Gene Delivery

Gold nanoparticles (AuNPs) provide non-toxic carriers for drug and gene delivery applications. With these systems, the gold core imparts stability to the assembly, while the monolayer allows tuning of surface properties such as charge and hydrophobicity. An additional attractive feature of AuNPs is their interaction with thiols, providing an effective and selective means of controlled intracellular release.

Gold nanoparticles (AuNPs) provide non-toxic carriers for drug and gene delivery applications. With these systems, the gold core imparts stability to the assembly, while the monolayer allows tuning of surface properties such as charge and hydrophobicity. An additional attractive feature of AuNPs is their interaction with thiols, providing an effective and selective means of controlled intracellular release.

Despite its long history and some preliminary work in tissue engineering nearly 30 years ago, electrospinning has not gained widespread interest as a potential polymer processing technique for applications in tissue engineering and drug delivery until the last 5e10 years. This renewed interest can be attributed to electrospinning's relative ease of use, adaptability, and the ability to fabricate fibers with diameters on the nanometer size scale. Furthermore, the electrospinning process affords the opportunity to engineer scaffolds with micro to nanoscale topography and high porosity similar to the natural extracellular matrix (ECM). The inherently high surface to volume ratio of electrospun scaffolds can enhance cell attachment, drug loading, and mass transfer properties. Various materials can be electrospun including: biodegradable, non-degradable, and natural materials. Electrospun fibers can be oriented or arranged randomly, giving control over both the bulk mechanical properties and the biological response to the scaffold. Drugs ranging from antibiotics and anticancer agents to proteins, DNA, and RNA can be incorporated into electrospun scaffolds. Suspensions containing living cells have even been electrospun successfully. The applications of electrospinning in tissue engineering and drug delivery are nearly limitless. This review summarizes the most recent and state of the art work in electrospinning and its uses in tissue engineering and drug delivery.

This work investigates the polyanion initiated gelation process in fabricating chitosan-TPP (tripolyphosphate) nanoparticles in the size range of 100-250 nm intended to be used as carriers for the delivery of gene or protein macromolecules. It demonstrates that ionic gelation of cationic chitosan molecules offers a flexible and easily controllable process for systematically and predictably manipulating particle size and surface charge which are important properties in determining gene transfection efficacy if the nanoparticles are used as non-viral vectors for gene delivery, or as delivery carriers for protein molecules. Variations in chitosan molecular weight, chitosan concentration, chitosan to TPP weight ratio and solution pH value were examined systematically for their effects on nanoparticle size, intensity of surface charge, and tendency of particle aggregation so as to enable speedy fabrication of chitosan nanoparticles with predetermined properties. The chitosan-TPP nanoparticles exhibited a high positive surface charge across a wide pH range, and the isoelectric point (IEP) of the nanoparticles was found to be at pH 9.0. Detailed imaging analysis of the particle morphology revealed that the nanoparticles possess typical shapes of polyhedrons (e.g., pentagon and hexagon), indicating a similar crystallisation mechanism during the particle formation and growth process. This study demonstrates that systematic design and modulation of the surface charge and particle size of chitosan-TPP nanoparticles can be readily achieved with the right control of critical processing parameters, especially the chitosan to TPP weight ratio.

Efficient delivery of therapeutic and diagnostic molecules to the cells and tissues is a difficult challenge. The cellular membrane is very effective in its role as a selectively permeable barrier. While it is essential for cell survival and function, also presents a major barrier for intracellular delivery of cargo such as therapeutic and diagnostic agents. In recent years, cell-penetrating peptides (CPPs), that are relatively short cationic and/or amphipathic peptides, received great attention as efficient cellular delivery vectors due to their intrinsic ability to enter cells and mediate uptake of a wide range of macromolecular cargo such as plasmid DNA (pDNA), small interfering RNA (siRNAs), drugs, and nanoparticulate pharmaceutical carriers. This review discusses the various uptake mechanisms of these peptides. Furthermore, we discuss recent advances in the use of CPP for the efficient delivery of nanoparticles, nanocarriers, DNA, siRNA, and anticancer drugs to the cells. In addition, we have been highlighting new results for improving endosomal escape of CPP-cargo molecules. Finally, pH-responsive and activable CPPs for tumor-targeting therapy have been described.

Nanotechnology has shown tremendous promise in target-specific delivery of drugs and genes in the body. Although passive and active targeted-drug delivery has addressed a number of important issues, additional properties that can be included in nanocarrier systems to enhance the bioavailability of drugs at the disease site, and especially upon cellular internalization, are very important. A nanocarrier system incorporated with stimuli-responsive property (e.g., pH, temperature, or redox potential), for instance, would be amenable to address some of the systemic and intracellular delivery barriers. In this review, we discuss the role of stimuli-responsive nanocarrier systems for drug and gene delivery. The advancement in material science has led to design of a variety of materials, which are used for development of nanocarrier systems that can respond to biological stimuli. Temperature, pH, and hypoxia are examples of "triggers" at the diseased site that could be exploited with stimuliresponsive nanocarriers. With greater understanding of the difference between normal and pathological tissues and cells and parallel developments in material design, there is a highly promising role of stimuli-responsive nanocarriers for drug and gene delivery in the future.

