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Abstract: To deliver nucleic acids including plasmid DNA (pDNA) and short interfering RNA (siRNA), polymeric gene carriers equipped with various functionalities have been extensively investigated. The functionalities of these polymeric vectors have been designed to overcome various extracellular and intracellular hurdles that nucleic acids and their carriers encounter during their journey from injection site to intracellular target site. This review briefly introduces known extracellular and intracellular issues of nucleic acid delivery and their solution strategies. We examine significant yet overlooked factors affecting nucleic acid delivery (e.g., microenvironmental pH, polymer/siRNA complexation, and pharmaceutical formulation) and highlight our reported approaches to solve these problems. [Copyright &y& Elsevier]

Abstract: Poly(lactide-co-glycolide) (PLGA) has most often been employed for the controlled release of protein formulations because of its safety profile with non-toxic degradation products. Nevertheless, such formulations have been plagued by a local acidic microenvironment and protein–polymer interactions, which result in chemical and physical denaturation of loaded proteins and often unfavorable release profiles. This study investigated the pH change of inner PLGA microsphere (MS) using charged (PLGA)n-b-branched polyethyleneimine (bPEI) micelles. The designed micelles can be transformed into either micelle or reverse micelle (RM) depending on the solvent and RM can form microspheres. In addition, (PLGA)n-b-bPEI can be modified into (PLGA)n-b-(carboxylated bPEI) via carboxylation of the primary amines. Cationic micelle (CM) or anionic micelle (AM) was complexed with counter-charged proteins leading to nanosized particles (approximately 100nm). In the micelle/protein complexes, the micelles mostly maintained their proton buffering capacity, and consequently, prevented or delayed the typical decrease in pH caused by degradation of PLGA in aqueous solution. Reconstitutable micelle/protein complexes allowed for increased and fine-tuned protein loading (~20wt.% when using CM1 (CM prepared from PLGA36kDa-b-bPEI25kDa)/insulin complexes) in PLGA MS. In CM2 (CM prepared from (PLGA36kDa)2-b-bPEI25kDa)/insulin (4 of weight ratio (WR) of micelle to protein; WR4)-loaded PLGA MS, CM2 strongly prevented the micellar nanoenvironmental pH (pH 6.6 within 5days and then approximately pH 8.5) to be acidified in PLGA MS for 9weeks, unlike CM2-free PLGA MS. In conclusion, our findings propose that the proton buffering capacity and protein loading in PLGA MS can be tuned by controlling the complexation ratios of micelles and proteins, polymeric architectures of (PLGA)n-b-bPEI copolymers and WR of micelle/protein complexes and PLGA (or RM). [Copyright &y& Elsevier]

Abstract: Ex vivo transfer of therapeutic genes to cells is one of the potential strategies to prolong the life span of cell transplants. However, relatively safe non-viral carriers have not been extensively investigated due to their lower transfection efficiency. In this study, poly(l-lysine)-g-sulfonylurea varying SU content (PLL-SU) was synthesized to promote gene delivery efficacy to an insulin secreting cell line, RINm5F, which is known to express sulfonylurea receptor (SUR). The polymer formed complexes with a model reporter gene of pCMV-Luc (DNA) and the size of resulting particles was around 100 nm. The transfection efficiency of a polymer synthesized with 5 mol% of SU in the reaction feed (PLL-SU5%) to RINm5F cell was at least 5 times higher than that of PLL. The cytotoxicity of PLL-SU5%/DNA complex was equivalent to that of PLL/DNA complex. PLL-SU5% showed less transfection efficiency than PLL to NIH3T3 and HepG2 cells which are SUR negative. In RINm5F cells, the addition of free SU decreased the transfection efficiency of PLL-SU5%/DNA complex, suggesting that the complex shares the same receptors for SU. The PLL-SU5%/DNA complex seems to be internalized via SUR-mediated endocytosis pathway as suggested by vacuolar ATPases inhibition by Bafilomycin A1. It is noted that RINm5F cells treated with PLL-SU5%/DNA complex secreted more insulin than control, untreated cells, suggesting the insulinotropic effect of SU in PLL-SU5%. In conclusion, PLL-SU may be useful for transfer of therapeutic genes into insulin secreting cells. [Copyright &y& Elsevier]

Sonodynamic therapy (SDT) has emerged as an effective therapeutic modality as it employs ultrasound (US) to eradicate deep-seated tumors noninvasively. However, the therapeutic efficacy of SDT in clinical settings remains limited owing to the low aqueous stability and poor pharmacokinetic properties of sonosensitizers. In this study, extracellular vesicles (EVs), which have low systemic toxicity, were used as clinically available nanocarriers to effectively transfer a sonosensitizer to cancer cells. Chlorin e6 (Ce6), a sonosensitizer, was conjugated to a mitochondria-targeting triphenylphosphonium (TPP) moiety and loaded into EVs to enhance the efficacy of SDT, because mitochondria are critical subcellular organelles that regulate cell survival and death. Additionally, piperlongumine (PL), a pro-oxidant and cancer-specific chemotherapeutic agent, was co-encapsulated into EVs to achieve efficient and selective anticancer activity. The EVs substantially amplified the cellular internalization of TPP-conjugated Ce6 (TPP-Ce6), resulting in the enhanced generation of intracellular reactive oxygen species (ROS) in MCF-7 human breast cancer cells upon US exposure. Importantly, EVs encapsulating TPP-Ce6 effectively destroyed the mitochondria under irradiation with US, leading to efficient anticancer activity. The co-encapsulation of pro-oxidant PL into EVs significantly enhanced the SDT efficacy in MCF-7 cells through the excessive generation of ROS. Moreover, the EV co-encapsulating TPP-Ce6 and PL [EV(TPP-Ce6/PL)] exhibited cancer-specific cell death owing to the cancer-selective apoptosis triggered by PL. In vivo study using MCF-7 tumor-xenograft mice revealed that EV(TPP-Ce6/PL) effectively accumulated in tumors after intravenous injection. Notably, treatment with EV(TPP-Ce6/PL) and US inhibited tumor growth significantly without causing systemic toxicity. This study demonstrated the feasibility of using EV(TPP-Ce6/PL) for biocompatible and cancer-specific chemo-SDT. [Display omitted] [ABSTRACT FROM AUTHOR]

Nucleotides (NTs), such as adenosine triphosphate (ATP) and guanosine triphosphate (GTP), are signaling and bioenergy molecules to mediate a range of cellular pathways. We recently reported their significant endosomolytic activity. To evaluate whether polymeric NTs keep endosomolytic and bioenergetic functions of NTs in drug delivery and cell survival, NTs were polymerized by a coupling reaction to form polynucleotides (pNTs: pATP and pGTP) with their molecular weights around 500 kDa. The cellular toxicity, indicated by IC 50 , of pNT was as low as that of corresponding monomeric NT. pNTs were degraded by an intracellular enzyme, alkaline phosphatase. Introduction of pNTs in a polycation-gene complex (polyplex) enhanced the extent of gene expression in cancerous, non-cancerous, and stem cells, up to 1500-fold higher than that of pNT-free polyplex. In addition, cells stored in a pATP solution resulted in a significantly higher survival rate (e.g., up to 20% increase) when exposed to low temperatures than pATP-free solution. The presence of pNT in polyplexes prevented the reduction of transfection efficiency induced by a low temperature. The findings in this study suggest that endosomolytic and bioenergetic pNTs serve as a non-toxic gene carrier component and protect cells from a cold shock or energy depletion. [ABSTRACT FROM AUTHOR]