 years), an important asset for biomedical applications. There is even the possibility to lyophilize the SLN carrier together with its DNA cargo, which represents an interesting pharmaceutical advantage of the SLN formulations over LF2k. These results reflect marked differences between the physicochemical properties of cationic liposomes and SLNs, the latter requiring more critical lipid-depending properties for effective 'packaging' of DNA but displaying a higher storage stability than cationic lipid based carriers like LF2k.

Adenovirus (Ad)-based vectors are considered a promising tool for effective gene transfer in a number of renal disease-specific applications. This opinion is based upon their natural ability to infect a broad array of both dividing and terminally differentiated, nondividing cell types, their capacity to deliver (transduce) large amounts of DNA, and the ease at which this vector platform can be mass produced. Furthermore, Ads remain the gene transfer vector of choice for numerous human clinical trials, as more clinical trials utilize Ad-based vectors than any other vector currently available. However, as with all types of gene transfer vectors, several limitations to the use of Ads have been delineated. The construction of advanced-generation Ad vectors with unique modifications of the viral genome has addressed many of these issues, although continued research will no doubt provide safer alternatives to the presently used viral vectors. In this chapter, we review the current state of Ad-based gene transfer, brief updates on advanced-generation Ad-based vectors, and provide a discussion of how these vectors infect various tissues. We then specifically focus on the kidney and discuss a multitude of techniques previously employed to deliver Ad-based vectors to various regions of the kidney and in the process, reveal many associated complexities. Furthermore, exciting new studies that use Ads to express immunosuppressive gene products have shown great promise in the area of transplantation and allograft survival. Based upon this summary, we confirm that Ad-based vectors currently offer multiple advantages for the study and potential treatment of a great variety of renal and urological diseases. Utilization of advanced-generation Ad vectors combined with novel insights into the complexities of Ad-based gene transfer in general, should allow numerous inroads to be made in the near term, relative to the use of Ad-based gene transfer to treat a variety of renal diseases.

Abstract: Toll-like receptors (TLRs) activate biochemical pathways that evoke activation of innate immunity, which leads to dendritic cell maturation and initiation of adaptive immune responses that provoke allograft rejection.

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The rate and character of superficial tissue regeneration after wounds, burns, and other traumas depend on cell proliferation within the damaged area. The acceleration of wound healing via the stimulation of cell proliferation and extracellular matrix synthesis is one of the most important tasks of modern medicine. There are gene therapy approaches to wound treatment, such as the transfer of genes that encode mitogenic growth factors to the wound area. The most important step in the development of the gene therapy approaches is the design of gene delivery tools. Despite the high efficiency of viral vectors, the nonviral approaches have some advantages (low toxicity, low immunogenity, safety, and the absence of side effects). Among the nonviral gene delivery tools molecular conjugates are the most popular due to their efficiency, simplicity, and the capacity for targeted gene transfer. In the present work, we have developed two molecular conjugates, NLS TSF7 and NLS TSF12, which consist of the modified signal of the nuclear localization of the T antigen of the SV40 virus (cationic part) and the peptide ligands of the mammalian transferrin receptor (ligand part). Those conjugates bind to plasmid DNA via the formation of polyelectrolytic complexes and are able to deliver plasmid DNA into cells that express transferrin receptors through receptor mediated endocy tosis. The transfer of the expression vector of the luciferase gene in the complex with the molecular conjugate NLS TSF7 to murine surface tissues led to about the 100 fold increase of luciferase activity in comparison with the transfer of the free expression vector. The treatment of mice with incised wounds with complexes of the expression vector of the synthetic human gene that encodes insulin like growth factor 1 with molecular conjugate NLS TSF7 led to the acceleration of wound healing in comparison with mice treated with the free expression vector. The obtained results confirm the high efficiency of the developed approach to regenerative gene therapy for treating the superficial tissue damage of mammals.

