Nanotechnology is an evolving field with enormous potential for biomedical applications. The growing interest to use inorganic nanoparticles in medicine is due to the unique size- and shape-dependent optoelectronic properties. Herein, we will focus on gold, silver and platinum nanoparticles, discussing recent developments for therapeutic applications with regard to cancer in terms of nanoparticles being used as a delivery vehicle as well as therapeutic agents. We will also discuss some of the key challenges to be addressed in future studies.

This perspective is to introduce a new pathway for non-viral gene delivery by taking advantage of nanofibrous scaffolds as gene storage devices, gene carriers and homing devices. During gene delivery to the target, the DNA has to be protected in order to pass through a set of barriers before reaching the nucleus. The DNA can form a complex with polycations, and numerous publications exist on how to stabilize the DNA fragments by natural and synthetic materials. Electrospun nanofibrous scaffolds can be used to store the DNA, especially in the form of a more stabilized polyplex, and then to deliver the DNA (polyplex) to cells that are attached to the scaffold. While each essential step has been tested experimentally, the overall yet untested process, especially for in vivo experiments, may lead to a promising specific approach for gene/drug storage and delivery. The pathway described herein is based mainly on our understanding of the physics and chemistry of gene storage and delivery processes, in contrast to using pure biological concepts. Novel biodegradable, biocompatible nanofibrous materials with imbedded DNA (e.g., in the polyplex form) can then be designed to fabricate an intelligent scaffold for gene delivery. To achieve the above goal, the first step is to stabilize the DNA so that it can be incorporated into nanofibrous scaffolds. In this respect, we shall discuss the different methods of DNA/gene condensation and complex formation, and then explain the strategy used to incorporate DNA into electrospun nanofibers. Solvent-induced DNA condensation and then encapsulation were achieved. However, the released naked DNA was not sufficiently protected for gene transfection in cells. The objective of the current perspective is to suggest that, instead of the solvent-induced DNA condensation, one can combine the recently developed polyplex formation by using branched polyethyleneimine (bPEI). More importantly, free bPEI can be incorporated into the nanofibers separately so that during the gene delivery step, the presence of a predesigned amount of free bPEI can greatly increase the gene transfection efficiency, as has been reported recently by Chi Wu and his coworkers. Thus, a physics/chemistry-based pathway that utilizes nanofibrous scaffolds for gene delivery is within reach.

Transfection is a widely used method in molecular biology for the introduction of foreign nucleic acids (DNA or RNA) into eukaryotic cells that permits to control intracellular processes, i.e. the induction or inhibition of protein expression. Nucleic acids alone cannot penetrate the cell membrane, therefore special carriers like cationic polymers or inorganic nanoparticles are required. Single-shell and multi-shell calcium phosphate nanoparticles were prepared and functionalized with DNA and siRNA. Thereby, the expression of enhanced green fluorescing protein (EGFP) can be induced (by using pcDNA3-EGFP) or silenced (by using siRNA). The single-shell nanoparticles were prepared by rapid mixing of aqueous solutions of calcium nitrate and diammonium hydrogen phosphate. The multi-shell nanoparticles were produced by adding further layers of calcium phosphate and DNA to protect DNA from the intracellular degradation by endonucleases. The size of the nanoparticles according to dynamic light scattering and electron microscopy was up to 100 nm with a zeta potential around -30 mV. The transfection efficiency of the nanoparticles was tested in vitro in cell culture.

Calcium phosphate has been used for over 30 years to deliver genetic material to mammalian cells. This vector has proven advantages over other transfection species such as viruses and dendrimers in terms of superior biocompatibility and reduced immune response. However, clinical application of calcium phosphate based transfection techniques is hampered by poor understanding of the key factors underlying its action. Despite widespread in vitro use, little attention has been given to the physico-chemical characteristics of the calcium phosphate particles mediating transfection. In this study parameters were optimised to produce calcium phosphate nanoparticles onto which plasmid DNA (pDNA) was adsorbed that were more effective than a commercial dendrimer vector in delivering pDNA to an osteoblastic cell line and compared favourably in a fibroblastic cell line without the need for special culture conditions such as cell cycle synchronization or glycerol shock treatment. Addition of the pDNA after nanoparticle synthesis allowed for characterisation of particle morphology, size, surface charge and composition. We found that the key parameters for effective calcium phosphate nanoparticle transfection were an optimal concentration of calcium and chloride ions and a nanosized non-agglomerated precipitate.

