Polyplexes
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To assist in overcoming the inherent instability of nucleic acid-containing polyplexes in physiological solutions, we have here set out to develop removable nanocoatings for modifying the surface of siRNA-based nanoparticles. Here, N-(2-hydroxypropyl)methacrylamide (HPMA) based copolymers containing carbonylthiazolidine-2-thione (TT) reactive groups in their side chains bound via disulfide spacers to the polymeric backbone were synthesized, and these copolymers were used to coat the surface of polyplexes formed by the self-assembly of anti-Luciferase siRNA with the polycations polyethylene imine (PEI) and poly(HPMA)-grafted poly(l-lysine) (GPL). The coating process was monitored by analyzing changes in the weight-average molecular weight (M(w)), the hydrodynamic radius (R(h)), and the zeta-potential (ζ) of the polyplexes, using both static (SLS) and dynamic (DLS) light scattering methods. The outlined methods resulted in the attachment of, on average, 28 polymer molecules to the surface of the polyplexes, forming a ∼5-nm-thick hydrophilic stealth coating. Initial efforts to develop RGD-targeted coated polyplexes are also described. Atomic force microscopy (AFM) showed an angular polyplex structure and confirmed the narrow size distribution of the coated nanoparticles. The stability of the polymer-coated and uncoated polyplexes was evaluated by gel electrophoresis and by turbidity measurements, and it was found that modifying the surface of the siRNA-containing polyplexes substantially improved their stability in physiological solutions. Using polymer-coated GPL-based polyplexes containing anti-Luciferase siRNA, we finally also obtained some initial proof-of-principle for time- and concentration-dependent target-specific gene silencing, suggesting that these systems hold significant potential for further in vitro and in vivo evaluation.
β-carotene is a natural compound with significant antioxidant activity. However, its poor solubility in water and low stability reduce its potential application. Innovative polyplexes based on the combination of amphiphilic chitosan assembled with DNA have been developed using a solvent-free, simple and low-cost method with the aim to load, retain and enhance the antioxidant capability of β-carotene. The polyplexes, with dimension about 100 nm, and excellent stability, were able to hold up to 400 μg of β-carotene per mg of the carrier, with minimal loss till two weeks. The antioxidant activity was significantly enhanced after loading, as demonstrated using two well known methods. Cytotoxicity assay confirmed the not toxicity of the system. The results suggest the polyplexes as an excellent candidate to develop formulation able to preserve and enhance the peculiarities of compounds which are used mainly in food, cosmetic and pharmaceutic but with still some limitations.
- MeSH
- antioxidancia chemie farmakologie toxicita MeSH
- beta-karoten chemie farmakologie toxicita MeSH
- bifenylové sloučeniny chemie MeSH
- buňky NIH 3T3 MeSH
- chitosan chemie MeSH
- DNA chemie MeSH
- hydrofobní a hydrofilní interakce * MeSH
- myši MeSH
- pikráty chemie MeSH
- rozpustnost MeSH
- stabilita léku MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
We report novel pH-reversibly surface-shielded polyplexes with enhanced gene transfer activity upon systemic administration. A four-arm-structured sequence-defined cationic oligomer KK[HK[(H-Sph-K)3HC]2]2 was designed and synthesized on solid-phase, containing additional lysine residues not only for improved pDNA polyplex stability, but also providing attachment points for subsequent polyplex functionalization with amine-reactive shielding polymers. Herein, the surface of polyplexes was shielded with hydrophilic polymers, monovalent PEG or monovalent and multivalent pHPMA, optionally attached to the polyplex via the acid-labile linker AzMMMan. Overall, surface modification with PEG or pHPMA resulted in a decrease in the zeta potential of polyplexes, consistent with the degree of surface shielding. At pH 6.0, only polyplexes modified via the acid-labile linkage showed an increase in zeta potential, consistent with a "deshielding" in acidic environment, expected as beneficial for endosomal escape. Shielding was more efficient for multivalent pHPMA (20kDa, 30kDa) as compared to monovalent pHPMA (10kDa, 20kDa, 30kDa) or PEG (5kDa). In vitro transfection studies revealed higher gene expression by the polyplexes with the acid-labile shield as compared to their irreversibly shielded counterparts. Intravenous administration of AzMMMan-pHPMA modified polyplexes in an in vivo tumor mouse model mediated enhanced gene expression in the subcutaneous tumor and reduced undesirable expression in the liver.
