Non-viral Gene Delivery Vectors Research Articles

MAGNETIC NANOPARTICLE MEDIATED GENE DELIVERY IN OLIGODENDROGLIAL CELLS: A COMPARISON OF DIFFERENTIATED CELLS VERSUS PRECURSOR FORMS

Magnetic nanoparticles (MNPs) have emerged as a major platform for the formulation of magnetic vectors for nonviral gene delivery. Notably the application of "magnetofection" strategies (use of magnetic fields to increase MNP–cell interactions) can significantly enhance MNP mediated gene transfer. Despite the potential of this approach, the use of MNPs and magnetofection for gene delivery to oligodendrocytes (the cells that make and maintain myelin, the insulating sheath around nerve fibers in the central nervous system) has never been tested. Here, we prove the feasibility of using MNPs in conjunction with applied static or oscillating gradient magnetic fields (the "magnetofection" method) to deliver genes to oligodendrocytes; all applied magnetic field conditions resulted in greater transfection than the no field condition but overall transfection levels obtained were typically low (ca. < 6%). Oligodendrocyte transfection levels under all magnetic field conditions were less than a third compared with their parent cell population, the oligodendrocyte precursor cells. Our results demonstrate for the first time that, within cells of a specific neural lineage, the amenability to transfection is dependent on the differentiation status of the cell.

Targeted gene delivery with noncovalent electrostatic conjugates of sgc‐8c aptamer and polyethylenimine

Several strategies have been shown to improve the transfection efficiency of polyethylenimine (PEI) as a nonviral gene delivery vector. In the present study, a nucleic acid aptamer specific for protein tyrosine kinase 7 (PTK7) surface marker, sgc-8c, was conjugated electrostatically to pre-formed 10-kDa PEI/plasmid DNA polyplexes, and the ability of the conjugate to transfer genetic material was evaluated in MOLT-4 human acute lymphoblastic leukemia T-cells, which express PTK7 on their surface. Polyplexes (plasmid DNA-vector conjugates), prepared using PEI-sgc-8c conjugate and pCMVLuc as a reporter gene, were characterized in terms of particle size, surface charge and the extent of DNA condensation. Polyplexes were also evaluated for cytotoxicity using the MTS colorimetric assay, as well as for transfection efficiency in MOLT-4 cells, and compared with the results obtained in U266 cells, which lack cell surface PTK7. Relative to pDNA/PEI, the size of pDNA/PEI/sgc-8c aptamer polyplexes increased with decreasing zeta potential. In MOLT-4 cells, pDNA/PEI/sgc-8c aptamer polyplexes exhibited an almost six- to eight-fold increase in transfection efficiency compared to that of pDNA/PEI polyplex, indicating that conjugation of sgc-8c aptamer to pre-formed 10-kDa PEI/plasmid DNA polyplexes achieved effective targeting without covalent attachment, whereas receptor-mediated conducted transfection was confirmed by performing a competitive transfection experiment and a cellular uptake study. The results of the present study provide an example of the usefulness of a nucleic acid aptamer in the form of noncovalent, electrostatic conjugates as an approach for enhancing the transfection efficiency of a polycation vector such as PEI without significant induced cytotoxicity.

Reduction biodegradable brushed PDMAEMA derivatives synthesized by atom transfer radical polymerization and click chemistry for gene delivery

Novel reducible and degradable brushed poly(2-(dimethylamino) ethyl methacrylate) (PDMAEMA) derivatives were synthesized and evaluated as non-viral gene delivery vectors. First, alkyne-functionalized poly(aspartic acid) with a disulfide linker between the propargyl group and backbone poly([(propargyl carbamate)-cystamine]-α,β-aspartamide) (P(Asp-SS-AL)) was synthesized. Second, linear low molecular weight (LMW) monoazido-functionalized PDMAEMAs synthesized via atom transfer radical polymerization were conjugated to the polypeptide side-chains of P(Asp-SS-AL) via click chemistry to yield high molecular weight (HMW) polyaspartamide-based disulfide-containing brushed PDMAEMAs (PAPDEs). The PAPDEs were able to condense plasmid DNA to form 100 to 200nm polyplexes with positive ζ-potentials. Moreover, in the presence of dithiothreitol the PAPDEs degraded into LMW PDAMEMA, resulting in disintegration of the PAPDE/DNA polyplexes and subsequent release of plasmid DNA. In vitro experiments revealed that the PAPDEs were less cytotoxic and more effective in gene transfection than control 25kDa poly(ethyleneimine) and HMW linear PDMAEMA. In conclusion, reducible and degradable polycations composed of LMW PDMAEMAs coupled to a polypeptide backbone via reduction-sensitive disulfide bonds are effective gene vectors with an excellent cytocompatibility.