Stem cells have the potential to be differentiated to a specific cell type through genetic manipulation and therefore, represent a new and versatile source of cell replacement in regenerative medicine. However, conventional ways of gene transfer to these progenitor cells, suffer from a number of disadvantages particularly involving safety and efficacy issues. We have recently reported on the development of a bio-functionalized DNA carrier of carbonate apatite by embedding fibronectin and E-cadherin chimera on the carrier, leading to its high-affinity interactions with embryonic stem cell surface and accelerated transgene delivery for subsequent expression. Here, we show the molecular basis of synthesizing highly functional composite particles utilizing DNA, cell-adhesive proteins and inorganic crystals, and finally establish a superior transfection system for a mouse stem cell line having potential applications in cell-based therapy.

PURPOSE Application of combinatorial chemistry and high throughput screening for the synthesis and evaluation of mini-library of novel biodegradable poly (beta-amino ester)s (PAE)s composed of gamma-aminopropyl-triethoxysilane (APES) and poly (ethylene glycol) diacrylate (PEGDA) for gene delivery efficiency and safety in 293T and HeLa cells in the presence of and absence of serum. MATERIALS AND METHODS PAEs were synthesized at different mole ratios of APES and PEGDA by Michael addition reaction and synthesis was confirmed by 1H nuclear magnetic resonance (1H-NMR). Ninety six ratios of polyplexes were evaluated for luciferase and MTS assay in 293T and HeLa cells in the presence of and absence of serum. Relationship between transfection efficiency and DNA binding ability of PAEs was studied by gel electrophoresis. Particle sizes and molecular weight of selected PAEs were measured by dynamic light scattering and gel permeation chromatography multi-angle light scattering, respectively. RESULTS 1H-NMR confirmed the synthesis of PAEs. In both cell lines, transfection efficiency and cell viability were increased for PAEs obtained from R106 (0.7:1, APES:PEGDA) to R121 (6:1, APES:PEGDA) with a marginal increase in APES concentration. Transfection pattern was uniform in the absence of and presence of serum. In both cell lines, PAE obtained from R121 demonstrated high transfection efficiency and low cytotoxicity as compared to polyethylenimine (25 KDa) and Lipofectamine. PAE obtained from R121 showed good DNA binding and condensation with average particle sizes of 133 nm. CONCLUSION Addition of PEGDA over APES resulted in a novel PAE which has high safety and transfection efficiency. Transfection and cytotoxicity are very sensitive to monomer ratios and mainly governed by concentration of amine monomer.

Increasing attention is being paid on synthetic DNA delivery systems considering some potential life-threatening effects of viral particles, for development of gene-based nanomedicine in the 21st century. In the current nonviral approaches, most of the efforts have been engaged with organic macromolecules like lipids, polymers, and peptides, but comparatively fewer attempts were made to evaluate the potential of inorganic materials for gene delivery. We recently reported that biodegradable nanoparticles of carbonate apatite are highly efficient in transfecting a wide variety of mammalian cells. Here we show that a number of parameters actively regulate synthesis of the nanoparticles and their subsequent transfection efficacy. Development of "supersaturation", which is the prerequisite for generation of such particles, could be easily modulated by reactant concentrations, pH of the buffered solution, and incubation temperatures, enabling us to establish a flexible particle generation process for highly productive trans-gene delivery. Carbonate incorporation into the particles have been proposed for generating nano-size particles resulting in cellular uptake of huge amount of plasmid DNA as well as endosome destabilization facilitating significant release of DNA from the endosomes.

Gene therapy is a promising therapeutic strategy to combat genetic or acquired diseases at their root cause rather than just treating symptoms. It is well recognised that there is an urgent need for non-toxic and efficient gene delivery vectors to fully exploit the current potential of gene therapy in molecular medicine. Cell-specific targeting of bioactive nucleotides is a prerequisite to attain the concentration of nucleic acids required for therapeutic efficacy in the target tissue. Many metal ions such as Mg2+, Mn2+, Ba2+ and, most importantly, Ca2+ have been demonstrated to have significant roles in gene delivery. These inorganic cations show low toxicity, good biocompatibility and promise for controlled delivery properties, thus presenting a new alternative to toxic and immunogenic carriers. Recently, inorganic nanoparticles alone, or in combination with a colloidal particulate system such as nanoliposome, an advanced approach to gene delivery, were found to exert a positive effect on gene transfer. In this report, the role of the divalent cations in nucleic acid delivery, particularly with respect to the potential improvement of transfection efficiency of nanolipoplexes, is reviewed.

