Vectors For Gene Transfection Research Articles

Gemini (dimeric) surfactants

A gemini surfactant (GS) consists of two conventional surfactant molecules chemically bonded together by a spacer. The two terminal hydrocarbon tails can be short or long; the two polar head groups can be cationic, anionic or nonionic; the spacer can be short or long, flexible or rigid. The GS need not be symmetrically disposed about the center of the spacer. GS can self-assemble at much lower concentrations and are superior in surface activity as compared to conventional surfactants. GS are very attractive for catalysis and adsorption applications, new synthetic vectors for gene transfection, analytical separations, solubilization processes, nanoscale technology, biotechnology, enhanced oil recovery and as paint additives.

Physicochemical optimisation of plasmid delivery by cationic lipids.

Non-viral gene therapy is based on the use of plasmid expression vectors and chemical or physical plasmid DNA delivery systems. This review discusses the roles of cationic lipids as vectors for gene transfection, reviews different strategies employed to improve cationic lipids for in vivo use, and provides original results on the physicochemistry of lipoplexes. Cationic lipid/DNA delivery vehicles have evolved considerably since their initial gene transfection experiments. Much work has been carried out to investigate their structure/activity relationships, methods of formulation and physicochemical properties. Further work has also focused on enhancing and prolonging their stability in a physiological environment as well as increasing their site-specific and tissue-specific interactions. Original data presented in this report confirm that cationic lipids associated to DNA form supramolecular lamellar structures, which protect DNA from serum DNAse degradation. The effect of formulation (and hence the size of the particles) on lipoplex in vivo circulation half-life and biodistribution is also discussed. A list of abbreviations can be found at the end of the review.

Degradable poly(amino alcohol esters) as potential DNA vectors with low cytotoxicity.

The synthesis of a new degradable polymer system, poly(amino alcohol esters) and the resulting polymers' potential for use in gene transfection vectors are reported. The polymerization proceeded in a one step reaction from commercially available bis(secondary amines) monomers (N,N'-dimethyl-1,3-propanediamine and N,N'-dimethyl-1,6-hexanediamine, respectively) through nucleophilic addition to the diglycidyl ester of dicarboxylic acid (diglycidyl adipate). Poly(amino alcohol ester) 1 and 2 were synthesized with a yield of 89% and 91% with Mn = 24,800 and Mn = 36,400, respectively. Poly(amino alcohol ester) 1 degraded hydrolytically in phosphate buffer at pH 7.4 with a half-life of approximately 5 days. Both polymers readily self-assembled with plasmid DNA into nanometer-sized DNA/polymer complexes less than 180 nm diameter and are significantly less cytotoxic than the commonly used DNA delivery polymer, poly(ethylene imine) (PEI).

Dextran‐spermine conjugate: an efficient vector for gene delivery

AbstractCationic Polysaccharides based on oligoamine‐dextran conjugates were synthesized and tested as vectors for gene transfection. Dextran with 40 kDa in average molecular weight was oxidized under mild conditions by potassium periodate to obtain the respective polyaldehydes in relatively high yields (∼90%). The oxidized dextran was reacted by reductive animation with various oligoamines of 2 to 4 amino groups to obtain the corresponding imine‐conjugates. These water‐soluble polymers were then reduced by excess of sodium borohydride to obtain the corresponding amine‐conjuagtes in 30‐40% overall yield. The electrostatic interactions of the representative polycations with plasmid DNA were evaluated as a function of charge ratio (+/−, polymer/DNA) and ionic strength of the medium applying the ethidium‐bromide quenching assay. Although most synthetic polycations formed stable complexes with Plasmid DNAs, only the dextran‐spermine conjugate of a defined amino content and molecular weight was able to transfect cells with high efficiency.

Gemini Surfactants: New Synthetic Vectors for Gene Transfection

AbstractFor Abstract see ChemInform Abstract in Full Text.

N-alkylated chitosan as a potential nonviral vector for gene transfection.

Alkylated chitosans (ACSs) were prepared by modifying chitosan (CS) with alkyl bromide. The self-aggregation of ACSs in acetic acid solution was characterized by fluorescence spectroscopy and dynamic light scattering method. The results indicate that introducing alkyl side chains leads to the self-aggregation of ACSs, and CS with a 99% deacetylation degree shows no aggregation due to the electrostatic repulsion. The electrophoresis experiment demonstrates that the complex between CS and DNA was formed at a charge ratio (+/-) of 1/1; ACS/DNA complexes were formed at a lower charge ratio (+/-) of 1/4. A small amount of alkylated chitosans play the same shielding role as chitosan in protecting DNA from DNase hydrolysis. Differential scanning calorimetry (DSC) and atomic force microscopy (AFM) were employed separately to investigate the thermodynamic behavior of dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)/CS and DPPC/ACS mixtures and the variation in topological structure of DPPC membrane induced by CS and ACS. It is shown that CS and ACS can cause the fusion of DPPC multilamellar vesicles as well as membrane destabilization. In contrast, the perturbation effect induced by ACS is more evident due to the hydrophobic interaction. CS and ACS were used to transfer plasmid-encoding CAT into C(2)C(12) cell lines. Upon elongating the alkyl side chain, the transfection efficiency is increased and levels off after the number of carbons in the side chain exceeds 8. It is proposed that the higher transfection efficiency of ACS is attributed to the increasing entry into cells facilitated by hydrophobic interactions and easier unpacking of DNA from ACS carriers due to the weakening of electrostatic attractions between DNA and ACS.

