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Dendritic peptide-conjugated polymeric nanovectors for non-toxic delivery of plasmid DNA and enhanced non-viral transfection of immune cells.

ABSTRACT: Plasmid DNA (pDNA) transfection is advantageous for gene therapies requiring larger genetic elements, including "all-in-one" CRISPR/Cas9 plasmids, but is limited by toxicity as well as poor intracellular release and transfection efficiency in immune cell populations. Here, we developed a synthetic non-viral gene delivery platform composed of poly(ethylene glycol)-b-poly(propylene sulfide) copolymers linked to a cationic dendritic peptide (DP) via a reduceable bond, PEG-b-PPS-ss-DP (PPDP). A library of self-assembling PPDP polymers was synthesized and screened to identify optimal constructs capable of transfecting macrophages with small (pCMV-DsRed, 4.6 kb) and large (pL-CRISPR.EFS.tRFP, 11.7 kb) plasmids. The optimized PPDP construct transfected macrophages, fibroblasts, dendritic cells, and T cells more efficiently and with less toxicity than a commercial Lipo2K reagent, regardless of pDNA size and under standard culture conditions in the presence of serum. The PPDP technology described herein is a stimuli-responsive polymeric nanovector that can be leveraged to meet diverse challenges in gene delivery.

SUBMITTER: Yi S

PROVIDER: S-EPMC9234717 | biostudies-literature | 2022 Jul

REPOSITORIES: biostudies-literature

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Dendritic peptide-conjugated polymeric nanovectors for non-toxic delivery of plasmid DNA and enhanced non-viral transfection of immune cells.

Yi Sijia S Kim Sun-Young SY Vincent Michael P MP Yuk Simseok A SA Bobbala Sharan S Du Fanfan F Scott Evan Alexander EA

iScience 20220608 7

Plasmid DNA (pDNA) transfection is advantageous for gene therapies requiring larger genetic elements, including "all-in-one" CRISPR/Cas9 plasmids, but is limited by toxicity as well as poor intracellular release and transfection efficiency in immune cell populations. Here, we developed a synthetic non-viral gene delivery platform composed of poly(ethylene glycol)-<i>b</i>-poly(propylene sulfide) copolymers linked to a cationic dendritic peptide (DP) via a reduceable bond, PEG-<i>b</i>-PPS-ss-DP ...[more]

PMID: 35769884

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Project description:BackgroundManagement of triple-negative breast cancer (TNBC) is still challenging because of its aggressive clinical behavior and limited targeted treatment options. Cisplatin represents a promising chemotherapeutic compound in neoadjuvant approaches and in the metastatic setting, but its use is limited by scarce bioavailability, severe systemic side effects and drug resistance. Novel site-directed aptamer-based nanotherapeutics have the potential to overcome obstacles of chemotherapy. In this study we investigated the tumor targeting and the anti-tumorigenic effectiveness of novel cisplatin-loaded and aptamer-decorated nanosystems in TNBC.MethodsNanotechnological procedures were applied to entrap cisplatin at high efficacy into polymeric nanoparticles (PNPs) that were conjugated on their surface with the epidermal growth factor receptor (EGFR) selective and cell-internalizing CL4 aptamer to improve targeted therapy. Internalization into TNBC MDA-MB-231 and BT-549 cells of aptamer-decorated PNPs, loaded with BODIPY505-515, was monitored by confocal microscopy using EGFR-depleted cells as negative control. Tumor targeting and biodistribution was evaluated by fluorescence reflectance imaging upon intravenously injection of Cyanine7-labeled nanovectors in nude mice bearing subcutaneous MDA-MB-231 tumors. Cytotoxicity of cisplatin-loaded PNPs toward TNBC cells was evaluated by MTT assay and the antitumor effect was assessed by tumor growth experiments in vivo and ex vivo analyses.ResultsWe demonstrate specific, high and rapid uptake into EGFR-positive TNBC cells of CL4-conjugated fluorescent PNPs which, when loaded with cisplatin, resulted considerably more cytotoxic than the free drug and nanovectors either unconjugated or conjugated with a scrambled aptamer. Importantly, animal studies showed that the CL4-equipped PNPs achieve significantly higher tumor targeting efficiency and enhanced therapeutic effects, without any signs of systemic toxicity, compared with free cisplatin and untargeted PNPs.ConclusionsOur study proposes novel and safe drug-loaded targeted nanosystems for EGFR-positive TNBC with excellent potential for the application in cancer diagnosis and therapy.

Project description:BackgroundWhilst traditional strategies to increase transfection efficiency of non-viral systems aimed at modifying the vector or the polyplexes/lipoplexes, biomaterial-mediated gene delivery has recently sparked increased interest. This review aims at discussing biomaterial properties and unravelling underlying mechanisms of action, for biomaterial-mediated gene delivery. DNA internalisation and cytoplasmic transport are initially discussed. DNA immobilisation, encapsulation and surface-mediated gene delivery (SMD), the role of extracellular matrix (ECM) and topographical cues, biomaterial stiffness and mechanical stimulation are finally outlined.Main textEndocytic pathways and mechanisms to escape the lysosomal network are highly variable. They depend on cell and DNA complex types but can be diverted using appropriate biomaterials. 3D scaffolds are generally fabricated via DNA immobilisation or encapsulation. Degradation rate and interaction with the vector affect temporal patterns of DNA release and transgene expression. In SMD, DNA is instead coated on 2D surfaces. SMD allows the incorporation of topographical cues, which, by inducing cytoskeletal re-arrangements, modulate DNA endocytosis. Incorporation of ECM mimetics allows cell type-specific transfection, whereas in spite of discordances in terms of optimal loading regimens, it is recognised that mechanical loading facilitates gene transfection. Finally, stiffer 2D substrates enhance DNA internalisation, whereas in 3D scaffolds, the role of stiffness is still dubious.ConclusionAlthough it is recognised that biomaterials allow the creation of tailored non-viral gene delivery systems, there still are many outstanding questions. A better characterisation of endocytic pathways would allow the diversion of cell adhesion processes and cytoskeletal dynamics, in order to increase cellular transfection. Further research on optimal biomaterial mechanical properties, cell ligand density and loading regimens is limited by the fact that such parameters influence a plethora of other different processes (e.g. cellular adhesion, spreading, migration, infiltration, and proliferation, DNA diffusion and release) which may in turn modulate gene delivery. Only a better understanding of these processes may allow the creation of novel robust engineered systems, potentially opening up a whole new area of biomaterial-guided gene delivery for non-viral systems.

Dataset Information

Dendritic peptide-conjugated polymeric nanovectors for non-toxic delivery of plasmid DNA and enhanced non-viral transfection of immune cells.

Publications

Dendritic peptide-conjugated polymeric nanovectors for non-toxic delivery of plasmid DNA and enhanced non-viral transfection of immune cells.

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