It has been suggested that macrophages and multinucleated giant cells are responsible for phagocytosis of resorbable calcium phosphate (CaP) compounds implanted in bone defects. However, function of macrophages around the CaP, if continuously exposed to various concentration of extracellular calcium ions ([Ca(2+)](o)), is still unknown. The present study showed that when resorbable octacalcium phosphate was implanted in mouse calvaria, macrophage-like cells were observed around the implant during bone formation. Then, experiments were designed to investigate whether secretion of bone morphogenetic protein 2 (BMP-2) is enhanced by [Ca(2+)](o) in a macrophage cell line (J774A.1) in vitro. The mRNA expression and the secretion of BMP-2 in J774A.1 cells were significantly increased when incubated in the medium with [Ca(2+)](o) up to 14mM. The promotion of mRNA expression was maintained even when incubated with a small amount of minute CaP crystals. The present results suggest that [Ca(2+)](o) above physiological concentration may stimulate macrophages to induce osteogenic cytokine, such as BMP-2, for bone formation by osteoblast.

Coated calcium phosphate nanoparticles were prepared for cell transfection. A calcium phosphate nanoparticle served as core which was then coated with DNA for colloidal stabilisation. The efficiency of transfection could be considerably increased by adding another layer of calcium phosphate on the surface, thereby incorporating DNA into the particle and preventing its degradation within the cell by lysosomes. A subsequent outermost layer of DNA on the calcium phosphate gave a colloidal stabilisation. The efficiency of such multi-shell particles was significantly higher than that of simple DNA-coated calcium phosphate nanoparticles. The transfection efficiency of EGFP-encoding DNA was tested with different cell lines (T-HUVEC, HeLa, and LTK). The dispersions were stable and could be used for transfection after 2 weeks of storage at 4 degrees C without loss of efficiency.

Extracellular matrix (ECM) plays important roles in tissue engineering because cellular growth and differentiation, in the two-dimensional cell culture as well as in the three-dimensional space of the developing organism, require ECM with which the cells can interact. Also, the development of new synthetic ECMs is very important because ECMs facilitate the localization and delivery of cells to the specific sites in the body. Therefore, the development of synthetic ECMs to replace the natural ECMs is increasingly essential and promising in tissue engineering. Recombinant genetic engineering method has enabled the synthesis of protein-based polymers with precisely controlled functionalities for the development of new synthetic ECMs. In this review, the design and construction of structure-based recombinant fusion proteins such as elastin-like polymers (ELPs) and silk-like polymers (SLPs), cell-bound growth factor-based recombinant fusion proteins such as basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF), hybrid system composed of recombinant protein and synthetic polymer, and E-cadherin-based fusion protein by recombinant genetic engineering were explained for application of the synthetic ECMs. Modulation of mechanical properties, stimuli-sensitivity, biodegradation and cell recognition can be achieved through precise control of sequence, length, hydrophobicity and cell binding domain by recombinant genetic engineering.

BACKGROUND To maintain pluripotency of human embryonic stem (huES) cells in feeder-free culture it has been necessary to provide a Matrigel substratum, which is a complex of poorly defined extracellular matrices and growth factors derived from mouse Engelbreth-Holm-Swarm sarcoma cells. Culture of stem cells under ill-defined conditions can inhibit the effectiveness of maintaining cells in a pluripotent state and reduce reproducibility of differentiation protocols. Moreover recent batches of Matrigel have been found to be contaminated with the single stranded RNA virus, Lactate Dehydrogenase Elevating Virus (LDEV), raising concerns regarding the safety of using stem cells that have been cultured on Matrigel in a therapeutic setting. To circumvent such concerns, we attempted to identify a recombinant matrix that could be used as an alternative to Matrigel for the culture of human pluripotent stem cells. huES and human induced pluripotent stem (hiPS) cells were grown on plates coated with a fusion protein consisting of E-cadherin and the IgG Fc domain using mTeSR1 medium. RESULTS Cells grown under these conditions maintained similar morphology and growth rate to those grown on Matrigel and retained all pluripotent stem cell features, including an ability to differentiate into multiple cell lineages in teratoma assays. We, therefore, present a culture system that maintains the pluripotency of huES and hiPS cells under completely defined conditions. CONCLUSIONS We propose that this system should facilitate growth of stem cells using good manufacturing practices (GMP), which will be necessary for the clinical use of pluripotent stem cells and their derivatives.

