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ABSTRACT: The objective of this study is to enhance the expression of a plasmid DNA for mesenchymal stem cells (MSC) by combination of 3-dimensional (3D) tissue engineered scaffolds and non-viral gene carrier. As a carrier of plasmid DNA, dextran-spermine cationic polysaccharide was prepared by means of reductive-amination between oxidized dextran and the natural oligoamine, spermine. As the MSC scaffold, collagen sponges reinforced by incorporation of poly(glycolic acid) (PGA) fibers were used. A complex of the cationized dextran and plasmid DNA of BMP-2 was impregnated into the scaffolds. MCS were seeded into each scaffold and cultured by a 3D culture method. When MSC were cultured in the PGA-reinforced sponge, the level of BMP-2 expression was significantly enhanced by the cationized dextran-plasmid DNA complex impregnated into the scaffold than by the cationized dextran-plasmid DNA complex in 2-dimensional (2D) (tissue culture plate) culture method. The alkaline phosphatase activity and osteocalcin content of transfected MSC cultured in the PGA-reinforced sponge were significantly higher compared with 2D culture method. We conclude that combination of cationized dextran plasmid DNA complex and 3D tissue engineered scaffold was promising to promote the in vitro gene expression for MSC.
Biomaterials 09/2006; 27(23):4269-78. · 7.40 Impact Factor
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ABSTRACT: Biodegradable water-soluble polysaccharide-spermine (SPM) polycation conjugates for nucleic acid delivery were synthesized by oxidizing polysaccharides using potassium periodate, followed by SPM conjugation. The polycations differ in their polysaccharide type, arabinogalactan (AG) or dextran (D), and/or in the IO(4)- /saccharide mole ratio used for polysaccharide oxidation (1:1, 1:3, or 1:5), resulting in either D(1:1)-SPM, AG(1:1)-SPM, D(1:3)-SPM, AG(1:3)-SPM, or AG(1:5)-SPM. Chemical structure of the conjugates was characterized for total nitrogen and primary amino groups. Surface pH and electrical surface potential were determined by means of spectral changes of covalently attached 7-hydroxycoumarin (HC, a pH- and electrical surface potential-sensitive fluorophore). The binding and the electrostatic neutralization of the polycations by plasmid DNA, as well as the relationship between chemical structure, physical parameters, and transfection of NIH3T3 cells, were also studied. D(1:1)-SPM, the only polycation that showed efficient cell transfection in culture, was shown to have: (1) high SPM content (2000 nmol/mg); (2) high levels of cross-linked SPM (39-51%); (3) at DNA P-/NH3+ ratio of 2.0, a plateau in neutralization of cationic groups (+48 mV, as determined by HC-labeled D(1:1)-SPM titration with DNA), and a drop in zeta-potential from +42 mV for the polymer alone to 0 mV for the polyplex, suggesting that some of the charges are hidden from the DNA; (4) pH(surface) value of 9.2, suggesting that at physiological bulk pH the polymer is only partially ionized, and therefore can act as a "proton sponge" in the endosome; and (5) high sensitivity to serum-rich growth medium. An oleyl derivative, N-oleyl-dextran-spermine (ODS), was synthesized and demonstrated improved transfection efficiency in serum-rich medium.
Biomaterials 04/2006; 27(8):1646-55. · 7.40 Impact Factor
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ABSTRACT: Gene delivery using self-assembled polyplexes, formed between negatively charged nucleic acids and cationic polymers, have several drawbacks including low transgene expression and toxicity effects related to their positive charge. Recently, a novel cationic polymer based on dextran-spermine (D-SPM) was developed for gene delivery. This polymer showed systemic biodistribution upon local administration (intramuscular (i.m.) and intranasal (i.n.)) followed by transgene expression in organs remote from the site of injection (liver and lungs). Polyplexes based on D-SPM were administered both i.m. and i.n. to BALB/c female mice. LacZ expression in the liver, lungs, and muscles was assessed using whole-mount 5-bromo-4-chloro-3-indolyl beta-d-galactopyranoside (X-gal) staining and paraffin sectioning. The local toxicity in these organs was evaluated from hematoxylin and eosin stained sections. The systemic toxicity of the polymer and polyplexes was estimated by comparing the mice total weight, major organ weights, blood counts (primarily white blood cells (WBC) and platelets), and serum transaminases, to those of control animals (which received PBS). Transgene expression using D-SPM polyplexes was dependent upon the dosage and the polyplexes (+/-) charge ratio. Using the i.m. and i.n. routes of administration the transfection occurred primarily in the bronchial epithelial cells, pneumocytes, and bronchial alveoli of the lungs; in the muscle's fibrocytes; and in the liver's hepatocytes. Histopathological assays revealed mild toxicity in muscle and no abnormal findings in liver and lung. No systemic toxicity was obtained, as we did not find decrease in WBC count or platelet and no increase in serum transaminases. In addition, mice body weights and major organ weights were similar to the control group at both 2 and 28 days post-administration. This study demonstrates systemic transfection efficacy upon local administration of D-SPM complexes with good tolerability and low toxicity.
