ABSTRACT
The success of gene therapy depends on the development of suitable carriers, and because of their architecture dendrimers are promising tools for gene delivery. This research concerns the use of second generation carbosilane dendrimers as carriers for anti-HIV oligodeoxynucleotides (ODNs). The aim was to characterize complexes formed by positively charged dendrimers and negatively charged oligonucleotides using a fluorescence method, laser Doppler electrophoresis, dynamic light scattering (DLS), atomic force microscopy (AFM), transmission electron microscopy (TEM) and molecular modeling. The zeta-potential of ODNs increased from -25 mV to positive values after the addition of dendrimers. DLS and TEM revealed that the diameters of dendriplexes ranged from 75 to 240 nm and from 50 to 260 nm, respectively, and this was dependent on the type of dendrimer and the molar ratios of the complexes formed; complexes were stable for between 100 and 300 minutes. AFM measurements and molecular modeling studies were carried out to determine the structure and size of dendriplexes. The physicochemical properties of the dendriplexes studied and data from previous research suggest that carbosilane dendrimers are good candidates for nucleic acid delivery.
Subject(s)
Dendrimers/chemistry , Drug Carriers/chemistry , Oligonucleotides, Antisense/administration & dosage , Oligonucleotides, Antisense/chemistry , Silanes/chemistry , Dendrimers/administration & dosage , Drug Carriers/administration & dosage , Microscopy, Atomic Force , Microscopy, Electron, Transmission , Models, Molecular , Oligonucleotides, Antisense/ultrastructure , Particle Size , Silanes/administration & dosage , ThermodynamicsABSTRACT
Smad3 mediates the intracellular signaling of TGF-beta1 superfamily and plays a critical role in the cellular proliferation, differentiation and elaboration of matrix pivotal to cutaneous wound healing. Smad3 antisense oligonucleotides (ASOs) impregnated polyelectrolyte complex (PEC) containing chitosan and sodium alginate was prepared for accelerated wound healing. Physicochemical properties of PEC were characterized by zeta potential, scanning electron microscopy and bioadhesive test. Full-thickness, excisional wounds were made on the dorsum of C57BL6 mice. Then, smad3 ASOs-PEC, PEC alone, smad3 ASOs and gauze dressing were applied to determine concentration of TGF-beta1 and collagen in tissues and observe the wound contraction and histology of tissues. Zeta potentials and bioadhesive strengths of ASOs-PEC were increased as the chitosan ratio in PEC. In smad3 ASOs-PEC, the healing process suggested by wound closure and histological observation was faster than other groups because collagen contents increased and level of TGF-beta1 decreased. These results demonstrate that the smad3 ASOs-PEC composed of chitosan and sodium alginate could be applied for accelerated wound healing.
Subject(s)
Alginates/metabolism , Chitosan/metabolism , Electrolytes/metabolism , Oligonucleotides, Antisense/pharmacology , Smad3 Protein/metabolism , Wound Healing/drug effects , Adhesiveness/drug effects , Animals , Cell Death/drug effects , Cell Proliferation/drug effects , Cell Survival/drug effects , Collagen/metabolism , Female , Fibroblasts/cytology , Fibroblasts/drug effects , Glucuronic Acid/metabolism , Hexuronic Acids/metabolism , Mice , Mice, Inbred C57BL , NIH 3T3 Cells , Oligonucleotides, Antisense/ultrastructure , Transforming Growth Factor beta1/metabolismABSTRACT
We measured the helical repeats of a non-natural nucleic acid, locked nucleic acid (LNA), by incorporating LNA strands into the outer arms of a DNA double crossover (DX) molecule; atomic force microscopy (AFM) imaging of the two-dimensional (2D) arrays self-assembled from these DX molecules allows us to derive the helical repeat of the LNA/DNA hetero-duplex to be 13.2 +/- 0.9 base pairs per turn.
Subject(s)
DNA/chemistry , DNA/ultrastructure , Oligonucleotides, Antisense/chemistry , Oligonucleotides, Antisense/ultrastructure , Base Sequence , Microscopy, Atomic Force , Molecular Sequence Data , Nucleic Acid Denaturation , Nucleic Acid Hybridization , Oligonucleotide Array Sequence Analysis , Oligonucleotides , TemperatureABSTRACT
Protamine, a polycationic peptide (mol. wt 4000-4500), was evaluated as a potential penetration enhancer for phosphodiester antisense oligonucleotides (ODNs). Unique complexes in the form of nanoparticles were spontaneously formed, which we call 'proticles'. The stability of the particles and the ODNs bound into the proticles was examined in foetal calf serum and cell culture medium. FITC-labelled ODNs bound to protamine showed an increased cellular uptake into human histiocytic lymphoma U 937 cells compared to free ODNs. Proticles significantly decreased cellular growth in a cell proliferation assay using ODNs against the c- myc proto-oncogene.
Subject(s)
Oligonucleotides, Antisense/administration & dosage , Protamines/administration & dosage , Cell Division , Drug Delivery Systems , Electrochemistry , Excipients , Genes, myc , Humans , Oligonucleotides, Antisense/chemistry , Oligonucleotides, Antisense/ultrastructure , Particle Size , Protamines/chemistry , Protamines/ultrastructure , Proto-Oncogene Mas , Tumor Cells, CulturedABSTRACT
c-myb antisense oligonucleotides (AS ODNs) were reversibly immobilized to a novel polymeric core shell nanosphere and their cellular uptake and inhibitory effect on HL60 leukemia cell proliferation studied. The nanosphere surface was so designed as to directly bind ODNs via ionic interactions and reversibly release them inside the cells. Compared with the cellular uptake of free oligonucleotide, the use of AS ODN (immobilized to the nanospheres) produced a 50-fold increase in the intracellular concentration. Specifically, a single dose of 320 nM of AS ODN immobilized to the nanospheres was capable of inhibiting HL60 cell proliferation with the same degree of efficiency obtained using a 50-fold higher dose of free AS ODN. Flow cytometric experiments with fluoresceinated ODNs showed a temperature-dependent uptake, which was detectable as early as 2 h after the beginning of treatment. The inhibitory effect on cell proliferation was maintained for up to 8 days of culture. Moreover, the level of c-Myb protein decreased by 24% after 2 days and by 60% after 4 days of treatment, thus indicating a continuous and sustained release of non-degraded AS ODN from the nanospheres inside the cells.