Various kinds of stimuli-responsive N-isopropylacrylamide copolymers are among an important class of bioengineering materials that have been the subject of many extensive investigations in the field of modern macromolecular bioengineering and biotechnology. These materials are of special interest in the fields of cell and enzyme immobilization, controlled drug delivery and gene delivery, bioconjugation, and protein dehydration process. This review summarizes the main advances published over the last 15 years, outlining the synthesis, structural phenomena, unique properties and application areas of the bioengineering copolymers of N-isopropyl acrylamide with various types of comonomers. These copolymers include linear, end functionalized and crosslinked binary and ternary copolymers, graft and block copolymers exhibiting stimuli-responsive behavior, and reversible phase transitions. Particular attention is paid to the effects of hydrogenbonding and complexing in copolymerization reactions, the formation of supramacromolecular structures, bioconjugates, physically and chemically crosslinkable copolymers, thermosensitive hydrogels and ion-exchange membrane systems, and other important bioengineering materials. r

The last decade has witnessed the development of cell-based therapy as a major biomedical research area, including the treatment of musculoskeletal diseases. Both differentiated and undifferentiated stem cells have been used as starting cell sources. In particular, the use of multipotent adult mesenchymal stem cells holds great promise for future therapeutic strategies. In addition to the cell type used, the cell delivery system is also of critical importance in cell-based therapy. Cell delivery may be achieved by direct cell injection or by grafting engineered constructs derived by cell seeding into natural or synthetic biomaterial scaffolds. While direct injection is the most direct and convenient means of cell delivery, the latter approach is capable of producing three-dimensional engineered tissues with mechanical properties compatible with those of various musculoskeletal tissues. This review will focus on the functional approach of using biomaterial scaffold materials as cell carriers for musculoskeletal applications, as well as the use of cell-based gene therapy for tissue engineering and regeneration.

The Michael addition reaction is a versatile synthetic methodology for the efficient coupling of electron poor olefins with a vast array of nucleophiles. This review outlines the role of the Michael addition reaction in polymer synthesis with attention to applications in emerging technologies including biomedical, pharmaceutical, optoelectronic, composites, adhesives, and coatings. Polymer architectures, which broadly range from linear thermoplastics to hyperbranched polymers and networks are achievable. The versatility of the Michael reaction in terms of monomer selection, solvent environment, and reaction temperature permits the synthesis of sophisticated macromolecular structures under conditions where other reaction processes will not operate. The utility of the Michael addition in many biological applications such as gene delivery, polymer drug conjugates, and tissue scaffolds is discussed in relation to macromolecular structure. q

Cholinergic neuron loss is a cardinal feature of Alzheimer disease. Nerve growth factor (NGF) stimulates cholinergic function, improves memory and prevents cholinergic degeneration in animal models of injury, amyloid overexpression and aging. We performed a phase 1 trial of ex vivo NGF gene delivery in eight individuals with mild Alzheimer disease, implanting autologous fibroblasts genetically modified to express human NGF into the forebrain. After mean follow-up of 22 months in six subjects, no long-term adverse effects of NGF occurred. Evaluation of the Mini-Mental Status Examination and Alzheimer Disease Assessment Scale-Cognitive subcomponent suggested improvement in the rate of cognitive decline. Serial PET scans showed significant (P < 0.05) increases in cortical 18-fluorodeoxyglucose after treatment. Brain autopsy from one subject suggested robust growth responses to NGF. Additional clinical trials of NGF for Alzheimer disease are warranted.

Mechanical alloying has been widely utilized to break down the clustered CNTs for incorporation in the metal matrix composites. However, the breakage of CNTs during the ball milling process degrades their effectiveness. Due to the challenges in collecting the CNTs for measurement, quantitative study of CNT breakage has been difficult. In this study, the CNTs with Al6061 powder were mechanically alloyed with high energy milling equipment. The CNTs from the surface of mechanically alloyed particles were collected and measured. Due to the difficulty in obtaining the CNTs embedded inside the particles, a mathematical model has been developed to predict the overall CNT length distribution in the composite. Significant CNT breakage occurred during the initial phase of the mechanical alloying due to the crushing of the clusters. The model predicted that no further change occurred in the overall CNT length during time greater than 1 h of mechanical alloying because most of the CNTs had already become embedded within the particles and were thus protected from further milling media impacts. A faster dispersion of CNTs and lower particle fracturing rate may help preserve the original CNTs.