N-cadherin is a cell-cell adhesion molecule and plays important roles in neural development. With conventionally used extracellular matrices (ECMs), maintenance of undifferentiated state of stem cells and regulation of their neural differentiation process is very difficult due to the colony formation through intercellular interactions. To overcome the above-mentioned problems, we developed a new artificial ECM to mimic N-cadherin-mediated cell adhesion. In this study, we constructed a chimeric protein (N-cadherin fused to IgG-Fc, abbreviated as N-cad-Fc), which contains extracellular domain of N-cadherin and Fc domain of IgG. We confirmed that N-cad-Fc can stably adsorb to hydrophobic surface. We checked maintenance of undifferentiated state and neural differentiation ability of stem cells cultured on N-cad-Fc-coated surface. Both P19 and MEB5 cells cultured on N-cad-Fc-coated surface showed scattering morphologies without colony formation and higher proliferating potency than conventional culture systems with maintenance of undifferentiated state. Both of two cell lines cultured on N-cad-Fc-coated surface differentiated into neural cells at a single cell level when induced with proper conditions. Furthermore, the expression of neuron-related gene Neurog1 in two cell lines cultured on N-cad-Fc-coated surface was promoted. Therefore, it will be expected that the constructed N-cad-Fc can be used as an artificial ECM for stem cells.

The design of artificial extracellular matrices (ECM) has attracted much attention in tissue engineering and regenerative medicine as well as in molecular biology research. A recombinant hepatocyte growth factor (HGF), fused to an immunoglobulin G (IgG) Fc region (abbreviated as AeHGF-Fc) was constructed and confirmed by Western blot assay. Almost similar amounts of HepG2 cells adhered to AeHGF-Fc-coated surface compared to collagen-coated one with large morphological changes. Immobilized AeHGF-Fc continuously activated Akt in HepG2 cells whereas Akt activation induced by soluble HGF rapidly decreased with time, indicating that immobilized AeHGF-Fc follows different signal transduction pathways compared to soluble HGF.

Embryonic stem (ES) cells are pluripotent-undifferentiated cells that have a great interest for the investigation of developmental biology. Murine ES cells maintain their pluripotency by the supplementation of the leukemia inhibitory factor (LIF). LIF is reported to act as a matrix-anchored form, and immobilized cytokines are useful to sustain their signaling on target cells. In this study, we used the immobilizable fusion protein composed of LIF and IgG-Fc region, which was used as a model of the matrix-anchored form of LIF to establish a novel system for ES cell culture and to investigate the effect of immobilized LIF on maintenance of ES cell pluripotency. Mouse ES cells maintained their undifferentiated state on the surface coated with LIF-Fc. Furthermore, when cultured on the co-immobilized surface with LIF-Fc and E-cadherin-Fc, mouse ES cells showed characteristic scattering morphologies without colony formation, and they could maintain their undifferentiated state and pluripotency without additional LIF supplementation. The activation of LIF signaling was sustained on the co-immobilized surface. These results indicate that immobilized LIF and E-cadherin can maintain mouse ES cells efficiently and that the immobilizable LIF-Fc fusion protein is useful for the investigation of signaling pathways of an immobilized form of LIF in the maintenance of ES cell pluripotency.

Increasing attention is being paid on synthetic DNA delivery systems considering some potential life-threatening effects of viral particles, for development of gene-based nanomedicine in the 21st century. In the current nonviral approaches, most of the efforts have been engaged with organic macromolecules like lipids, polymers, and peptides, but comparatively fewer attempts were made to evaluate the potential of inorganic materials for gene delivery. We recently reported that biodegradable nanoparticles of carbonate apatite are highly efficient in transfecting a wide variety of mammalian cells. Here we show that a number of parameters actively regulate synthesis of the nanoparticles and their subsequent transfection efficacy. Development of "supersaturation", which is the prerequisite for generation of such particles, could be easily modulated by reactant concentrations, pH of the buffered solution, and incubation temperatures, enabling us to establish a flexible particle generation process for highly productive trans-gene delivery. Carbonate incorporation into the particles have been proposed for generating nano-size particles resulting in cellular uptake of huge amount of plasmid DNA as well as endosome destabilization facilitating significant release of DNA from the endosomes.