Autophagy and formation of tubulovesicular autophagosomes provide a barrier against nonviral gene delivery

Cationic liposome (lipoplex) and polymer (polyplex)-based vectors have been developed for nonviral gene delivery. These vectors bind DNA and enter cells via endosomes, but intracellular transfer of DNA to the nucleus is inefficient. Here we show that lipoplex and polyplex vectors enter cells in endosomes, activate autophagy and generate tubulovesicular autophagosomes. Activation of autophagy was dependent on ATG5, resulting in lipidation of LC3, but did not require the PtdIns 3-kinase activity of PIK3C3/VPS34. The autophagosomes generated by lipoplex fused with each other, and with endosomes, resulting in the delivery of vectors to large tubulovesicular autophagosomes, which accumulated next to the nucleus. The tubulovesicular autophagosomes contained autophagy receptor protein SQSTM1/p62 and ubiquitin, suggesting capture of autophagy cargoes, but fusion with lysosomes was slow. Gene delivery and expression from both lipoplex and polyplex increased 8-fold in atg5−/− cells unable to generate tubulovesicular autophagosomes. Activation of autophagy and capture within tubulovesicular autophagosomes therefore provides a new cellular barrier against efficient gene transfer and should be considered when designing efficient nonviral gene delivery vectors.

Novel hyperbranched polyethyleneimine conjugate as an efficient non-viral gene delivery vector

In this study, we developed polyethyleneimine-methyl acrylate-polyethyleneimine (PMP) conjugate as a novel, non-viral gene carrier based on low molecular weight polyethyleneimine, and proved PMP derivative exhibit high gene transfection efficiency and low toxicity. We linked methyl acrylate (MA) on polyethyleneimine (PEI) and then grafted PEI onto the PEI-MA. The synthesized PMP was characterized by 1H NMR and FTIR. When added to DNA solution, PMP condensed with DNA at weight ratios over 1:1 (polymer:DNA) to form 96–108 nm polyplexes with 37 mV of zeta potential. In cytotoxicity assay, PMP turned out less toxic than PEI 25 kDa. In vitro transfection assays showed that the gene transfection of PMP/pDNA complexes was similar or slightly higher than that of the PEI 25 kDa/pDNA complexes in 293, HeLa, and HepG2 cells. Our data suggest that PMP-based gene delivery system is economical, effective, and biocompatible, and it may be a promising gene delivery vector for gene therapies. Open image in new window

Enhanced Transfection Efficiency and Reduced Cytotoxicity of Novel Lipid–Polymer Hybrid Nanoplexes

The present study reports the development, characterization, and evaluation of novel polyelectrolytes stabilized lipoplexes as a nonviral vector for gene delivery. In order to achieve the advantage of both DOTAP (1,2-dioleoyl-3-trimethylammonium propane) and PEI (high transfection efficiency) a system was hypothesized in which DOTAP/phosphatidyl choline (PC) lipoplexes were electrostatically coated with anionic poly(acrylic acid) (PAA) and cationic polyethylenimine (PEI) alternatively to finally shape a robust structure PEI-PAA-DOTAP/PC-lipoplexes (nanoplexes). The nanoplexes were found to have size of 242.6 ± 9.4 nm and zeta potential of +23.1 ± 1.5 mV. Following development nanoplexes were evaluated for cellular uptake, nuclear colocalization, transfection efficiency, and cellular toxicity in MCF-7, HeLa, and HEK-293 cell lines. In support of our hypothesis nanoplexes exhibited higher uptake and nuclear colocalization in comparison with DOTAP/PC, DOTAP/DOPE lipoplexes, and PEI polyplexes. Nanoplexes also exhibited 50-80, 11-12, 6-7, and 5-6 fold higher transfection efficiency in comparison with DOTAP/PC-lipoplexes, DOTAP/DOPE-lipoplexes, PEI-polyplexes, and lipofectamine, respectively, and significantly lower toxicity in comparison with DOTAP/PC, DOTAP/DOPE lipoplexes, PEI polyplexes, and commercial lipofectamine.

