Reducible DNA nanoparticles enhance in vitro gene transfer via an extracellular mechanism.
ABSTRACT: We developed polylysine based DNA nanoparticles (DNA NPs) that contain disulfide linkage in the carrier and demonstrated that this reducible DNA NP enhances in vitro gene transfer via an extracellular mechanism. Polylysine was conjugated through an N-terminal cysteine to a polyethylene glycol chain (PEG) by either a disulfide bond (SS) or a thioether bond (CS), and the resulting PEG-peptide conjugates were used to compact plasmid DNA into reducible SS-DNA NPs or non-reducible CS-DNA NPs with identical physical properties. SS-DNA NPs mediated more than 10-fold higher in vitro gene transfer. Others have suggested that disulfide bonds in synthetic gene carriers undergo cleavage in the reducing environment inside the cell, allowing increased intracellular DNA release. In this study, however, both higher cellular uptake of SS-DNA NPs and inhibition of SS-DNA NP mediated in vitro gene transfer by blocking extracellular free thiols suggested an extracellular mechanism. DePEGylation of SS-DNA NPs by extracellular thiols caused aggregation which might lead to higher cellular uptake and higher transgene expression. A series of SS-DNA NPs prepared with stabilized disulfide bonds survived the extracellular environment without aggregation but lost the superior gene transfer ability, indicating that, in our system, intracellular mechanisms are not involved. These results provided further insight into the mechanisms of in vitro gene transfer enhancement by introducing reducible linkages, contributing to the rational design of more efficient non-viral gene delivery systems.
Project description:Biodegradability can be incorporated into cationic polymers via use of disulfide linkages that are degraded in the reducing environment of the cell cytosol. In this work, N-(2-hydroxypropyl)methacrylamide (HPMA) and methacrylamido-functionalized oligo-l-lysine peptide monomers with either a non-reducible 6-aminohexanoic acid (AHX) linker or a reducible 3-[(2-aminoethyl)dithiol] propionic acid (AEDP) linker were copolymerized via reversible addition-fragmentation chain transfer (RAFT) polymerization. Both of the copolymers and a 1:1 (w/w) mixture of copolymers with reducible and non-reducible peptides were complexed with DNA to form polyplexes. The polyplexes were tested for salt stability, transfection efficiency, and cytotoxicity. The HPMA-oligolysine copolymer containing the reducible AEDP linkers was less efficient at transfection than the non-reducible polymer and was prone to flocculation in saline and serum-containing conditions, but was also not cytotoxic at charge ratios tested. Optimal transfection efficiency and toxicity were attained with mixed formulation of copolymers. Flow cytometry uptake studies indicated that blocking extracellular thiols did not restore transfection efficiency and that the decreased transfection of the reducible polyplex is therefore not primarily caused by extracellular polymer reduction by free thiols. The decrease in transfection efficiency of the reducible polymers could be partially mitigated by the addition of low concentrations of EDTA to prevent metal-catalyzed oxidation of reduced polymers.
Project description:A novel amphiphilic cholesterol-based block copolymer comprised of a polymethacrylate bearing cholesterol block and a polyethylene glycol block with reducible disulfide bonds (PC5MA-SS-PEO) was synthesized and evaluated as a redox-sensitive nanoparticulate delivery system. The self-assembled PC5MA-SS-PEO nanoparticles (SS-NPs) encapsulated the anticancer drug doxorubicin (DOX) with high drug loading (18.2% w/w) and high encapsulation efficiency (94.9%). DOX-encapsulated PC5MA-SS-PEO self-assembled nanoparticles (DOX-encapsulated SS-NPs) showed excellent stability and exhibited a rapid DOX release in response to dithiothreitol reductive condition. Importantly, following internalization by lung cancer cells, the reducible DOX-encapsulated SS-NPs achieved higher cytotoxicity than the non-reducible thioester NPs whereas blank nanoparticles were non-cytotoxic. Furthermore, in vivo imaging studies in tumor-bearing severe combined immunodeficiency (SCID) mice showed that the nanoparticles preferentially accumulated in tumor tissue with remarkably reduced accumulation in the healthy non-target organs. The results indicated that the SS-NPs may be a promising platform for cancer-cell specific delivery of hydrophobic anticancer drugs.The use of nanocarriers for drug delivery against tumors has been under intense research. One problem of using carrier system is the drug release kinetics at tumor site. In this article, the authors continued their previous study in the development of an amphiphilic cholesterol-based block copolymer with redox-sensitive modification, so that the payload drug could be released in response to the microenvironment. The interesting results should provide a new direction for designing future novel nanocarrier systems.