The roles of growth factors and extracellular matrices (ECMs) in regulation of hepatocyte behaviors are very important for the establishment of liver-tissue engineering. Especially, collaboration between growth factors and ECMs is a big concern for liver-tissue engineering. In this study, the hepatocyte responses by hepatocyte growth factor (HGF) were compared between natural ECMs and a synthetic galactose-carrying polymer: poly(N-p-vinylbenzyl-4-O-beta-D-galactopyranosyl-D-gluconamide)(PVLA). Hepatocytes underwent proliferation on type I collagen- and fibronectin-coated surfaces in the presence of HGF, whereas hepatocytes formed spheroid on laminin-1-, PVLA-, and poly-L-lysine (PLL)-coated surfaces in the presence of HGF without the activation of proliferation. HGF accelerated ECM deposition, especially laminin-10/11, beneath the hepatocytes cultured on PVLA- and PLL-coated surfaces and the deposited laminin-10/11 activated integrin signaling to collaborate with HGF signaling. Therefore, the deposited ECM molecules should be focused to clear the mechanism of hepatocyte behaviors in the presence of HGF.

Embryonic stem (ES) cell differentiation is regulated by cytokines and growth factors, as well as small-compound chemicals incorporated into cells by transporter proteins. Little is known regarding the effect of transporters on ES cell differentiation. This study focused on the effect of transporters during the neural-lineage differentiation of ES cells. Among the 27 types of SLC family transporters, MCT8 expression was coincident with that of neural stem cell markers, and the overexpression of MCT8 accelerated the differentiation into neural cells. These results suggested that the transporters and their substrates also play a crucial role in the regulation of ES cell differentiation.

It was believed for a long time that mRNA is very unstable, and can not be used for therapeutic purposes. In the last decade, however, many research groups proved its transfection feasibility along with advantages and applications. Our investigation is aimed at establishing a potent and efficient mRNA delivery system. We previously reported that an inorganic-organic hybrid carrier by exploiting the advantages of inorganic nano apatite particles onto organic carrier DOTAP {N-[1-(2,3-dioleoloxy)propyl]-N,N,N-trimethyl ammonium chloride} and showed potential effect of carbonate apatite particles on each of the mRNA delivery steps in dividing and non-dividing cell. Here, we report on the development of a more efficient mRNA carrier by complexing ECM protein, fibronectin with the DOTAP-apatite carrier. The carrier showed enhanced uptake of luciferase mRNA both qualitatively and quantitatively. Accelerated cellular endocytosis rate was evaluated using labeled endosome. Finally expression of lucifearse mRNA was higher for fibronectin complexed carrier in compared to the uncoated one.

Nano-structured calcium phosphate (NanoCaP) particles have been proven to be a powerful means of non-viral gene delivery. In order to better understand the mechanisms through which NanoCaPs-mediated mammalian cell transfection is achieved, we have sought to define the intracellular trafficking pathways involved in the cellular uptake and intracellular processing of these particles. Previous work has indicated that NanoCaP-DNA complexes are most likely internalized via endocytosis, however the subsequent pathways involved have not been determined. Through the use of specific inhibitors, we show that endocytosis of NanoCaP particles is both clathrin- and caveolae-dependent, and suggest that the caveolaer mechanism is the major contributor. We demonstrate colocalization of NanoCaP-pDNA complexes with known markers of both clathrin-coated and caveolar vesicles. Furthermore, through the use of quantitative flow cytometry, we present the first work in which the percent internalization of CaP-DNA complexes into cells is quantified. The overall goal of this research is to foster the continued improvement of NanoCaP-based gene delivery strategies.

Gene therapy provides a unique approach to medicine as it can be adapted towards the treatment of both inherited and acquired diseases. Recently, calcium phosphate vectors as a new generation of the non viral gene delivery nano carriers have been studied because of their biocompatibility and DNA condensation and gene transfer ability. Substituting cations, like magnesium, affects physical and chemical properties of calcium phosphate nano particles. In this study, Mg(2+) substituted calcium phosphate nano particles have been prepared using the simple sol gel method. X-ray diffraction analysis, Fourier transform infra red spectroscopy, transmission electron microscopy, specific surface area analysis, zeta potential measurement and ion release evaluation were used for characterization of the samples. It was concluded that presence of Mg ions decrease particle size and crystallinity of the samples and increase positive surface charge as well as beta tricalcium phosphate fraction in chemical composition of calcium phosphate. These properties result in increasing the DNA condensation ability, specific surface area and dissolution rate of the samples which make them suitable particles for gene delivery application.

