had similar or greater success. cellular uptake of naked dna is generally inefficient. research efforts focusing on improving the efficiency of naked dna uptake have yielded several novel methods, such as electroporation, sonoporation, and the use of a "gene gun", which shoots dna coated gold particles into the cell using high pressure gas. electroporation electroporation is a method that uses short pulses of high voltage to carry dna across the cell membrane. this shock is thought to cause temporary formation of pores in the cell membrane, allowing dna molecules to pass through. electroporation is generally efficient and works across a broad range of cell types. however, a high rate of cell death following electroporation has limited its use, including clinical applications. more recently a newer method of electroporation, termed electron-avalanche transfection, has been used in gene therapy experiments. by using a high-voltage plasma discharge, dna was efficiently delivered following very short (microsecond) pulses. compared to electroporation, the technique resulted in greatly increased efficiency and less cellular damage. gene gun the use of particle bombardment, or the gene gun, is another physical method of dna transfection. in this technique, dna is coated onto gold particles and loaded into a device which generates a force to achieve penetration of the dna into the cells, leaving the gold behind on a "stopping" disk. sonoporation sonoporation uses ultrasonic frequencies to deliver dna into cells. the process of acoustic cavitation is thought to disrupt the cell membrane and allow dna to move into cells. magnetofection in a method termed magnetofection, dna is complexed to magnetic particles, and a magnet is placed underneath the tissue culture dish to bring dna complexes into contact with a cell monolayer. hydrodynamic delivery hydrodynamic delivery involves rapid injection of a high volume of a solution into vasculature (such as into the inferior vena cava, bile duct, or tail vein). the solution contains molecules that are to be inserted into cells, such as dna plasmids or sirna, and transfer of these molecules into cells is assisted by the elevated hydrostatic pressure caused by the high volume of injected solution. oligonucleotides the use of synthetic oligonucleotides in gene therapy is to deactivate the genes involved in the disease process. there are several methods by which this is achieved. one strategy uses antisense specific to the target gene to disrupt the transcription of the faulty gene. another uses small molecules of rna called sirna to signal the cell to cleave specific unique sequences in the mrna transcript of the faulty gene, disrupting translation of the faulty mrna, and therefore expression of the gene. a further strategy uses double stranded oligodeoxynucleotides as a decoy for the transcription factors that are required to activate the transcription of the target gene. the transcription factors bind to the decoys instead of the promoter of the faulty gene, which reduces the transcription of the target gene, lowering expression. additionally, single stranded dna oligonucleotides have been used to direct a single base change within a mutant gene. the oligonucleotide is designed to anneal with complementarity to the target gene with the exception of a central base, the target base, which serves as the template base for repair. this technique is referred to as oligonucleotide mediated gene repair, targeted gene repair, or targeted nucleotide alteration. lipoplexes look up lipoplex in wiktionary, the free dictionary. to improve the delivery of the new dna into the cell, the dna must be protected from damage and positively charged. initially, anionic and neutral lipids were used for the construction of lipoplexes for synthetic vectors. however, in spite of the facts that there is little toxicity associated with them, that they are compatible with body fluids and that there was a possibility of adapting them to be tissue specific; they are complicated and time-consuming to produce so attention was turned to the cationic versions. cationic lipids, due to their positive charge, were first used to condense negatively charged dna molecules so as to facilitate the encapsulation of dna into liposomes. later it was found that the use of cationic lipids significantly enhanced the stability of lipoplexes. also as a result of their charge, cationic liposomes interact with the cell membrane, endocytosis was widely believed as the major route by which cells uptake lipoplexes. endosomes are formed as the results of endocytosis, however, if genes can not be released into cytoplasm by breaking the membrane of endosome, they will be sent to lysosomes where all dna will be destroyed before they could achieve their functions. it was also found that although cationic lipids themselves could condense and encapsulate dna into liposomes, the transfection efficiency is very low due to the lack of ability in terms of "endosomal escaping". however, when helper lipids (usually electroneutral lipids, such as dope) were added to form lipoplexes, much higher transfection efficiency was observed. later on, it was discovered that certain lipids have the ability to destabilize endosomal membranes so as to facilitate the escape of dna from endosome, therefore those lipids are called fusogenic lipids. although cationic liposomes have been widely used as an alternative for gene delivery vectors, a dose dependent toxicity of cationic lipids were also observed which could limit their therapeutic usages. the most common use of lipoplexes has been in gene transfer into cancer cells, where the supplied genes have activated tumor suppressor control genes in the cell and decrease the activity of oncogenes. recent studies have shown lipoplexes to be useful in transfecting respiratory epithelial cells. polymersomes polymersomes are synthetic versions of liposomes (vesicles with a lipid bilayer), made of amphiphilic block copolymers. they