The Michael addition reaction is a versatile synthetic methodology for the efficient coupling of electron poor olefins with a vast array of nucleophiles. This review outlines the role of the Michael addition reaction in polymer synthesis with attention to applications in emerging technologies including biomedical, pharmaceutical, optoelectronic, composites, adhesives, and coatings. Polymer architectures, which broadly range from linear thermoplastics to hyperbranched polymers and networks are achievable. The versatility of the Michael reaction in terms of monomer selection, solvent environment, and reaction temperature permits the synthesis of sophisticated macromolecular structures under conditions where other reaction processes will not operate. The utility of the Michael addition in many biological applications such as gene delivery, polymer drug conjugates, and tissue scaffolds is discussed in relation to macromolecular structure. q

Chitosan-DNA nanoparticles were prepared using a complex coacervation process. The important parameters for the nanoparticle synthesis were investigated, including the concentrations of DNA, chitosan and sodium sulfate, temperature of the solutions, pH of the buffer, and molecular weights of chitosan and DNA. At an amino group to phosphate group ratio (N / P ratio) between 3 and 8 and a chitosan concentration of 100 mg / ml, the size of particles was optimized to |100-250 nm with a narrow distribution, with a composition of 35.6 and 64.4% by weight for DNA and chitosan, respectively. The surface charge of these particles was slightly positive with a zeta potential of 112 to 118 mV at pH lower than 6.0, and became nearly neutral at pH 7.2. The chitosan-DNA nanoparticles could partially protect the encapsulated plasmid DNA from nuclease degradation as shown by electrophoretic mobility analysis. The transfection efficiency of chitosan-DNA nanoparticles was cell-type dependent. Typically, it was three to four orders of magnitude, in relative light units, higher than background level in HEK293 cells, and two to ten times lower than that achieved by LipofectAMINEE-DNA complexes. The presence of 10% fetal bovine serum did not interfere with their transfection ability. Chloroquine could be co-encapsulated in the nanoparticles at 5.2%, but with negligible enhancement effect despite the fact that chitosan only showed limited buffering capacity compared with PEI. The present study also developed three different schemes to conjugate transferrin or KNOB protein to the nanoparticle surface. The transferrin conjugation only yielded a maximum of four-fold increase in their transfection efficiency in HEK293 cells and HeLa cells, whereas KNOB conjugated nanoparticles could improve gene expression level in HeLa cells by 130-fold. Conjugation of PEG on the nanoparticles allowed lyophilization without aggregation, and without loss of bioactivity for at least 1 month in storage. The clearance of the PEGylated nanoparticles in mice following intravenous administration was slower than unmodified nanoparticles at 15 min, and with higher depositions in kidney and liver. However, no difference was observed at the 1-h time point.

Gene therapy has emerged as a promising approach for the treatment or prevention of acquired and genetic diseases. At the present time, major somatic gene transfer approaches employ either viral or non-viral vectors. Viral vectors show high gene transfer efficiency, but are deficient in several areas, including the induction of a host inflammatory and immune response. Some of these problems can be circumvented by employing non-viral vehicles, such as cationic liposomes or polymers. The complexes they form with DNA are defined as "lipoplexes" or "polyplexes, respectively, and constitute the most promising alternative to the use of viral vectors for gene therapy. Here we review the interactions between the vectors and DNA leading to complex formation, the supramolecular structures of lipoplexes and polyplexes, and their mechanisms of DNA transfer. Our objective is to provide a framework for the future design and synthesis of optimal non-viral vectors for gene therapy. The structure, charge and formulation of these vehicles are also related to the stability, and consequently to the efficiency of gene transfection. While lipids that facilitate transformation of lipoplexes to non-bilayer phases mediate high transfection activity in vitro, lipids, like cholesterol, that confer stability in serum, are more suitable for gene delivery in vivo. The efficiency of polyplex-mediated transfection depends on the ability of the polymer to condense DNA, while allowing it to dissociate once inside the cell.

Nanotechnology has shown tremendous promise in target-specific delivery of drugs and genes in the body. Although passive and active targeted-drug delivery has addressed a number of important issues, additional properties that can be included in nanocarrier systems to enhance the bioavailability of drugs at the disease site, and especially upon cellular internalization, are very important. A nanocarrier system incorporated with stimuli-responsive property (e.g., pH, temperature, or redox potential), for instance, would be amenable to address some of the systemic and intracellular delivery barriers. In this review, we discuss the role of stimuli-responsive nanocarrier systems for drug and gene delivery. The advancement in material science has led to design of a variety of materials, which are used for development of nanocarrier systems that can respond to biological stimuli. Temperature, pH, and hypoxia are examples of "triggers" at the diseased site that could be exploited with stimuliresponsive nanocarriers. With greater understanding of the difference between normal and pathological tissues and cells and parallel developments in material design, there is a highly promising role of stimuli-responsive nanocarriers for drug and gene delivery in the future.

For gene therapies, viral vectors (infection) are by far the most specific, efficient and effective means of DNA delivery, due to highly evolved, and specialized, viral proteins and peptides. However, enthusiasm for viral-vector mediated DNA delivery, unfortunately, has been tempered by growing concerns over the drug carriers --viral vectors. Non-viral vectors, on the other hand, have much better safety profiles although they are still generally inferior to most viral vectors. A hybrid system that combines both viral and non-viral advantages may overcome the major barriers to DNA delivery safely and efficiently.