Genetic manipulation of human cells through delivery of a functional gene or a gene-silencing element is an attractive approach to treat critical diseases very precisely and effectively. Extensive research on the genetic basis of human diseases with complete sequencing of human genome has revealed many vital genes as possible targets in gene therapy programs. On the other hand, to facilitate cell- or tissue-directed delivery of genes and gene-silencing nucleic acid sequences, both genetic and chemical engineering approaches have led to the generation of various viral and nonviral carriers. However, considering the issues of both safety and efficacy, none of the existing vectors is an ideal candidate for clinical use. We recently established pH-sensitive inorganic nanocrystals of carbonate apatite with capability of efficient intracellular delivery and release of associated DNA molecules for subsequent protein expression. Here we show a new synthetic approach for carbonate apatite crystals with stronger affinity toward DNA, leading to significant increment in both transgene delivery and expression. Moreover, CaCl(2) and NaCl, existing as the major electrolytes in the bicarbonate-buffered solution, dose-dependently govern particle size and eventually internalization and expression of particle-associated DNA.

BACKGROUND Recently, particle bombardment has become increasingly popular as a transfection method, because of a reduced dependency on target cell characteristics. In this study, we evaluated in vitro gene transfer by particle bombardment. METHODS gWIZ luciferase and gWIZ green fluorescent protein (GFP) plasmids were used as reporter genes. Mammalian cell lines HEK 293, MCF7 and NIH/3T3 were used in the transfection experiments. Transfection was performed by bombardment of the cells with gene-coated gold particles using the Helios Gene Gun. The technology was assessed by analyzing gene expression and cell damage. Cell damage was evaluated by MTT assay. RESULTS This technology resulted in efficient in vitro transfection, even in the cells which are difficult to transfect. The gene expression was dependent on the gene gun's helium pressure, the sizes of the gold particles, the amount of the particles and DNA loading, while cell viability was mostly dependent on helium pressure and amount of the gold particles. CONCLUSIONS This technology was useful to transfection of cells. Optimal transfection conditions were determined to be between 75 and 100 psi of helium pressure, 1.0 to 1.6 mum gold particle size and 0.5 mg of gold particle amount with a loading ratio of 4 microg DNA/mg gold particles. GENERAL SIGNIFICANCE These findings will be useful in the design of gene gun device, and bring further improvements to the in vitro and in vivo transfection studies including gene therapy and vaccination.

BACKGROUND Non-viral methods of gene delivery, especially using polyethylenimine (PEI), have been widely used in gene therapy or DNA vaccination. However, the PEI system has its own drawbacks, which limits its applications. METHODS We have developed a novel non-viral delivery system based on PEI coated on the surface of bacterial magnetic nanoparticles (BMPs). The ability of BMPs-PEI complexes to bind DNA was determined by retardation of plasmid DNA in agarose gel electrophoresis. The transfection efficiency of BMPs-PEI/DNA complexes into eukaryotic cells was determined by flow cytometric analysis. The MTT assay was invited to investigate the cytotoxicity of BMPs-PEI/DNA complexes. The expression efficiency in vivo of BMPs-PEI bound to the plasmid pCMVbeta encoding beta-galactosidase was evaluated intramuscularly inoculated into mice. The immune responses of in vivo delivery of BMPs-PEI bound plasmid pcD-VP1 were determined by MTT assay for T cell proliferation and ELISA for detecting total IgG antibodies. RESULTS BMPs-PEI complexes could bind DNA and provide protection from DNase degradation. The transfection efficiency of BMPs-PEI/DNA complexes was higher than that in PEI/DNA complexes. Interestingly, in contrast to PEI, the BMPs-PEI complex was less cytotoxic to cells in vitro. We further demonstrated that the BMPs-PEI system can deliver an exogenous gene to animals and allow it to be expressed in vivo. Such expression resulted in higher levels of humoral and cellular immune responses against the target antigen compared to controls. CONCLUSIONS We have developed a novel BMPs-PEI gene delivery system with a high transfection efficiency and low toxicity, which presents an attractive strategy for gene therapy and DNA vaccination.