can encapsulate either hydrophilic or hydrophobic contents and can be used to deliver cargo such as dna, proteins, or drugs to cells. advantages of polymersomes over liposomes include greater stability, mechanical strength, blood circulation time, and storage capacity. polyplexes look up polyplex in wiktionary, the free dictionary. complexes of polymers with dna are called polyplexes. most polyplexes consist of cationic polymers and their fabrication is based on self-assembly by ionic interactions. one important difference between the methods of action of polyplexes and lipoplexes is that polyplexes cannot directly release their dna load into the cytoplasm. as a result, co-transfection with endosome-lytic agents such as inactivated adenovirus was required to facilitate nanoparticle escape from the endocytic vesicle made during particle uptake. however, a better understanding of the mechanisms by which dna can escape from endolysosomal pathway, i.e. proton sponge effect, has triggered new polymer synthesis strategies such as incorporation of protonable residues in polymer backbone and has revitalized research on polycation-based systems. due to their low toxicity, high loading capacity, and ease of fabrication, polycationic nanocarriers demonstrate great promise compared to their rivals such as viral vectors which show high immunogenicity and potential carcinogenicity, and lipid-based vectors which cause dose dependence toxicity. polyethyleneimine and chitosan are among the polymeric carriers that have been extensively studied for development of gene delivery therapeutics. other polycationic carriers such as poly(beta-amino esters) and polyphosphoramidate are being added to the library of potential gene carriers. in addition to the variety of polymers and copolymers, the ease of controlling the size, shape, surface chemistry of these polymeric nano-carriers gives them an edge in targeting capability and taking advantage of enhanced permeability and retention effect. dendrimers a dendrimer is a highly branched macromolecule with a spherical shape. the surface of the particle may be functionalized in many ways and many of the properties of the resulting construct are determined by its surface. in particular it is possible to construct a cationic dendrimer, i.e. one with a positive surface charge. when in the presence of genetic material such as dna or rna, charge complementarity leads to a temporary association of the nucleic acid with the cationic dendrimer. on reaching its destination the dendrimer-nucleic acid complex is then taken into the cell via endocytosis. in recent years the benchmark for transfection agents has been cationic lipids. limitations of these competing reagents have been reported to include: the lack of ability to transfect some cell types, the lack of robust active targeting capabilities, incompatibility with animal models, and toxicity. dendrimers offer robust covalent construction and extreme control over molecule structure, and therefore size. together these give compelling advantages compared to existing approaches. producing dendrimers has historically been a slow and expensive process consisting of numerous slow reactions, an obstacle that severely curtailed their commercial development. the michigan-based company dendritic nanotechnologies discovered a method to produce dendrimers using kinetically driven chemistry, a process that not only reduced cost by a magnitude of three, but also cut reaction time from over a month to several days. these new "priostar" dendrimers can be specifically constructed to carry a dna or rna payload that transfects cells at a high efficiency with little or no toxicity. inorganic nanoparticles inorganic nanoparticles, such as gold, silica, iron oxide (ex. magnetofection) and calcium phosphates have been shown to be capable of gene delivery. some of the benefits of inorganic vectors is in their storage stability, low manufacturing cost and often time, low immunogenicity, and resistance to microbial attack. nanosized materials less than 100 nm have been shown to efficiently trap the dna or rna and allows its escape from the endosome without degradation. inorganics have also been shown to exhibit improved in vitro transfection for attached cell lines due to their increased density and preferential location on the base of the culture dish. quantum dots have also been used successfully and permits the coupling of gene therapy with a stable fluorescence marker. engineered organic nanoparticles are also under development, which could be used for co-delivery of genes and therapeutic agents. cell-penetrating peptides cell-penetrating peptides (cpps), also known as peptide transduction domains (ptds), are short peptides (< 40 amino acids) that efficiently pass through cell membranes while being covalently or non-covalently bound to various molecules, thus facilitating these molecules' entry into cells. cell entry occurs primarily by endocytosis but other entry mechanisms also exist. examples of cargo molecules of cpps include nucleic acids, liposomes, and drugs of low molecular weight. cpp cargo can be directed into specific cell organelles by incorporating localization sequences into cpp sequences. for example, nuclear localization sequences are commonly used to guide cpp cargo into the nucleus. for guidance into mitochondria, a mitochondrial targeting sequence can be used; this method is used in protofection (a technique that allows for foreign mitochondrial dna to be inserted into cells' mitochondria). hybrid methods due to every method of gene transfer having shortcomings, there have been some hybrid methods developed that combine two or more techniques. virosomes are one example; they combine liposomes with an inactivated hiv or influenza virus. this has been shown to have more efficient gene transfer in respiratory epithelial cells than either viral or liposomal methods alone. other methods involve mixing other viral vectors with cationic lipids or hybridising viruses.