Gemini Surfactants: New Synthetic Vectors for Gene Transfection

The superior surfactant properties of cationic gemini surfactants are applied to the complex problem of introducing genes into cells. Of almost 250 new compounds tested, of some 20 different structural types, a majority showed very good transfection activity in vitro. The surfactant is shown to bind and compact DNA efficiently, and structural studies and calculations provide a working picture of the "lipoplex" formed. The lipoplex can penetrate the outer membranes of many cell types, to appear in the cytoplasm encapsulated within endosomes. Escape from the endosome--a key step for transfection--may be controlled by changes in the aggregation behavior of the lipoplex as the pH falls. The evidence suggests that DNA may be released from the lipoplex before entry into the nucleus, where the new gene can be expressed with high efficiency.

Cationic Polysaccharides for Gene Delivery

Cationic polysaccharides based on spermine−dextran conjugates were synthesized and tested as vectors for gene transfection. Dextrans of 10−380 kDa were oxidized under mild conditions by potassium periodate to obtain the respective polyaldehydes in 90% overall yield. The oxidized dextrans were reacted by reductive amination with increasing amounts of spermine, and the efficacy of conjugation between the oligoamine and polysaccharides was studied as a function of spermine/aldehyde mole ratio, pH, and temperature of medium. The optimal conjugation yields were obtained at 1.25 mole ratio (spermine/aldehyde groups) and pH 11 at room temperature. Under these conditions, ∼2 μmol/mg (spermine/polysaccharide) conjugation was achieved with 25−30% of the spermine moieties were conjugated in both sides to form branched polymers. The water-soluble polymers obtained were interacted with pCMV-GFP plasmid to form nanoparticles that were introduced to HEK293 and NIH3T3 cells in vitro for transfection efficacy assessment. ...

Chitosan and its derivatives--a promising non-viral vector for gene transfection.

The ability of a vector to ferry gene into targeted cells is the premise of improving gene-transfer efficiency. Chitosan, a naturally occurring cationic polysaccharide, has been shown to excel in transcellular transport. This attribute has been fully reflected in chitosan-mediated gene transfection systems. The objective of this review is to summarize the recent encouraging advances in unveiling the mechanism of cell entry and application of chitosan and its derivatives as novel non-viral vectors. It is our belief that researchers will uncover more truth about chitosan-based vector and realize the long-term goal of gene transfection--produce the desired clinical effect.

Aminoglycoside-derived cationic lipids as efficient vectors for gene transfection in vitro and in vivo.

Cationic lipids are at present very actively investigated for gene transfer studies and gene therapy applications. Basically, they rely on the formation of DNA/lipid aggregates via electrostatic interactions between their cationic headgroup and the negatively charged DNA. Although their structure/activity relationships are not well understood, it is generally agreed that the nature of the positive headgroup impacts on their transfection activity. Thus, we have directed our efforts toward the development of cationic lipids with novel cationic moieties. In the present work, we have explored the transfection potential of the lipophilic derivatives of the aminoglycoside kanamycin A. Indeed, aminoglycosides, which are natural polyamines known to bind to nucleic acids, provide a favorable scaffold for the synthesis of a variety of cationic lipids because of their structural features and multifunctional nature. We report here the synthesis of a cationic cholesterol derivative characterized by a kanamycin A headgroup and of its polyguanidinylated derivative. The amino-sugar-based cationic lipid is highly efficient for gene transfection into a variety of mammalian cell lines when used either alone or as a liposomal formulation with the neutral phospholipid dioleoylphosphatidylethanolamine (DOPE). Its polyguanidinylated derivative was also found to mediate in vitro gene transfection. In addition, colloidally stable kanamycin-cholesterol/DOPE lipoplexes were found to be efficient for gene transfection into the mouse airways in vivo. These results reveal the usefulness of cationic lipids characterized by headgroups composed of an aminoglycoside or its guanidinylated derivative for gene transfection in vitro and in vivo.

235. Aminoglycoside-Derived Cationic Lipids Are Efficient Vectors for Gene Transfection In Vitro and In Vivo

235. Aminoglycoside-Derived Cationic Lipids Are Efficient Vectors for Gene Transfection In Vitro and In Vivo

Early gene therapy-induced apoptotic response in BT4C gliomas by magnetic resonance relaxation contrast T1 in the rotating frame.