Project description:This paper addresses how to evaluate the efficacy of the growing inventory of thiol-reactive inhibitors of mammalian protein disulfide Isomerase (PDI) enzymes under realistic concentrations of potentially competing thiol-containing peptides and proteins. For this purpose, we introduce a variant of the widely-used reductase assay by using a commercially-available cysteine derivative (BODIPY FL L-Cystine; BD-SS) that yields a 55-fold increase in fluorescence (excitation/emission; 490/513nm) on scission of the disulfide bond. This plate reader-compatible method detects human PDI down to 5-10nM, can utilize a range of thiol substrates (including 5µM dithiothreitol, 10µM reduced RNase thiols, and 5mM glutathione; GSH), and can operate from pH 6-9.5 in a variety of buffers. PDI assays often employ low micromolar levels of substrates leading to ambiguities when thiol-directed inhibitors are evaluated. The present work utilizes 5mM GSH for both pre-incubation and assay phases to more realistically reflect the high concentration of thiols that an inhibitor would encounter intracellularly. Extracellular PDI faces a much lower concentration of potentially competing thiols; to assess reductase activity under these conditions, the pre-reduced PDI is treated with inhibitor and then fluorescence increase upon reduction of BD-SS is followed in the absence of additional competing thiols. Both assay modes were tested with four mechanistically diverse PDI inhibitors. Two reversible reagents, 3,4-methylenedioxy-?-nitrostyrene (MNS) and the arsenical APAO, were found to be strong inhibitors of PDI in the absence of competing thiols, but were ineffective in the presence of 5mM GSH. A further examination of the nitrostyrene showed that MNS not only forms facile Michael adducts with GSH, but also with the thiols of unfolded proteins (Kd values of 7 and <0.1µM, respectively) suggesting the existence of multiple potential intracellular targets for this membrane-permeant reagent. The inhibition of PDI by the irreversible alkylating agent, the chloroacetamide 16F16, was found to be only modestly attenuated by 5mM GSH. Finally, the thiol-independent flavonoid inhibitor quercetin-3-O-rutinoside was found to show equal efficacy in reoxidation and turnover assay types. This work provides a framework to evaluate inhibitors that may target the CxxC motifs of PDI and addresses some of the complexities in the interpretation of the behavior of thiol-directed reagents in vivo.
Project description:Redox-responsive polyplexes represent a promising class of non-viral gene delivery vectors. The reducible disulfide bonds in the polyplexes undergo intracellular reduction owing to the presence of high concentrations of reduced glutathione (GSH). Available evidence suggests improved transfection activity of redox-sensitive polyplexes upon artificial modulation of intracellular GSH. This study investigates the effect of innate differences in GSH concentration in a panel of human pancreatic cancer cell lines on activity of reducible polyplexes of the four major classes of nucleic acid therapeutics: plasmid DNA (pDNA), messenger RNA (mRNA), antisense oligodeoxynucleotides (AON) and siRNA. In general, reducible polyplexes of linear poly(amido amines) (PAA) show improved activity compared to non-reducible polyplexes of PAA. Results demonstrate that increased GSH levels are associated with improved transfection of mRNA polyplexes but no clear trend is observed for pDNA, AON and siRNA polyplexes.