The effectiveness, cytotoxicity, and intracellular trafficking of nonviral vectors for gene delivery to bone mesenchymal stem cells

Nonviral gene delivery that enables exogenous gene expression in bone mesenchymal stem cells could accelerate clinical application of cell-based gene therapy. This study systematically investigated and compared the potential of polyethylenimine and Lipofectamine 2000 as gene carriers to modify bone mesenchymal stem cells including transfection efficiency, cytotoxicity, intracellular trafficking as well as cell membrane damage and apoptosis/necrosis. Polyethylenimine at its optimal N/P ratio of 10 demonstrated the same toxic effects but lower transfection efficiency (17.1% vs 39.5%) compared to Lipofectamine. Intracellular trafficking resulted in over 80% of bone mesenchymal stem cells that were able to take up polyethylenimine polyplexes, but only 20.69% showed nuclear uptake; however, for Lipofectamine, about half bone mesenchymal stem cells were found to uptake lipoplexes but about 30% displayed nuclear localization. Moreover, the percentages of nuclear localization of both vectors were in close relationship with their transfection efficiency. We concluded that for bone mesenchymal stem cell transfection, polyethylenimine displayed high cellular uptake but Lipofectamine was more effective in delivering genes into the nucleus, which was likely the underlying basis for a more efficient gene expression. Further structure modification of polyethylenimine such as improving its nuclear entry ability will eventually make it a better candidate for bone mesenchymal stem cells’ in vitro gene delivery.

Biophysical study of novel oligoelectrolyte‐based nonviral gene delivery systems for mammalian cells

Gene therapy is an important treatment for genetic and acquired diseases. The success of gene therapy is largely dependent on the development of suitable vectors for gene transfer. Vectors are needed to overcome cellular barriers and to achieve efficient DNA delivery with low cytotoxicity. In the present study, we synthesized and characterized a novel comb-like oligoelectrolyte nanocarrier, BG-2, as a nonviral gene delivery vector. A novel surface-active oligoelectrolyte of comb-like structure was synthesized via controlled radical copolymerization using oligoperoxide Cu(+2) coordinating complex as a multisite initiator of graft copolymerization. The critical micellar concentration was determined by Nile Red fluorescence. Complex formation of DNA with BG-2 was determined by YOYO-1 fluorescence. The physicochemical properties of DNA in complex with BG-2 have been investigated by electrophoresis, dynamic light scattering and fluorescence spectroscopy. The BG-2/DNA complex was demonstrated by scanning electron microscopy. Interactions between BG-2/DNA complex and model membranes were also studied. The sensitivity of the DNA molecule, complexed with BG-2, against deoxyribonuclease I and serum nucleases was assessed by agarose gel electrophoresis. BG-2 efficiency in the transfection of HeLa cells was determined by measuring luciferase gene expression using a luminometer and cytotoxicity was also evaluated. BG-2 oligoelectrolyte was successful in overcoming cellular barriers as a result of forming stable and small sized complexes with DNA, interacting with model membranes in a desirable manner and protecting DNA from nuclease. The transfection efficiency was quite high and cytotoxicity was low. BG-2 appears to be a promising nonviral vector with low cytotoxicity and efficient transfection properties.

Down‐regulated lysosomal processing improved pegylated lipopolyplex‐mediated gene transfection

Nonviral lipid-based gene delivery vectors have been shown to possess better stability and a longer circulation time after surface poly(ethylene glycol) PEG modification. However, surface PEGylation may decrease the transfection efficiency dramatically. In the present study, we addressed the hypothesis that down-regulating lysosomal processing with a clinical available proton pump inhibitor omeprazole might decrease the sequestration of PEGylated Lipid-Mu-DNA (LMD) in intracellular organelles, thereby increasing their transfection efficiencies. LMD nanoparticles were prepared by the self-assembling of cationic liposomes, peptide Mu and plasmid DNA. The characteristics of LMD lipopolyplexes were detected by scanning electron microscopy, photon correlation spectroscopy and DNA gel electrophoresis. They were added to cultured cells with or without omeprazole pretreatment. The detailed cellular uptake and subcellular distribution were followed by flow cytometry and confocal microscopy. Gene transfection efficiencies were evaluated in four cell lines, as well as in xenograft tumor models. Lysosome staining revealed that 0.1 mg/ml (290 μM) omeprazole raised the pH value of intracellular acidic organelles and induced alterations in the lysosomal compartments. Confocal microscopy showed that more gene materials distributed from intracellular organelles to cytoplasms with omeprazole treatment. A luciferase gene transfection assay showed that omeprazole (probably at a nontoxic concentration) increased PEGylated LMD transfection efficiency significantly in human (NCI-H1299, HT-1080 and A375) and mouse (4T1) cell lines, as well as in tumors of H1299 xenograft tumor models (p < 0.05). Omeprazole might be used as a helper to improve gene transfection efficiencies of some PEGylated gene delivery vectors both in vitro and in vivo.

