The current nonviral siRNA delivery systems in the literature face many problems such as, cellular entry, endosomal escape and efficient siRNA release. Considering this motive, we developed gold nanoparticles (AuNPs) and temperature/pH responsive pentablock copolymer based siRNA delivery systems to address these problems. The temperature and pH responsive cationic and amphiphilic pentablock copolymers, which were consisted of the temperature responsive Pluronic F127 middle block constructed by PEO-PPO-PEO ((poly(ethyleneoxide)-block-poly(propyleneoxide)-block-poly(ethyleneoxide))) blocks contributing cellular entry through temperature responsive micellization and pH responsive cationic PDEAEM (poly(2-diethylaminoethyl methacrylate)) end blocks facilitating nucleic acid condensation and endosomal escape, were used for the first time in the development of polyplex and AuNP based multicomponent siRNA delivery systems (MCSs). The results indicated that systems managed to protect siRNA from external effects, maintain the system stability, facilitate cellular entry and enhance endosomal escape. It was noted that the transfection efficiency of the MCSs, which were boosted by the presence of cleavable disulfide bond, was ~15% higher than the commercial product RNAiMax while the efficacy of polyplexes alone were similar to the RNAiMax. In addition, a hydrogel system based on these responsive copolymers were also used as depot for MCSs to provide sustained siRNA release, which is another current issue to be overcome. As an alternative to responsive pentablock copolymers, TAT-HA2 fusion peptide consisting of influenza A virus hemagglutinin protein (HA2) sequence providing endosomal escape and cell permeable HIV Trans-Activator of Transcription (TAT) protein transduction domain (PTD) enabling cellular entry, was used to develop AuNP based MCSs, peptideplex and conjugate siRNA delivery systems. The results indicated that each system provided good siRNA protection and stability, cellular uptake and enhanced endosomal escape properties. The siRNA activity of siRNA-peptide conjugates bearing cleavable disulfide bonds with 70% siRNA efficiency were found to be 5% more efficient than that MSCs and peptideplex systems. As a parallel research, the lack of detailed information about the potential effects of surface modified or bare AuNPs on cell division and replication steps, programmed cell death and DNA damage motivated us to pursue a further detailed study. We have investigated the influence of the PEG coated gold nanoparticles (AuNPs) not only on cellular uptake and toxicity but also on cell cycle phase arrest, apoptosis and DNA damage against different cells. The overall results illustrated that it is possible to minimize the alteration in cell cycle upon exposure to nanoparticles by changing PEG grafting density and hydrodynamic volume. It seems that an optimum PEG grafting density (~0.65 g/nm2) exists at which particles do not severely alter the cell cycle phases.
Another aspect of our ongoing research involves the development of functional biodegradable particles and films/conduits for controlled release of nerve growth factors targeting the problems in peripheral nerve regeneration. In this study, the nerve growth factor (β-NGF) encapsulated amphiphilic and biodegradable polyanhydride microparticles with different compositions (50:50 CPTEG-CPH and 20:80 CPTEG-CPH microparticles (1,6-bis-(p-carboxyphenoxy)hexane (CPH) and 1,8-bis-(p-carboxyphenoxy)-3,6-dioxaocatane (CPTEG))) were prepared and distributed in porous poly-L-lactic acid (PLLA) films with longitudinal micro-patterns on the surface to manipulate and provide sustained β-NGF release. The results indicated that the initial burst NGF release from the film surface was further followed by a sustained NGF release from the polyanhydride particles distributed in the film matrix. The release characteristics changed with respect to particle type and encapsulated NGF amount. The cumulative released NGF amount showed enhanced neurite extension activity on PC12 cells. As an alternative, transdifferentiated mesenchymal stem cells (tMSCs) capable of continuously secreting β-NGF were used along with the lumen of different conduits (gelatin, PLLA, PLGA etc.) with different morphologies to provide sustained NGF release and activity in vitro and in vivo. Our preliminary experiments showed very promising results indicating the importance of conduit morphology.
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