In recent years, attention has been focused on the possibility of utilizing DNA vaccines in fish aquaculture. A successful regime for intramuscular injection of naked DNA into fish has been developed and novel methods to deliver this DNA to fish are under investigation. The potential of chitosan as a polycationic gene carrier for oral administration has been explored since 1990s. The present study examines the potential efficacy of DNA vaccine against Vibrio anguillarum through oral route using chitosan nanoparticles encapsulation. The porin gene of V. anguillarum was used to construct DNA vaccine using pcDNA 3.1, a eukaryotic expression vector and the construct was named as pVAOMP38. The chitosan nanoparticles were used to deliver the constructed plasmid. In vitro and in vivo expression of porin gene was observed in sea bass kidney cell line (SISK) and in fish, respectively by fluorescent microscopy. The cytotoxicity of chitosan encapsulated DNA vaccine construct was analyzed by MTT assay and it was found that the cytotoxicity of pVAOMP38/chitosan was quite low. Distribution of gene in different tissues was studied in fish fed with the DNA (pVAOMP38) encapsulated in chitosan by using immunohistochemistry. The results indicate that DNA vaccine can be easily delivered into fish by feeding with chitosan nanoparticles. After oral vaccination Asian sea bass were challenged with Vibrio anguillarum by intramuscular injection. A relative percent survival (RPS) rate of 46% was recorded. The results indicate that Sea bass (Lates calcarifer) orally vaccinated with chitosan-DNA (pVAOMP38) complex showed moderate protection against experimental V. anguillarum infection.

Lentiviral vectors have emerged over the last decade as powerful, reliable and safe tools for stable gene transfer in a wide variety of mammalian cells. Unlike other vectors derived from oncoretroviruses, they allow for stable gene delivery into most nondividing primary cells, including neurons. This is why LVs are becoming the most useful and promising tools in the field of neuroscience, not only for research, but also for future gene and cell therapy approaches. Lentivectors (LVs) derived from HIV-1 have gradually evolved to display many desirable features aimed at increasing both their safety and their versatility. These latest designs are reviewed in this unit. This unit also describes protocols for production and titration of LVs that can be implemented in a research laboratory setting, with an emphasis on standardization to improve transposability of results between laboratories.

a b s t r a c t Lentiviral vectors (LV) are promising tools for gene and cell therapy. They are presently used in several clinical trials as in vivo or ex vivo gene delivery vectors. However their mass production remains a challenge and might limit their potential therapeutic use. New robust and scalable processes are required for industrial production of these vectors.

Nanoparticles formulated from biodegradable polymers such as poly(lactic acid) (PLA) and poly(lactide-co-glycolide) (PLGA) are being extensively investigated as non-viral gene delivery systems due to their controlled release characteristics and biocompatibility. PLGA nanoparticles for DNA delivery are mainly formulated by an emulsion-solvent evaporation technique using PVA as a stabilizer generating negatively charged particles and heterogeneous size distribution. The objective of the present study was to formulate cationically modified PLGA nanoparticles with defined size and shape that can efficiently bind DNA. An Emulsion-diffusion-evaporation technique to make cationic nanospheres composed of biodegradable and biocompatible copolyester PLGA has been developed. PVA-chitosan blend was used to stabilize the PLGA nanospheres. The nanospheres were characterized by atomic force microscopy (AFM), photon-correlation spectroscopy (PCS), and Fourier transform infrared spectroscopy (FTIR). Zeta potential and gel electrophoresis studies were also performed to understand the surface properties of nanospheres and their ability to condense negatively charged DNA. The designed nanospheres have a zeta potential of 10 mV at pH 7.4 and size under 200 nm. From the gel electrophoresis studies we found that the charge on the nanospheres is sufficient to efficiently bind the negatively charged DNA electrostatically. These cationic PLGA nanospheres could serve as potential alternatives of the existing negatively charged nanoparticles. r

The last decade has witnessed enormous research focused on cationic polymers. Cationic polymers are the subject of intense research as non-viral gene delivery systems, due to their flexible properties, facile synthesis, robustness and proven gene delivery efficiency. Here, we review the most recent scientific advances in cationic polymers and their derivatives not only for gene delivery purposes but also for various alternative therapeutic applications. An overview of the synthesis and preparation of cationic polymers is provided along with their inherent bioactive and intrinsic therapeutic potential. In addition, cationic polymer based biomedical materials are covered. Major progress in the fields of drug and gene delivery as well as tissue engineering applications is summarized in the present review.

Despite their relatively lower efficiency, nonviral approaches are emerging as safer alternatives in gene therapy to viral
vectors. Delivery of nucleic acids to the target site is an important factor for effective gene expression (plasmid DNA) or knockdown (siRNA) with minimal side effects. Direct deposition at the target site by physical methods, including ultrasound, electroporation and gene gun, is one approach for local delivery. For less accessible sites, the development of carriers that can home into the target tissue is required. Cationic peptides, lipoplexes, polyplexes and nanoplexes have been used as carriers for delivery of nucleic acids. Targeting
ligands, such as cell targeting peptides, have also been applied to decorate delivery vehicles in order to enhance their efficacy. This review focuses on delivery strategies and recent progress in non-viral carriers and their modifications to improve their performance in targeting and transfection.