The design and evaluation of therapeutic gene transfection protocols and vectors are under extensive development. Magnetic resonance imaging (MRI) techniques can aid considerably in the development of experimental treatment approaches, as well as in determining treatment response by observing gross tissue morphology. However, through a unique set of contrast parameters, namely T1, T2, and diffusion, more information about tissue status can be obtained while delineating and classifying tumor characteristics in more detail. We show here that T1 relaxation in the rotating frame, T1rho, provides unique in vivo MRI contrast. Ganciclovir treatment of HSV-tk+BT4C gliomas, which effectively eradicates these tumors, resulted in significantly prolonged T1rho relaxation times in MRI already after 3 days of treatment, whereas conventional contrast parameters were elevated after 6-8 days of therapy. Interestingly, the prolonged T1rho values were observed while an increase in tumor volume was still taking place. The regions of elevated T1rho relaxation coincided with high apoptotic activity as determined by histology, suggesting that T1rho MRI contrast could be used as a novel early indicator of cytotoxic cell damage in gliomas.

Supramolecular Ultrathin Film Strategies for DNA Assemblies: Substrates for Optobioelectronics, Gene Therapy, and Microarrays

ABSTRACTWe describe our strategies and results in the preparation of supramolecularly ordered ultrathin films of DNA assemblies using the layer-by-layer (LbL) alternate polyelectrolyte adsorption technique. The properties of DNA are intimately associated with their polyelectrolyte behavior in solution. Deposition at interfaces is governed by conformation, orientation, and charge density of these biomolecules in relation to the physisorption phenomena in oppositely charged surfaces. Thus, controlling the nature of surfaces (polymer charge density, ionic strength, other non-covalent interactions, etc.) is important in modifying the adsorption phenomena. In this work, differences in adsorption and incorporation of DNA with dyes, linear polymers and dendrimers are highlighted. A number of surface sensitive spectroscopic and microscopic techniques were used to probe the adsorption and multilayer assembly phenomena, e.g. surface plasmon resonance spectroscopy (SPS), AFM, quartz crystal microbalance (QCM) and ellipsometry. These studies are important for future applications such as the use of polycations as non-viral gene transfection vectors for drug-delivery and DNA adsorption on microarray surfaces. By combining with the alternate assembly of azobenzene and phthalocyanine dyes, we have been able to prepare optobiolelectronic substrates where the phenomena of irradiation and electrochemistry can be used to probe the ordering and response of these films.

Retroviral transfection of the lacZ gene from Liz-9 packaging cells to glioma spheroids

Despite the development of numerous vectors for gene transfection to gliomas, patient survival length remains unaffected in clinical trials. For glioma gene therapy to be successful, the extent of gene transfer to the solid tumor tissue has to be high. In the present work we review some of the vector types and strategies so far utilized in experimental and clinical glioma gene therapy. Since gene transfer efficacy into solid glioma tissue is unknown for many vectors, we studied the gene transfer efficacy into multicellular spheroids derived from a human glioma cell line GaMg as well as into spheroids derived from human glioma biopsies (glioblastoma multiforme, GBM). A replication deficient retroviral vector from the Liz 9 packaging cell line was used for transfer of the bacterial β-galactosidase lacZ gene into the target tissue. Gene transfer was obtained by adding medium containing virus from the producer cells to the target tissue. The experiments were also conducted with EGF (epidermal growth factor) added to the medium. The data show that the transfection rate ranged from 0–4.5% where the transfection efficacy was higher in spheroids after the addition of EGF. Most of the transfected cells were found at the surface, but transfected cells could also be observed in the center of the spheroids. We conclude that using this vector system, the transfection efficacy was low, even if the number of replicating cells was increased by adding EGF. The findings are consistent, and may partly explain, the lack of effect using this vector system during in vivo studies.

Introduction of a Disulfide Bond into a Cationic Lipid Enhances Transgene Expression of Plasmid DNA

We have introduced a convenient method of synthesis for disulfide-containing cationic lipids. The lipid, 1,2-dioleoyl-sn-glycero-3-succinyl-2-hydroxyethyl disulfide ornithine conjugate (DOGSDSO), was synthesized and used to prepare liposomes in combination with DOPE. The rationale behind the selection of the disulfide bond was to produce a lipid which could be selectively destabilized within the cytosol of the cell. The disulfide bond of DOGSDSO was shown to be cleaved by reductive media leading to destabilization of the liposome/DNA complex, thus increasing the release of pDNA compared to a non-disulfide-containing analog. The introduction of a disulfide bond increases the transfection activity using model animal cell lines. The transfection activity and toxicity of DOTAP, DOGSDSO and its analog in three cell lines were compared. The amount of transgene (e.g. luciferase) produced with the use of DOGSDSO/DOPE was greater than that of DOTAP/DOPE and up to 50 times more than that of its non-disulfide analog. The results indicate disulfide-containing cationic liposomes may act as excellent vectors for gene transfection.