Project description:Calcium phosphates (CaPs), constituents of the inorganic phase of natural bone, are highly biocompatible and biodegradable. Strontium (Sr) regulates the formation and resorption of bone. Incorporation of Sr into CaPs may target genes of interest to bone cells while regulating their function. In this work, we developed a single-step synthesis method to prepare Sr-doped CaP nanoparticles (SrCaP-DNA NPs) by using DNA as a template for controlling the mineralization and the stability of the colloidal solution. The resulting nanoparticles were monodispersed with well-controlled size, morphology, and composition. By using this method, we were able to fabricate CaP NPs with varying contents of Sr2+. We demonstrated that the stability of CaP NPs in extracellular environments increased when Sr2+ partially replaced Ca2+ in CaP NPs. We showed that the cellular uptake of SrCaP-DNA NPs and the efficiency of gene transfer and alkaline phosphatase activity in human fetal osteoblastic cell line (hFOB1.19) were dependent on the content of Sr2+ in NPs. Together with other studies, our results suggest SrCaP-DNA NPs can be optimized for targeted gene transfer to regulate function of bone cells, enabling applications such as bone tissue engineering and treating bone diseases.
Project description:Polycations have been successfully used as gene transfer vehicles both in vitro and in vivo; however, their cytotoxicity has been associated with increasing molecular weight. Polymers that can be rapidly degraded after internalization are typically better tolerated by mammalian cells compared to their non-degradable counterparts. Here, we report the use of a dibromomaleimide-alkyne (DBM-alkyne) linking agent to reversibly bridge cationic polymer segments for gene delivery and to provide site-specific functionalization by azide-alkyne cycloaddition chemistry. A panel of reducible and non-reducible, statistical copolymers of (2-dimethylamino)ethyl methacrylate (DMAEMA) and oligo(ethylene glycol)methyl ether methacrylate (OEGMA) were synthesized and evaluated. When complexed with plasmid DNA, the reducible and non-reducible polymers had comparable DNA condensation properties, sizes, and transfection efficiencies. When comparing cytotoxicity, the DBM-linked, reducible polymers were significantly less toxic than the non-reducible polymers. To demonstrate polymer functionalization by click chemistry, the DBM-linked polymers were tagged with an azide-fluorophore and were used to monitor cellular uptake. Overall, this polymer system introduces the use of a reversible linker, DBM-alkyne, to the area of gene delivery and allows for facile, orthogonal, and site-specific functionalization of gene delivery vehicles.
Project description:A unique pH/redox dual-sensitive cationic unimolecular nanoparticle (NP) enabling excellent endosomal/lysosomal escape and efficient siRNA decomplexation inside the target cells was developed for tumor-targeted delivery of siRNA. siRNA was complexed into the cationic core of the unimolecular NP through electrostatic interactions. The cationic core used for complexing siRNA contained reducible disulfide bonds that underwent intracellular reduction owing to the presence of high concentrations of reduced glutathione (GSH) inside the cells, thereby facilitating the decomplexation of siRNA from the unimolecular NPs. The cationic polymers were conjugated onto the hyperbranched core (H40) via a pH-sensitive bond, which further facilitated the decomplexation of siRNA from the NPs. In vitro studies on the siRNA release behaviors showed that dual stimuli (pH=5.3, 10mM GSH) induced the quickest release of siRNA from the NPs. In addition, the imidazole groups attached to the cationic polymer segments enhanced the endosomal/lysosomal escape of NPs via the proton sponge effect. Intracellular tracking studies revealed that siRNA delivered by unimolecular NPs was efficiently released to the cytosol. Moreover, the GE11 peptide, an anti-EGFR peptide, enhanced the cellular uptake of NPs in MDA-MB-468, an EFGR-overexpressing triple negative breast cancer (TNBC) cell line. The GE11-conjugated, GFP-siRNA-complexed NPs exhibited excellent GFP gene silencing efficiency in GFP-MDA-MB-468 TNBC cells without any significant cytotoxicity. Therefore, these studies suggest that this smart unimolecular NP could be a promising nanoplatform for targeted siRNA delivery to EFGR-overexpressing cancer cells.
