Intracellular Drug Delivery Nanocarriers of Glutathione-Responsive Degradable Block Copolymers Having Pendant Disulfide Linkages

Self-assembled micelles of amphiphilic block copolymers (ABPs) with stimuli-responsive degradation (SRD) properties have a great promise as nanotherapeutics exhibiting enhanced release of encapsulated therapeutics into targeted cells. Here, thiol-responsive degradable micelles based on a new ABP consisting of a pendant disulfide-labeled methacrylate polymer block (PHMssEt) and a hydrophilic poly­(ethylene oxide) (PEO) block were investigated as effective intracellular nanocarriers of anticancer drugs. In response to glutathione (GSH) as a cellular trigger, the cleavage of pendant disulfide linkages in hydrophobic PHMssEt blocks of micellar cores caused the destabilization of self-assembled micelles due to change in hydrophobic/hydrophilic balance. Such GSH-triggered micellar destabilization changed their size distribution with an appearance of large aggregates and led to enhanced release of encapsulated anticancer drugs. Cell culture results from flow cytometry and confocal laser scanning microscopy for cellular uptake as well as cell viability measurements for high anticancer efficacy suggest that new GSH-responsive degradable PEO-b-PHMssEt micelles offer versatility in multifunctional drug delivery applications

<i>miR-200c</i> modulates proinflammatory mediators in human preosteoblasts.

<p><b>A</b> and <b>B:</b> the transcripts of IL-6 <b>(A)</b> and IL-8 (<b>B</b>) in non-treated HEPM cells and the cells with <i>miR-200c</i> or scrambled <i>miRs</i> cultured in DMEM supplemented with LPS at 0, 1, 5 and 10 μg/mL after 24 hours; *:p<0.05 vs non-treated; <b>C:</b> the amounts of IL-8 secreted by HEPM cells with <i>miR-200c</i> or scrambled <i>miRs</i> cultured in DMEM supplemented with or without LPS at different time points; *: p<0.05 vs cells with scrambled miRs; <b>D</b> and <b>E:</b> the amounts of IL-6 (<b>D</b>) and CCL-5 (<b>E</b>) secreted by HEPM cells with <i>miR-200c</i> or <i>scrambled miRs</i> cultured in DMEM supplemented with or without LPS after 24 hrs; <b>F:</b> the amounts of OPG secreted by HEPM cells with different <i>miRs</i> cultured in DMEM supplemented with or without LPS after 32 hours. *: p<0.05.</p

Intracellular delivery of <i>miR-200c</i> using PEI nanoparticles to human primary periodontal ligament fibroblasts and bone marrow MSCs.

<p><b>A</b>: TEM image of PEI-<i>miR-200c</i> nanoplexes. <b>B</b> and <b>C:</b> Fold change of the transcript of <i>miR-200c</i> in non-treated human periodontal ligament fibroblasts (<b>B</b>) and bone marrow MSCs (<b>C</b>) and the cells transfected with empty vector (EV) (10μg/per well) and <i>miR-200c</i> (1, 5, 10μg/per well).</p

<i>miR-200c</i> delivered using PEI nanoparticles inhibits IL-6, IL-8, and CCL-5 in primary human periodontal ligament fibroblasts.

<p><b>A-C</b>: The transcripts of IL-6 (<b>A</b>), IL-8 (<b>B</b>), and CCL-5 (<b>C</b>) in the cells with <i>miR-200c</i> or empty vector cultured in DMEM supplemented with LPS after 24 hours; <b>D</b> and <b>E</b>: the amounts of IL-6 (<b>D</b>), IL-8 (<b>E</b>), and CCL-5 (<b>F</b>) secreted by the cells with miR-200c or empty vector cultured in DMEM supplemented with LPS after 12 and 32 hrs, respectively. *: p<0.05 vs empty vector with the same amount.</p

Enhancement of osteogenic differentiation of human bone marrow MSCs with overexpression of <i>miR-200c</i> using PEI nanoparticles.

<p><b>A</b>: Images of ALP and von-Kossa staining in MSCs overexpressing <i>miR-200c</i>, one and two weeks after treatment with osteogenic medium. <b>B</b> and <b>C</b>: the transcripts of ALP (<b>B</b>) and Runx2 (<b>C</b>) in MSCs overexpressing <i>miR-200c</i>, one week after treatment with osteogenic medium. <b>D</b> and <b>E</b>: Quantitative measurement of ALP levels (<b>D</b>) and calcium content (<b>E</b>) in MSCs overexpressing <i>miR-200c</i>, one and two week after treatment with osteogenic medium. Each measurement was made in triplicate. *: p<0.05.</p

<i>miR-200c</i> increases osteogenic biomarkers in human preosteoblasts.

<p><b>A</b> and <b>B</b>: the amounts of the transcript of OCN (<b>A</b>) and calcium content (<b>B</b>) in non-treated HEPM cells and the cells with <i>miR-200c</i> or scrambled <i>miRs</i> cultured in DMEM supplemented <i>β</i>-glycerophosphate and ascorbic acid after 1 and 2 weeks, respectively. *: p<0.05.</p

<i>miR-200c</i> overexpression in HEPM cells and the effects on their proliferation.

<p><b>A</b>: Microphotographs of HEPM cells and the cells with <i>miR-200c</i> or scrambled <i>miRs</i> under phase-contrast. Bar = 10μm. <b>B</b>: Fold change of <i>miR-200c</i> expression in non-treated HEPM cells and the cells with <i>miR-200c</i> and scrambled <i>miRs</i>. <b>C</b>: The doubling time of non-treated HEPM cells and the cells in the context of <i>miR</i> infection. **: p<0.01.</p

<i>PMIS-200c</i> reduces binding activity of <i>miR-200c</i> to the <i>3’UTR</i> of IL-6, IL-8, and CCL-5 and the function of <i>miR-200c</i>.

<p><b>A-C</b>: Normalized luciferase activities of the <i>3’ UTR</i> IL-6, IL-8, and CCL-5-luciferase reporters and their <i>3’UTR</i>-mutated-luciferase reporters co-treated with <i>miR-200c</i> and <i>PMIS-EV</i> or PMIS-200c at different ratios of concentration. <b>D-F</b>: the transcripts of IL-6 (<b>D</b>), IL-8 (<b>E</b>), and CCL-5 (<b>F</b>) in the cells co-treated with <i>miR-200c</i> and <i>PMIS-EV</i> or <i>PMIS-200c</i> cultured in DMEM supplemented with LPS after 24 hours; *: p<0.05.</p