The Copolymer of Poly(2‐dimethylaminoethyl methacrylate) and Methacrylated Chondroitin Sulfate with Low Cytotoxicity for Gene Delivery

Poly(2-dimethylaminoethyl methacrylate) (PDMAEMA) is one of the most potent synthetic nonviral gene-delivery vectors because of its high transfection efficiency. However, the cytotoxicity of PDMAEMA is a major concern for its clinical applications. An anionic crosslinker is synthesized based on a natural polysaccharide, chondroitin sulfate (CS), by introducing methacrylate groups (CSMA). By systematically adjusting the substitution degree of methacrylation on CS and the weight percent of CSMA and PDMAEMA, sol-type copolymers are obtained as a gene-delivery vector. The combination of CS and PDMAEMA is expected not only to reduce the cytotoxicity of PDMAEMA, but also to facilitate better transfection efficiency than PDMAEMA because of the recognition of CS by CD44 receptors on cell surfaces. Two CSMA-modified PDMAEMA copolymers with different CSMA constituents are selected and their polyplexes prepared with plasmid DNA. The cytotoxicity and gene transfection efficiency of the polyplexes are tested and compared with those of PDMAEMA/pDNA. The copolymers of CSMA and PDMAEMA show significantly improved cell viability as compared with PDMAEMA. Their formed polyplexes with pDNA also show lower cytotoxicity than does PDMAEMA/pDNA. The transfection efficiency remarkably increases as the CSMA-modified PDMAEMA/pDNA polyplex is prepared at a weight ratio of 2.4. The internalization mechanism of CSMA-modified PDMAEMA/pDNA in HEK 293T cells is mainly based on caveolae-mediated endocytosis. However, both caveolae-mediated and CD44-mediated endocytosis mechanisms are involved in U87 cells.

Linear polycations by ring-opening polymerization as non-viral gene delivery vectors

For a clinically effective non-viral gene delivery system, a non-toxic and highly efficient vector is of great importance. A series of linear cationic polymers were synthesized by the ring-opening polymerization between diglycidyl ethers and diamines. Their structure–activity relationships as gene delivery vectors were systematically studied. Besides the amino groups with various densities, these polymers have uniform distribution of hydroxyl groups, which were formed in the polymerization and may benefit their biocompatibility and serum-tolerance. These polymers have good DNA binding ability and could condense DNA into nanoparticles with proper sizes and zeta-potentials. MTT assay revealed that polyplexes formed from title polymers have lower cytotoxicity than that derived from PEI. Most of the polymers have higher transfection efficiency than 25 kDa PEI in the in vitro transfection experiments. Polymers prepared from diglycidyl ethers with less or no N atom (2a and 2b) gave dramatically decreased TE, indicating that secondary amine on the backbone is highly required for efficient gene transfection, and compound 2 may be a good building block in the design of cationic polymers for gene delivery. More importantly, these polymers showed much better serum tolerance. Unlike PEI, the transfection mediated by P5 was seldom affected by the presence of 10% serum. Cellular uptake and intracellular distribution studies also confirmed the good performance of P5 in the transfection process with serum.

The Headgroup Evolution of Cationic Lipids for Gene Delivery

Cationic lipids are one of the most widely used nonviral vectors for gene delivery and are especially attractive because they can be easily synthesized and extensively characterized. Additionally, they can best facilitate the elucidation of structure-activity relationships by modifying each of their constituent domains. The polar hydrophilic headgroups enable the condensation of nucleic acids by electrostatic interactions with the negatively charged phosphate groups of the genes, and further govern transfection efficiency. The headgroups of cationic lipids play a crucial role for gene delivery; they can be quaternary ammoniums, amines, aminoacids or peptides, guanidiniums, heterocyclic headgroups, and some unusual headgroups. This review summarizes recent research results concerning the nature (such as the structure and shape of cationic headgroup) and density (such as the number and the spacing of cationic headgroup) of head functional groups for improving the design of efficient cationic lipids to overcome the critical barriers of in vitro and in vivo transfection.