The use of the food-grade bacterium Lactococcus lactis as a DNA delivery vehicle at the mucosal level is an attractive DNA vaccination strategy. Previous experiments showed that recombinant L. lactis expressing the Listeria monocytogenesis inlA gene can deliver a functional gene into mammalian cells. Here, we explored the potential use of non-invasive L. lactis strains as DNA delivery vehicle. We constructed two shuttle Escherichia coli-L. lactis plasmids, pLIG:BLG1 and pLIG:BLG2, containing a eukaryotic expression cassette with the cDNA of bovine beta-lactoglobulin (BLG). The greatest BLG expression after transfection of Cos-7 cells was obtained with pLIG:BLG1, which was then used to transform L. lactis MG1363. The resulting L. lactis MG1363(pLIG:BLG1) strain was not able to express BLG. The potential of L. lactis as a DNA delivery vehicle was analyzed by detection of BLG in human colon carcinoma Caco-2 cells after 3 hours co-incubation with: i) purified pLIG:BLG1, ii) MG1363(pLIG:BLG1), iii) a mix of MG1363(pLIG) and purified pLIG:BLG1 and iv) MG1363. Both BLG cDNA and BLG expression were only detected in Caco-2 cells co-incubated with MG1363(pLIG:BLG1). There was a decrease in BLG cDNA in Caco-2 cells between 24 and 48 h after co-incubation. BLG expression by Caco-2 cells started at 24 h and increased between 24 and 72 h. BLG secretion by Caco-2 cells started 48 h after co-incubation with MG1363(pLIG:BLG1). We conclude that lactococci can deliver BLG cDNA into mammalian epithelial cells, demonstrating their potential to deliver in vivo a DNA vaccine.

Two unique and fascinating properties of carbonate apatite which are well-known in hard tissue engineering, have been unveiled, for the first time, for the development of the simplest, but most efficient non-viral gene delivery device - ability of preventing the growth of crystals needed for high frequency DNA transfer across a plasma membrane and a fast dissolution rate for effective release of DNA during endosomal acidification, leading to a remarkably high transgene expression (5 to 100-fold) in mammalian cells compared to the widely used transfecting agents. Moreover, by modulating the crystal dissolution rate of carbonate apatite through incorporation of fluoride or strontium into it, transfection activity could be dramatically controlled, thus shedding light on a new barrier in the non-viral route, which was overlooked so far. Thus we have developed an innovative technology with significant insights, that would come as a promising tool for both basic research laboratories and clinical settings.

DNA can be delivered into the cell nucleus either using physical means or specific carriers that carry the genes into the cells for gene expression). Various carriers for delivering genes have been investigated which can be divided into two main groups: viral carriers where the DNA to be delivered is inserted into a virus, and cationic molecular carriers that form electrostatic interactions with DNA). Successful gene therapy depends on the efficient delivery of genetic materials into the cells nucleus and its effective expression within these cells). Although at present the in vivo expression levels of synthetic molecular gene vectors are lower than for viral vectors and gene expression is transient, these vehicles are likely to present several advantages including safety, low-immunogenicity, capacity to deliver large genes and large-scale production at low-cost). The two leading classes of synthetic gene delivery systems that have been mostly investigated are cationic lipids and cationic polymers). This review discusses recent developments in viral vectors, physical means and molecular gene carriers). The last part focuses on our recent studies in developing a new series of biodegradable polycations for in vitro and in vivo gene transfection).

The recent rapid development of molecular biology together with the steady progress of genome projects has given us some essential and revolutionary informations of gene to elucidate all the biological phenomena at the molecular level. Under these circumstances, gene transfection has become a fundamental technology indispensable to the basic research of medicine and biology. On the other hand, the technology of gene transfection is also important for gene therapy of several diseases. Some human gene therapies have been performed with a plasmid DNA alone or virus vectors but are clinically limited by the poor gene expression of plasmid DNA and the adverse effects of virus itself, such as immunogenicity and toxicity or the possible mutagenesis of cells transfected. Therefore, several non-viral vectors of synthetic materials have been explored to enhance the transfection efficiency of gene into mammalian cells both in vitro and in vivo. In this paper, the researches about non-viral vectors and recent research trials about the controlled release of plasmid DNA are briefly reviewed to emphasize the significance of gene delivery technology in basic biology and medicine as well as clinical medicine. A new system of gene release based on biodegradable hydrogel is introduced.