Retroviral vectors from both oncoretroviral and lentiviral origins have a great potential as gene delivery vehicles. A number of research groups have devoted considerable effort to the development of large-scale production strategies for retroviral vectors. However, the manufacturing of clinical-grade vectors for gene therapy, especially for in vivo applications, additionally requires scaleable purification strategies to remove the contaminants present in the harvested supernatants while preserving the functionality of the vectors. In this article, we review recent advances made in the field of downstream processing of retroviral vectors. The methods currently described in the literature for clarification, concentration and purification of retroviral vectors will be presented, with special emphasis on novel chromatography methods that open up the possibility to selectively and efficiently purify retroviruses on a large-scale. Problems associated with stability and quantification of retroviral particles will be outlined and future challenges will be discussed. D

The selective activation of a relatively non-toxic prodrug by an enzyme present only in the tumour should enhance the drug concentration at the tumour site and result in a better anti-tumour effect and a reduction in systemic toxicity as compared to conventional chemotherapy. β-Glucuronidase is such an enzyme. It is normally expressed in the lysosomes of cells. In larger tumours, however, high levels of the enzyme are present in necrotic areas. Several glucuronide prodrugs have been synthesised that can be activated by βglucuronidase. They are relatively non-toxic due to their hydrophilic nature, which prevents them from entering cells and thus from contact with lysosomal β-glucuronidase. The main problem of glucuronide prodrugs for clinical use is their fast renal clearance. Special attention should be paid to the development of new less hydrophilic prodrugs with slower clearance, as this would result in a prolonged exposure to βglucuronidase at the site of the tumour and a reduction of the amount of prodrug needed. A number of interesting anthracyclin-based glucuronide prodrugs have been synthesised and have shown favourable therapeutic effects compared to treatment with the parent drug. The tumoural levels of β-glucuronidase can even be enhanced by two-step approaches, in which exogenous enzyme is targeted to the tumour by an antibody (ADEPT) or by the gene encoding the enzyme in transduced tumour cells (GDEPT). The ADEPT and GDEPT approaches in combination with glucuronide prodrugs have shown enhanced efficacy in experimental tumour models. Further improvement of ADEPT and GDEPT is warranted to optimise the tumour uptake and retention of antibody-enzyme fusion proteins and the efficiency and safety of current gene delivery methods. In conclusion, it is clear that glucuronide prodrugs hold promise for future use in the treatment of cancer in patients as monotherapy. Enhancement of the therapeutic effects of glucuronide prodrugs, also in patients with small tumour lesions, may possibly be achieved by techniques that target β-glucuronidase specifically to the site of the tumour.

Carbon nanotubes and carbon nanofibers have long been investigated for applications in composite structural materials, semiconductor devices, and sensors. With the recent well-documented ability to chemically modify nanofibrous carbon materials to improve their solubility and biocompatibility properties: a whole new class of bioactive carbon nanostructures has been created for biological applications. This review focuses on the latest applications of carbon nanofibers and carbon nanotubes in regenerative medicine.

Solid lipid nanoparticles (SLNs) have been proposed in the 1990s as appropriate drug delivery systems, and ever since they have been applied in a wide variety of cosmetic and pharmaceutical applications. In addition, SLNs are considered suitable alternatives as carriers in gene delivery. Although important advances have been made in this particular field, fundamental knowledge of the underlying mechanisms of SLN-mediated gene delivery is conspicuously lacking, an imperative requirement in efforts aimed at further improving their efficiency. Here, we address recent advances in the use of SLNs as platform for delivery of nucleic acids as therapeutic agents. In addition, we will discuss available technology for conveniently producing SLNs. In particular, we will focus on underlying molecular mechanisms by which SLNs and nucleic acids assemble into complexes and how the nucleic acid cargo may be released intracellularly. In discussing underlying mechanisms, we will, when appropriate, refer to analogous studies carried out with systems based on cationic lipids and polymers, that have proven useful in the assessment of structure–function relationships. Finally, we will give suggestions for improving SLN-based gene delivery systems, by pointing to alternative methods for SLNplex assembly, focusing on the realization of a sustained nucleic acid release.

Current tissue engineering strategies are focused on the restoration of pathologically altered tissue architecture by transplantation of cells in combination with supportive scaffolds and biomolecules. In recent years, considerable attention has been given to chitosan (CS)-based materials and their applications in the field of orthopedic tissue engineering. Interesting characteristics that render chitosan suitable for this purpose are a minimal foreign body reaction, an intrinsic antibacterial nature, and the ability to be molded in various geometries and forms such as porous structures, suitable for cell ingrowth and osteoconduction. Due to its favorable gelling properties chitosan can deliver morphogenic factors and pharmaceutical agents in a controlled fashion. Its cationic nature allows it to complex DNA molecules making it an ideal candidate for gene delivery strategies. The ability to manipulate and reconstitute tissue structure and function using this material has tremendous clinical implications and is likely to play a key role in cell and gene therapies in coming years. In this paper we will review the current applications and future directions of CS in articular cartilage, intervertebral disk and bone tissue engineering. r