Biomaterials 04/2006; 27(8):1636-45. · 7.40 Impact Factor
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ABSTRACT: Cationic polysaccharides were synthesized by conjugation of various oligoamines to oxidized polysaccharides by reductive amination and tested for antiprion activity. Polycations of dextran, pullulan and arabinogalactan grafted with oligoamines of 2 to 4 amino groups were investigated for their ability to eliminate PrP(Sc), the protease-resistant isoform of the prion protein, from chronically infected neuroblastoma cells, ScN2a-M. The proteinase K (PK)-resistant PrP elimination depends on both the concentration of the reagent and the duration of exposure. The most potent compound was found to be dextran-spermine that caused depletion of PrP(Sc) to undetectable levels at concentration of 31 ng/mL after 4 days of exposure. Activity analysis revealed that grafted oligoamine indentity of the polycation plays a significant role in elimination of PK-resistant PrP from chronically infected N2a-M cells, regardless of the polysaccharide used. Dextran-spermine conjugates were modified with oleic acid and with methoxypoly(ethylene glycol) (MPEG) at various degrees of substitution for further studies and their antiprion activity was examined. Substitution of dextran-spermine with MPEG or oleic acid slightly decreases its activity as a function of MPEG/oleic acid content. These findings confirm previous reports that polycations are effective in eliminating PrP(Sc) in vitro.
Journal of Medicinal Chemistry 04/2005; 48(5):1414-20. · 5.25 Impact Factor
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ABSTRACT: 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).
Current Drug Delivery 05/2004; 1(2):165-93.
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ABSTRACT: Dextran polysaccharide was grafted by reductive-amination with mixtures of spermine and other natural/synthetic oligoamines of two to four amine groups. The transfection efficiencies of the polycations thus obtained were assessed in various cell lines, and found to depend on the spermine contents. Higher spermine ratios of grafted oligoamines resulted in high gene expression, whereas low to negligible expressions were obtained with lower spermine contents. The effect was explained by spermine residues which exhibit altered buffering capacity in comparison to other substituted oligoamines. Hydrophobization of dextran-spermine (D-SPM) was achieved by treating the polymer with N-hydroxysuccinimide derivatives of cholesterol and fatty acids in a mixture of water/THF. The degree of hydrophobization was in the range of 1-30% mol/mol (hydrophobic moieties/primary amine) and the coupling yields were >95% as determined by (1)H-NMR. The oleate-modified D-SPM remarkably enhanced the gene expression in serum rich media, in marked contrast to unmodified D-SPM which resulted with a drastic decrease in the transfection yields. Modified D-SPM derivatives of other fatty acids and cholesterol showed improved transfection yields in comparison to unmodified D-SPM, but to a lower extent when compared to oleate modification. The improvement in cell transfection was attributed to oleate residues which probably play a role in increasing stability and uptake of polycation-DNA complexes.
Journal of Controlled Release 04/2004; 96(2):309-23. · 5.73 Impact Factor
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ABSTRACT: Malignant primary and metastatic brain tumors have remained fatal in spite of major advances in diagnostic tools and the improvement of conventional therapies. Recent discoveries in the molecular basis of the disease have allowed increased understanding of the events that lead to the development of brain tumors and have also brought a new spectrum of alternatives for treatment. By using gene therapy, brain tumors can be treated by targeting their fundamental molecular defects, delivering gene-drugs to the malignant cells. The possible targets for this type of treatment are progressively increasing but abundant clinical success has yet to be obtained, in part due to imperfect delivery systems. In this review, the genetic fundamentals of various cerebral neoplasms and neurogenetic syndromes, different strategies used for gene therapy, various available DNA delivery systems, status of ongoing clinical trials, and possible prospects for the future are discussed.
Expert Review of Neurotherapeutics 09/2003; 3(5):685-701.
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ABSTRACT: 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. Out of about 50 different polymer structures, only spermine−dextran of 6000−8000 Da, spermine content of 2 μmol/mg, and degree of branching of 25−30% was active in transfecting about 50% of the cells while all other polymers were significantly less active.
12/2002;
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ABSTRACT: This work describes a versatile and universal polycation system based on oligoamines grafted on natural polysaccharides that is capable of complexing various plasmids and administering them into various cells in high yield to produce a desired protein. These polycations are expected to better meet the requirements for effective complexation and delivery of plasmid or an antisense and to biodegrade into nontoxic components at a controlled rate. The developed biodegradable polycations are based on spermine, a natural tetramine, conjugated to dextran or arabinogalactan. These polycations were prepared by reductive amination of oxidized polysaccharides with the desired oligoamines. The Schiff base conjugates thus obtained were reduced to the stable amine conjugates by sodium borohydride. Over 300 different polycations were prepared starting from various polysaccharides and oligoamines, mainly oligoamines of two to four amino groups. Although most of these conjugates formed stable complexes with various plasmids as determined by turbidity experiments, only a few polycations were found to be active in transfecting cells. This work indicates that the structure of the polycation plays a significant role in the transfection activity of polycations.
Journal of Medicinal Chemistry 05/2002; 45(9):1817-24. · 5.25 Impact Factor
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ABSTRACT: Cationic polysaccharides were synthesized by conjugation of various monoquaternary (MQ) ammonium oligoamines to oxidized dextran by reductive amination and tested for gene transfection. Polycations of dextran grafted with MQ ammonium oligoamines of two to four amino groups were investigated for their ability to cause pCMV-GFP encoding for green fluorescence protein and beta-Gal encoding for beta-galactosidase protein transfection on EPC and HEK-293 cell lines. These polycations were expected to strongly complex DNA due to increased surface cationic charge of the carrier, which may result in a higher transfection yield. However, the transfection yields were much lower compared to the parent vector, dextran-spermine conjugate, which was highly effective both in vitro and in vivo.
Bioconjugate Chemistry 16(5):1196-203. · 4.93 Impact Factor