Novel imidazole-functionalized cyclen cationic lipids: Synthesis and application as non-viral gene vectors

A series of novel 1,4,7,10-tetraazacyclododecanes (cyclen)-based cationic lipids bearing histidine imidazole group 10a–10e were synthesized. These amphiphilic molecules have different hydrophobic tails (long chain, cholesterol or α-tocopherol) and various type of linking groups (ether, carbamate or ester). These molecules were used as non-viral gene delivery vectors, and their structure–activity relationships were investigated. As expected, the imidazole group could largely improve the buffering capabilities comparing to cyclen. The liposomes formed from 10 and dioleoylphosphatidyl ethanolamine (DOPE) could bind and condense plasmid DNA into nanoparticles with proper size and zeta-potentials. Comparing with Lipofectamine 2000, the formed lipoplexes gave lower transfected cells proportion, but higher fluorescence intensity, indicating their good intracellular delivering ability. Furthermore, results indicate that transfection efficiency of the cationic lipids is influenced by not only the hydrophobic tails but also the linking group. The cyclen-based cationic lipid with α-tocopherol hydrophobic tail and an ester linkage could give the highest transfection efficiency in the presence of serum.

Application of quantum dots as vectors in targeted survivin gene siRNA delivery

Gene silencing using short interfering RNA (siRNA) is becoming an attractive approach for probing gene function in mammalian cells. This study evaluated the specificity and efficiency of quantum dots (QDs) as non-viral gene vectors for delivery of survivin siRNA and downregulation of survivin gene expression in oral squamous cell carcinoma Tca8113 cells. Water-dispersible cationically-modified QDs were electrostatically attached to anionic siRNA molecules and complexed with siRNA for downregulating expression of the survivin gene. Cellular uptake and allocation of QD–siRNA complexes in Tca8113 cells were monitored using confocal laser scanning microscopy. Real-time polymerase chain reaction (PCR) was used to quantify survivin messenger RNA (mRNA) levels. CdSe QDs were observed with high intensity fluorescence under confocal laser scanning microscopy. Tca8113 cells were successfully transfected by QDs with survivin siRNA, and the red fluorescence from CdSe QDs and green fluorescein amidite fluorescence from siRNA could both be easily observed after 6 hours of incubation. The release of siRNA into the cytoplasm was verified through real-time PCR quantification that showed reduced survivin mRNA levels. In this study, survivin siRNA successfully complexed with water-soluble CdSe QDs and exhibited excellent fluorescent properties and downregulated the expression of the survivin gene in oral squamous cell carcinoma Tca8113 cells. QDs are a novel non-viral gene delivery vector.

Complexation of DNA with poly-(L-lysine) and its copolymers in dimethylformamide

The polyelectrolyte complexes formed by DNA and polycations have been widely used as non-viral vectors for gene delivery. The DNA complex prepared in aqueous solutions is usually controlled by kinetics. In this work, we demonstrated, by using salmon testes DNA (2000 bp), poly-(L-lysine) (PLL35), and poly(ethylene oxide)-b-Poly-(L-lysine) (PEO45-b-PLL35), that the DNA complexes formed in dimethylformamide (DMF) was milder than those formed in aqueous solutions. In DMF, no precipitation of DNA complex is observed even at the stoichiometric charge ratio. Taking advantage of the weak complexation and better solubility of complex in DMF, we premixed DNA and PLL at higher concentration in DMF, followed by quenching the mixture into a large amount of aqueous solution. This method alleviates the kinetic control to a large extent, and the complex obtained is smaller in size and lower in molar mass than those prepared solely in aqueous solution. The morphology of the complex, as studied by AFM, is also different.

A pH-responsive cyclodextrin-based hybrid nanosystem as a nonviral vector for gene delivery

The absence of safe, efficient, cost-effective, and easily scalable delivery platforms is one of the most significant hurdles and critical issues that limit the bench to bedside translation of oligonucleotides-based therapeutics. Acid-labile materials are of special interest in developing nonviral vectors due to their capability of intracellularly delivering therapeutic payload. In this study, a nanovector was designed by integrating a pH-responsive cyclodextrin material and low molecular weight polyethylenimine (PEI). Antisense oligonucleotide (ASON) Bcl-xl could be encapsulated into this hybrid nanosystem with extremely high loading efficiency by a nanoemulsion technique. The developed pH-responsive ASON nanotherapeutics could be efficiently transfected into human lung adenocarcinoma cells in a time- and dose-dependent manner, resulting in effective cell growth inhibition, significant suppression on the expression of Bcl-xl mRNA/protein, and efficient cell apoptosis. Importantly, the new nanovector showed drastically higher efficacy and lower cytotoxicity when compared with PLGA-based counterpart and commonly used cationic vectors like branched PEI (25,000 Da) and Lipofectamine 2000. This pH-responsive hybrid nanosystem may serve as a safe and efficient nonviral vector that may find wide applications in gene therapy.