Project description:Synthetic vectors based on reducible polycations consisting of histidine and polylysine residues (HIS RPCs) were evaluated for their ability to deliver nucleic acids. Initial experiments showed that RPC-based vectors with at least 70% histidine content mediated efficient levels of gene transfer without requirement for the endosomolytic agent chloroquine. Significant gene transfer was observed in a range of cell types achieving up to a 5-fold increase in the percentage of transfected cells compared to 25 kDa PEI, a gold standard synthetic vector. In contrast to 25 kDa PEI, HIS RPCs also mediated efficient transfer of other nucleic acids, including mRNA encoding green fluorescent protein in PC-3 cells and siRNA directed against the neurotrophin receptor p75(NTR) in post-mitotic cultures of rat dorsal root ganglion cell neurons. Experiments to elevate intracellular glutathione and linear profiling of cell images captured by multiphoton fluorescent microscopy highlighted that parameters such as the molecular weight and rate of cleavage of HIS RPCs were important factors in determining transfection activity. Altogether, these results demonstrate that HIS RPCs represent a novel and versatile type of vector that can be used for efficient cytoplasmic delivery of a broad range of nucleic acids. This should enable different or a combination of therapeutic strategies to be evaluated using a single type of polycation-based vector.
Project description:The precision oncology significantly relies on the development of multifunctional agents to integrate tumor targeting, imaging and therapeutics. In this study, a first small-molecule theranostic probe, RhoSSCy is constructed by conjugating 5'-carboxyrhodamines (Rho) and heptamethine cyanine IR765 (Cy) using a reducible disulfide linker and pH tunable amino-group to realize thiols/pH dual sensing. In vitro experiments verify that RhoSSCy is highly sensitive for quantitative analysis and imaging intracellular pH gradient and biothiols. Furthermore, RhoSSCy shows superb tumor targeted dual-modal imaging via near-infrared fluorescence (NIRF) and photoacoustic (PA). Importantly, RhoSSCy also induces strongly reactive oxygen species for tumor photodynamic therapy (PDT) with robust antitumor activity both in vitro and in vivo. Such versatile small-molecule theranostic probe may be promising for tumor targeted imaging and precision therapy.
Project description:Brusatol (Bru) exhibits promising anticancer effects, with both proliferation inhibition and chemoresistance amelioration activity. However, the poor solubility and insufficient intracellular delivery of Bru greatly restrict its application. Herein, to simultaneously utilize the advantages of Pluronics as drug carriers and tumor microenvironment-responsive drug release profiles, a flexible amphiphilic copolymer with a polymer skeleton, that is, Pluronic<sup>®</sup> F68 grafting with linoleic acid moieties by redox-reducible disulfide bonds (F68-SS-LA), was synthesized. After characterization by <sup>1</sup>H-nuclear magnetic resonance and Fourier transform infrared spectroscopy, the redox-sensitive F68-SS-LA micelles were self-assembled in a much lower critical micelle concentration than that of the unmodified F68 copolymer. Bru was loaded in micelles (Bru/SS-M) with high loading efficiency, narrow size distribution, and excellent storage stability. The redox-sensitive Bru/SS-M exhibited rapid particle dissociation and drug release in response to a redox environment. Based on the enhanced cellular internalization, Bru/SS-M achieved higher cytotoxicity in both Bel-7402 and MCF-7 cells compared with free Bru and nonreducible micelles. The improved anticancer effect was attributed to the remarkably decreased mitochondrial membrane potential and increased reactive oxygen species level as well as apoptotic rate. These results demonstrated that F68-SS-LA micelles possess great potential as an efficient delivery vehicle for Bru to promote its anticancer efficiency via an oxidation pathway.