Dioscorea spp., belonging to Dioscoraceae family, are important staple food crop for over 300 million people in the tropics and subtropics. They are mostly grown for their starchy tubers (both cultivated and wild) and for medicinal properties. Cultivated yams are vegetatively propagated while wild yams are sexually propagated and comprise both diploid and polyploid species. Although yam is an economically

The purpose of this review is to discuss and summarize some of the interesting findings and applications of cyclodextrins (CDs) and their derivatives in different areas of drug delivery, particularly in protein and peptide drug delivery and gene delivery. The article highlights important CD applications in the design of various novel delivery systems like liposomes, microspheres, microcapsules, and nanoparticles. In addition to their well-known effects on drug solubility and dissolution, bioavailability, safety, and stability, their use as excipients in drug formulation are also discussed in this article. The article also focuses on various factors influencing inclusion complex formation because an understanding of the same is necessary for proper handling of these versatile materials. Some important considerations in selecting CDs in drug formulation such as their commercial availability, regulatory status, and patent status are also summarized. CDs, because of their continuing ability to find several novel applications in drug delivery, are expected to solve many problems associated with the delivery of different novel drugs through different delivery routes.

Biosynthesis of gold nanoparticles with small size and biostability is very important and used in various biomedical applications. There are lot of reports for the synthesis of gold nanoparticles by the addition of reducing agent and stabilizing agent. In the present study we have synthesized gold nanoparticles, with a particle size ranging from 5 to 15 nm, using Zingiber officinale extract which acts both as reducing and stabilizing agent. Z. officinale extract is reported to be a more potent anti-platelet agent than aspirin. Therefore, green synthesis of gold nanoparticles with Z. officinale extract, as an alternative to chemical synthesis, is beneficial from its biological and medical applications point of view, because of its good blood biocompatibility and physiological stability. The formation and size distribution of gold nanoparticles were confirmed by dynamic light scattering (DLS), UV-vis spectrophotometer and transmission electron microscopy (TEM). Gold nanoparticles synthesized using citrate and Z. officinale extract demonstrated very low protein adsorption. Both nanoparticles were non platelet activating and non complement activating on contact with whole human blood. They also did not aggregate other blood cells, however, nanoparticles synthesised with Z. officinale extract was highly stable at physiological condition compared to citrate capped nanoparticles, which aggregated. Thus the usage of nanoparticles, synthesized with Z. officinale extract, as vectors for the applications in drug delivery, gene delivery or as biosensors, where a direct contact with blood occurs is justified.

The use of nano-delivery systems formed through assembly of synthetic amphiphilic block copolymers (ABCs) in experimental medicine and pharmaceutical sciences is experiencing rapid development. This rapid development is driven by a crucial need in improving the performance of existing therapeutic agents, as well as the necessity for the development of advanced delivery systems for complex new entities such as genes, proteins and other cellular components. The flexibility in the construction of appropriate carriers for the delivery requirements of these complex new "drugs" offered by versatile polymer chemistry provides an undeniable advantage for polymer based nano-delivery systems compared to other colloids in this regard. With seven formulations already in different stages of clinical trials, polymeric micelles are in the front line of drug development among different ABC-based nano-carriers. The success in rapid advancement of polymeric micelles from bench to bedside is owed to the rational engineering of core/shell structure so that the polymeric micellar carrier can meet the requirements for optimum delivery of specific drug(s) in certain disease condition(s). The engineering efforts in this regard have mostly been aimed at providing efficient drug loading, micellar stabilization, and sustained and/or site specific drug release. The objective of this review is to provide an update on different engineering strategies employed to achieve optimum polymeric micellar formulations.

The world is driving in to the era of transformation
of chemical therapeutic molecules to biological genetic material
therapeutics, and that is where the biological drugs
especially “genes” come into existence. These genes worked
as “magical bullets” to specifically silence faulty genes responsible
for progression of diseases. Viral gene delivery
research is far ahead of nonviral gene delivery technique.
However, with more advancement in polymer science, new
ways are opening for better and efficient nonviral gene
delivery. But efficient delivery method is always considered
as a bottleneck for gene delivery as success of which will
decide the fate of gene in cells. During the past decade, it
became evident that extracellular as well as intracellular barriers
compromise the transfection efficiency of nonviral vectors.
The challenge for gene therapy research is to pinpoint the
rate-limiting steps in this complex process and implement
strategies to overcome the biological physiochemical and
metabolic barriers encountered during targeting. The synergy
between studies that investigate the mechanism of breaking in
and breaking out of nonviral gene delivery carrier through
various extracellular and intracellular barriers with desired
characteristics will enable the rational design of vehicles and
revolutionize the treatment of various diseases