Colloidal-chemical characteristics of block/branched cationic and non-ionic polyamphiphiles containing poly(fluorine-alkyl methacrylate) (poly(FMA)) block and their intermolecular complexes with biopolymers were studied. The dependences of their surface activity and micelle size on the length of hydrophobic and hydrophilic blocks, as well as the length of side fluorine-alkyl branches were established. Poly(FMA)-block-poly(DMAEMA) was used for formation of interpolyelectrolyte complexes with plasmid DNA (pDNA) via their electrostatic interaction. Novel non-viral polyplexes were tested as gene delivery systems for mammalian cells. The results of DLS, TEM and MALDI-ToF studies demonstrated disaggregation of lysozyme (LYZ) aggregates in the presence of poly(FMA)-block-poly(NVP) and formation of the polyamphiphile…LYS complex possessing antibacterial action.
- MeSH
- DNA chemie metabolismus MeSH
- fluor chemie MeSH
- hydrofobní a hydrofilní interakce MeSH
- methakryláty chemie MeSH
- micely MeSH
- muramidasa chemie metabolismus MeSH
- plazmidy chemie metabolismus MeSH
- polyethylenglykoly chemie MeSH
- polymery chemie metabolismus MeSH
- technika přenosu genů MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Chitosan nanoparticles modified with 10 and 30% urocanic acid (CUA) via carbodiimide crosslinking were examined for an efficient gene delivery carrier. The CUA gene carrier was characterized by FTIR, TEM, SEM and the in vitro transfection efficiency CUA polyplex was tested with HeLa and 3T3 cells. The loading efficiency of CUA complexes with DNA was assessed at different N/P ratio of 1, 2, 4, 6, 8, and 10. The DNA loading efficiency was found be to >85% for chitosan, CUA10 and CUA30% and the DNA protection ability of CUA10 and CUA30 nanoparticle complexes was confirmed upon incubation with NheI and HindIII. The cell toxicity and cell viability results have supported the non-toxic nature of CUA10 and CUA30 nanoparticles. In vitro transfection efficiency of CUA10 and CUA30 polyplex was tested for EGFP expression in 3T3 and HeLa cells and a relative maximum % transfection of about 10% was confirmed by CUA10 and CUA30 after 96h transfection. The feasibility and biocompatibility of CUA gene carrier in transgenic chickens was also demonstrated. The in vitro transfection and in vivo embryonic viability studies further confirmed the CUA as promising gene carrier because of the improved biocompatibility and DNA protection ability.
- MeSH
- buněčná smrt MeSH
- buňky 3T3 MeSH
- chitosan chemie MeSH
- DNA metabolismus MeSH
- endonukleasy metabolismus MeSH
- geneticky modifikovaná zvířata MeSH
- HeLa buňky MeSH
- kuřecí embryo MeSH
- kyselina urokanová chemie MeSH
- lidé MeSH
- myši MeSH
- nanočástice chemie MeSH
- ninhydrin chemie MeSH
- plazmidy metabolismus MeSH
- restrikční mapování MeSH
- spektroskopie infračervená s Fourierovou transformací MeSH
- statická elektřina MeSH
- technika přenosu genů * MeSH
- transfekce MeSH
- velikost částic MeSH
- viabilita buněk MeSH
- zvířata MeSH
- Check Tag
- kuřecí embryo MeSH
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