Caproic acid grafted chitosan cationic nanocomplexes for enhanced gene delivery: Effect of degree of substitution

This work was designed to investigate the effect of the degree of substitutions of caproic acid on plasmid DNA (pDNA) binding, cellular uptake, biocompatibility, and transfection efficiency of caproic acid grafted chitosan (CGC). The CGC with three substitution degrees (CGC-5, CGC-15, and CGC-25) were synthesized by coupling caproic acid with chitosan. Chemical characterization of graft polymers was performed using FTIR, NMR, and elemental analysis. The CGC polymers showed good pDNA condensing capacity and efficient protection of pDNA from DNase I. The nanosized CGC/pDNA polyplexes exhibited well-defined spherical shapes and stability in serum. Isothermal titration calorimetry demonstrated reduction in CGC-pDNA binding constant with increase in the degree of caproic acid substitution. Caproic acid substitution resulted in 2-7-fold higher cellular uptake in HEK 293 cells mainly via the clathrin-mediated pathway without affecting biocompatibility. In vitro transfection study suggested a dependence of transfection efficiency on the variability of caproic acid substitution. The CGC-15 polymer exhibited 31-fold and 1.33-fold higher gene expression compared to chitosan and the marketed non-viral vector FuGENEHD, respectively. These finding suggests that the CGC-15 graft polymer is a promising non-viral gene delivery vector due to its superior transfection efficiency and biocompatibility.

PAMAM Dendrimers Conjugated with L-Arginine and γ-Aminobutyric Acid as Novel Polymeric Gene Delivery Carriers

In this study, we synthesized functional dendrimer derivatives as nonviral gene delivery vectors. Poly(amidoamine) dendrimer (PAMAM, generation 4) was modified to possess functional amino acids to enhance gene transfection efficiency. PAMAM G4 derivatives conjugated with L-arginine (Arg) and γ-aminobutyric acid (GABA) showed higher transfection efficiency and lower cytotoxicity compared to the native PAMAM G4 dendrimer. The polyplex of the PAMAM G4 derivative/pDNA was evaluated using an agarose gel retardation assay and Picogreen reagent assay. Additionally, the MTT assay was performed to examine the cytotoxicity of synthesized polymers. All PAMAM G4 derivatives showed lower cytotoxicity than PEI25kD. Particularly, PAMAM G4-GABA-Arg displayed enhanced transfection efficiency compared to the native PAMAM G4 dendrimer.

Non-viral gene delivery via membrane-penetrating, mannose-targeting supramolecular self-assembled nanocomplexes.

Supramolecular self-assembled nanocomplexes (SSANs) capable of mannose receptor-mediated endocytosis and permeable to cellular and endosomal membranes are developed via the assembly of multiple rationally designed, function-specific materials. As a unique non-viral gene delivery vector, SSANs outperform commercial transfection reagents, including LPF2000, PEI, and jetPEI, by up to 2 orders of magnitude.

Preparation and Chemical Characterization of Eco-friendly ORMOSIL Nanoparticles of Potential Application in DNA Gene Therapy

One focus in nanotechnology is the development and use of nonviral vectors for safe and efficient gene delivery. Inorganic and organically modified silica nanoparticles are chemical and biologically inert, optically transparent and can be doped with imaging agents and/or functionalized to promote its conjugation with different therapeutic molecules. Silica/ORMOSIL nanoparticles can be engineered to improve diagnosis, treatment and follow-up of diseases. A combination of diagnosis devices and therapeutics (theranostics) would be beneficial for patients. In this work, ORMOSIL nanoparticles as non-viral vectors for gene delivery were prepared via a modified Stober sol-gel process directly with 3-aminopropyltriethoxysilane (APTES), methyltriethoxysilane (MTES), vinyltrimethoxysilane (VTES), N 1 - (3-(trimethoxysilyl)-propyl)diethylenetriamine (DETA), and tetraethylorthosilicate (TEOS) as precursors. Dynamic light scattering, transmission electron microscopy and Fourier transformed infrared spectroscopy were used to characterize the ORMOSIL nanospheres. Synthesis has been optimized and monodisperse spherical nanoparticles with desired size have been obtained. Nanoparticle-DNA com- plexes were successfully obtained at different ratios (nanoparticle/pDNA) and confirmed by agarose gel electrophoresis and ethidium bromide exclusion test.