Cationic lipid-mediated gene transfer and delivery still attract great attention of many gene therapy laboratories. From the point of view of the most important characteristics of lipoplex particles, e.g. its charge and size, we reviewed recent studies available. In general, the paper deals with non-viral systems of gene transfer into eukaryotic cell based on various lipids. Having usually less efficiency in gene transfer, lipid-based gene transfer vehicles (lipoplexes/genosomes) are characterized with certain advantages even over viral ones: they are less toxic and immunogenic, could be targetable and are easy for large-scale production, a size of transferred DNA being quite high. Conditions of DNA condensation during interactions with lipids are described. Results of the studies of mechanism of DNA -lipid complex interactions with the cell membrane and their transport into the nucleus are summarized. Dependence of efficiency of gene transfer on lipoplex structure and physical -chemical properties is reviewed. Advantages and disadvantages of different macromolecule complexes from the point of view of transfection efficiency, possibility of use in vivo, cytotoxicity and targeted gene transfer in certain organs and tissues are also discussed. Results of transfection of different cells using neutral, anion and cation liposomes are reviewed. The conclusion reached was that efficiency and specificity of gene transfer may grow considerably when mixed macromolecule lipid systems including polycations and glycolipids are used.

Advanced therapies combating acute and chronic skin wounds are likely to be brought about using our knowledge of regenerative medicine coupled with appropriately tissue-engineered skin substitutes. At the present time, there are no models of an artificial skin that completely replicate normal uninjured skin. Natural biopolymers such as collagen and fibronectin have been investigated as potential sources of biomaterial to which cells can attach. The first generation of degradable polymers used in tissue engineering were adapted from other surgical uses and have drawbacks in terms of mechanical and degradation properties. This has led to the development of synthetic degradable gels primarily as a way to deliver cells and/or molecules in situ, the so-called smart matrix technology. Tissue or organ repair is usually accompanied by fibrotic reactions that result in the production of a scar. Certain mammalian tissues, however, have a capacity for complete regeneration without scarring; good examples include embryonic or foetal skin and the ear of the MRL/MpJ mouse. Investigations of these model systems reveal that in order to achieve such complete regeneration, the inflammatory response is altered such that the extent of fibrosis and scarring is diminished. From studies on the limited examples of mammalian regeneration, it may also be possible to exploit such models to further clarify the regenerative process. The challenge is to identify the factors and cytokines expressed during regeneration and incorporate them to create a smart matrix for use in a skin equivalent. Recent advances in the use of DNA microarray and proteomic technology are likely to aid the identification of such molecules. This, coupled with recent advances in non-viral gene delivery and stem cell technologies, may also contribute to novel approaches that would generate a skin replacement whose materials technology was based not only upon intelligent design, but also upon the molecules involved in the process of regeneration.

In vitro studies of non-viral gene delivery vectors are typically not performed at physiological conditions, and thus may not provide meaningful results for in vivo investigations. We determine if polycation-plasmid DNA complexes (polyplexes) exploited for in vitro studies behave similarly to variants more applicable to in vivo use by examining their cellular uptake and trafficking. Branched polyethylenimine (25 kDa) or a linear bcyclodextrin-containing polymer are each used to formulate polyplexes, which can be PEGylated (PEG: poly(ethylene glycol)) to create particles stable in physiological salt concentrations. Particle size, cellular uptake, intracellular trafficking, and reporter gene expression are reported for polyplexes and for their PEGylated variants. PEGylation confers salt stability to particles but produced a reduction in luciferase expression. Examination of in vitro particle internalization by transmission electron microscopy shows unmodified polyplexes entering cells as large aggregates while PEGylated particles remain small and discrete, both outside and within cells. Unmodified and PEGylated particles enter cells through the endocytic pathway and accumulate in a perinuclear region. Immunolabeling reveals unpackaged exogenous DNA in the cytoplasm and nuclei. It appears all particle types traffic towards the nucleus within vesicles and undergo degradation in vesicles and/or cytoplasm, and eventually some exogenous DNA enters the nucleus, where it is transcribed. In comparing polyplexes and their PEGylated variants, significant differences in particle morphology, cellular uptake, and resultant expression suggest that in vitro studies should be conducted with particles prepared for physiological conditions if the results are to be relevant to in vivo performance.

In recent years, attention has been focused on the possibility of utilizing DNA vaccines in fish aquaculture. A successful regime for intramuscular injection of naked DNA into fish has been developed and novel methods to deliver this DNA to fish are under investigation. The potential of chitosan as a polycationic gene carrier for oral administration has been explored since 1990s. The present study examines the potential efficacy of DNA vaccine against Vibrio anguillarum through oral route using chitosan nanoparticles encapsulation. The porin gene of V. anguillarum was used to construct DNA vaccine using pcDNA 3.1, a eukaryotic expression vector and the construct was named as pVAOMP38. The chitosan nanoparticles were used to deliver the constructed plasmid. In vitro and in vivo expression of porin gene was observed in sea bass kidney cell line (SISK) and in fish, respectively by fluorescent microscopy. The cytotoxicity of chitosan encapsulated DNA vaccine construct was analyzed by MTT assay and it was found that the cytotoxicity of pVAOMP38/chitosan was quite low. Distribution of gene in different tissues was studied in fish fed with the DNA (pVAOMP38) encapsulated in chitosan by using immunohistochemistry. The results indicate that DNA vaccine can be easily delivered into fish by feeding with chitosan nanoparticles. After oral vaccination Asian sea bass were challenged with Vibrio anguillarum by intramuscular injection. A relative percent survival (RPS) rate of 46% was recorded. The results indicate that Sea bass (Lates calcarifer) orally vaccinated with chitosan-DNA (pVAOMP38) complex showed moderate protection against experimental V. anguillarum infection.

The last decade has witnessed enormous research focused on cationic polymers. Cationic polymers are the subject of intense research as non-viral gene delivery systems, due to their flexible properties, facile synthesis, robustness and proven gene delivery efficiency. Here, we review the most recent scientific advances in cationic polymers and their derivatives not only for gene delivery purposes but also for various alternative therapeutic applications. An overview of the synthesis and preparation of cationic polymers is provided along with their inherent bioactive and intrinsic therapeutic potential. In addition, cationic polymer based biomedical materials are covered. Major progress in the fields of drug and gene delivery as well as tissue engineering applications is summarized in the present review.

An electrical model for biological cells predicts that for pulses with durations shorter than the charging time of the outer membrane, there is an increasing probability of electric field interactions with intracellular structures. Experimental studies in which human cells were exposed to pulsed electric fields of up to 300-kV/cm amplitude, with durations as short as 10 ns, have confirmed this hypothesis. The observed effects include the breaching of intracellular granule membranes without permanent damage to the cell membrane, abrupt rises in intracellular free calcium levels, and enhanced expression of genes. At increased electric fields, the application of submicrosecond pulses induces apoptosis (programmed cell death) in biological cells, an effect that has been shown to reduce the growth of tumors. Possible applications of the intracellular electroeffect are enhancing gene delivery to the nucleus, controlling cell functions that depend on calcium release (causing cell immobilization), and treating tumors.

Abstract
Gene therapy as a modern therapeutic approach has not yet advanced to a globally-approved therapeutic approach. Lack of adequate reliable gene delivery system seems to be one of the major reasons from the pharmaceutical biotechnology point of view. Main obstacles delaying successful application of human gene therapy are presented in this review. The unique advantages of non-biological gene carriers as compared to their biological counterparts make them ideal alternatives for  overcoming extra- and intracellular barriers in a more safely manner. We, therefore, highlight the significant contributions in non-biological gene delivery and favorable characteristics of different design attitudes with focus on in vivo approaches. Bypassing the rapid extracellular enzymatic degradation of genetic materials is covered in extracellular segment of this review with emphasis on PEGylated and targeted formulations. The successful approaches to pave the rest of the way from cellular uptake to intracellular transfer and gene expression of unpacked DNA are also discussed. From these approaches, we emphasize more on optimization of cationic-based polymers and dendrimers, developing newly designed membrane-effective components, and adjusting the hydrophilic-hydrophobic balance of the synthesized vectors

Water-soluble, biodegradable, polymeric, polyelectrolyte complex dispersions (PECs) have evolved because of the limitations, in terms of toxicity, of the currently available systems. These aqueous nanoparticulate architectures offer a significant advantage for products that may be used as drug delivery systems in humans. PECs are created by mixing oppositely charged polyions. Their hydrodynamic diameter, surface charge, and polydispersity are highly dependent on concentration, ionic strength, pH, and molecular parameters of the polymers that are used. In particular, the complexation between polyelectrolytes with significantly different molecular weights leads to the formation of water-insoluble aggregates. Several PEC characteristics are favorable for cellular uptake and colloidal stability, including hydrodynamic diameter less than 200 nm, surface charge of >30 mV or <j30 mV, spherical morphology, and polydispersity index (PDI) indicative of a homogeneous distribution. Maintenance of these properties is critical for a successful delivery vehicle. This review focuses on the development and potential applications of PECs as multifunctional, site-specific nanoparticulate drug/gene delivery and imaging devices.

A common viral immune evasion strategy involves mutating viral surface proteins in order to evade host neutralizing antibodies. Such immune evasion tactics have not previously been intentionally applied to the development of novel viral gene delivery vectors that overcome the critical problem of anti-vector immunity. Recombinant, replication-incompetent adenovirus serotype 5 (rAd5) vector-based vaccines for human immunodeficiency virus type 1 and other pathogens have proved highly immunogenic in preclinical studies 1,2 but will probably be limited by the high prevalence of pre-existing anti-Ad5 immunity in human populations, particularly in the developing world 3-6 . Here we show that rAd5 vectors can be engineered to circumvent anti-Ad5 immunity. We constructed novel chimaeric rAd5 vectors in which the seven short hypervariable regions (HVRs) on the surface of the Ad5 hexon protein were replaced with the corresponding HVRs from the rare adenovirus serotype Ad48. These HVR-chimaeric rAd5 vectors were produced at high titres and were stable through serial passages in vitro. HVR-chimaeric rAd5 vectors expressing simian immunodeficiency virus Gag proved comparably immunogenic to parental rAd5 vectors in naive mice and rhesus monkeys. In the presence of high levels of pre-existing anti-Ad5 immunity, the immunogenicity of HVR-chimaeric rAd5 vectors was not detectably suppressed, whereas the immunogenicity of parental rAd5 vectors was abrogated. These data demonstrate that functionally relevant Ad5-specific neutralizing antibodies are focused on epitopes located within the hexon HVRs. Moreover, these studies show that recombinant viral vectors can be engineered to circumvent pre-existing anti-vector immunity by removing key neutralizing epitopes on the surface of viral capsid proteins. Such chimaeric viral vectors may have important practical implications for vaccination and gene therapy.

The present study aimed at developing a new approach in gene delivery of calcium phosphate nanoparticles through simulated body fluid (CaP-SBF). The physicochemical and biological characteristics of the CaP-SBF nanoparticles were compared with those made in pure water (CaP-water) via a similar procedure. The CaP-SBF and CaP-water solutions were then adjusted to two different pH values of 7.4 and 8.0, mixed with plasmid DNA (pDNA), and added in varying amounts to human embryonic kidney (HEK 293T) cells. The transfection efficiency and cell viability were studied in vitro by reporter gene (luciferase and Enhanced Green Fluorescent Protein) expression and the resazurin reduction assay, respectively, 24 and 48 h after the incubation with the nanoparticles. Our results indicated considerably high in vitro transfection efficiency for CaP-SBF/DNA complexes at physiological pH (7.4) with high amounts of CaP. Additionally, the SBF solution exhibited the ability to reduce the rapid growth of CaP particles over time, leading to higher transfection efficiency of CaP-SBF/DNA complexes than those made in water (CaP-water/DNA).

Efficient delivery of therapeutic and diagnostic molecules to the cells and tissues is a difficult challenge. The cellular membrane is very effective in its role as a selectively permeable barrier. While it is essential for cell survival and function, also presents a major barrier for intracellular delivery of cargo such as therapeutic and diagnostic agents. In recent years, cell-penetrating peptides (CPPs), that are relatively short cationic and/or amphipathic peptides, received great attention as efficient cellular delivery vectors due to their intrinsic ability to enter cells and mediate uptake of a wide range of macromolecular cargo such as plasmid DNA (pDNA), small interfering RNA (siRNAs), drugs, and nanoparticulate pharmaceutical carriers. This review discusses the various uptake mechanisms of these peptides. Furthermore, we discuss recent advances in the use of CPP for the efficient delivery of nanoparticles, nanocarriers, DNA, siRNA, and anticancer drugs to the cells. In addition, we have been highlighting new results for improving endosomal escape of CPP-cargo molecules. Finally, pH-responsive and activable CPPs for tumor-targeting therapy have been described.

The dihydrofolate reductase-deficient Chinese hamster ovary (CHO) cell line DG44 is the dominant mammalian host for recombinant protein manufacturing, in large part because of the availability of a well-characterized genetic selection and amplification system. However, this cell line has not been studied at the cytogenetic level. Here, the first detailed karyotype analysis of DG44 and several recombinant derivative cell lines is described. In contrast to the 22 chromosomes in diploid Chinese hamster cells, DG44 has 20 chromosomes, only seven of which are normal. In addition, four Z group chromosomes, seven derivative chromosomes, and 2 marker chromosomes were identified. For all but one of the 16 DG44-derived recombinant cell lines analyzed, a single integration site was detected by fluorescence in situ hybridization regardless of the gene delivery method (calcium phosphate-DNA coprecipitation or microinjection), the topology of the DNA (circular or linear), or the integrated plasmid copy number (between 1 and 51). Chromosomal aberrations, observed in more than half of the cell lines studied, were mostly unbalanced with examples of aneuploidy, deletions, and complex rearrangements. The results demonstrate that chromosomal aberrations are frequently associated with the establishment of recombinant CHO DG44 cell lines. Noteworthy, there was no direct correlation between the stability of the genome and the stability of recombinant protein expression.

Lipoplexes and polyplexes, electrostatic complexes between a plasmid DNA and cationic lipids or polymers are chemical systems that are developed for gene delivery. Considerable efforts have been done to delineate the exact knowledge of their entry mechanisms and the intracellular routing of the plasmid DNA that are of major importance for the designing of these gene delivery systems. While the uptake of lipoplexes made with several types of cationic lipids proceeds mainly by the clathrin-dependent pathway, it appears that for polyplexes the uptake pathway is more dependent on the polymer and the cell types. So, after an overview on the current knowledge of different endocytic pathways, we present here a selection of current reports related to the entry mechanisms and intracellular routing of plasmid DNA complexed with select cationic polymers. The review includes the role of glycosaminoglycans, cell polarization and cell cycle in the polyplex uptake and their transfection efficiency. We also report current data showing that the insertion of specific B motifs in the nucleic acid sequence provides an increase of the plasmid import into the nucleus. This has been demonstrated by fluorescence methods suitable to investigate the intracellular trafficking of pDNA.