Enhanced siRNA Delivery Using a Combination of an Arginine-Grafted Bioreducible Polymer, Ultrasound, and Microbubbles in Cancer Cells

RNAi-based gene therapy for cancer treatment has not shown significant clinical impact due to poor siRNA delivery to the target site. Here, we design a nonviral siRNA gene carrier using a combination of an arginine-grafted bioreducible polymer (ABP), microbubbles (MB), and ultrasound (US), for targeting vascular endothelial growth factor (VEGF) in a human ovarian cancer cell line. Newly designed MBs with a perfluorocrownether gas core show higher stability compared to controls. Further, MBs in combination with polyplexes show a significant higher loading capacity compared to naked siRNA. Lastly, only siRNA-ABP-MB (SAM) complexes in combination with US show significant VEGF knock down in A2780 human ovarian cancer cell line compared to naked siRNA when incubated for a short time after sonication treatment

Evaluation of Histidylated Arginine-Grafted Bioreducible Polymer To Enhance Transfection Efficiency for Use as a Gene Carrier

To increase cellular uptake and endosomal escape efficiency, various methods have been studied to efficiently deliver plasmid DNA (pDNA) into the cell. Here, we designed a histidylated arginine-grafted bioreducible polymer (HABP) as a nonviral gene carrier using different ratios of histidine and arginine-grafted bioreducible poly­(cystaminebis­(acrylamide)-diaminohexane) (poly­(CBA-DAH)), known as ABP, to increase cellular uptake and endosomal escape efficiency. HABPs consist of arginine (cell penetrating functionality), histidine (endosome buffering functionality), and a disulfide bond backbone (bioreducible functionality in cytoplasm). These components result in the following: (1) polyplexes are easily taken up by cells, (2) polyplexes can easily escape from the endosome into the cytosol, and (3) pDNA can dissociate from polyplexes in reducing environments such as the cytoplasm. HABPs showed increased buffering capacity over histidine-ungrafted ABP, and HABPs formed nanosized polyplexes with pDNA. These polyplexes were about 90 nm in size and had positive charges of about of 30–40 mV. HABPs/pDNA polyplexes showed enhanced transfection efficiency and no significant cytotoxicity in comparison with polyethylenimine 25 kDa (PEI 25k), histidine-ungrafted ABP, and Lipofectamine (commercial reagent) in human cervical carcinoma (HeLa), rat cardiomyocytes (H9C2), and colon carcinoma (CT26) cells

Tuning Surface Charge and PEGylation of Biocompatible Polymers for Efficient Delivery of Nucleic Acid or Adenoviral Vector

As an effective and safe strategy to overcome the limits of therapeutic nucleic acid or adenovirus (Ad) vectors for in vivo application, various technologies to modify the surface of vectors with nonimmunogenic/biocompatible polymers have been emerging in the field of gene therapy. However, the transfection efficacy of the polymer to transfer genetic materials is still relatively weak. To develop more advanced and effective polymers to deliver not only Ad vectors, but also nucleic acids, 6 biocompatible polymers were newly designed and synthesized to different sizes (2k, 3.4k, or 5k) of poly­(ethylene) glycol (PEG) and different numbers of amine groups (2 or 5) based on methoxy poly­(ethylene glycol)-<i>b</i>-poly­{<i>N</i>-[<i>N</i>-(2-aminoethyl)-2-aminoethyl]-l-glutamate (PNLG). We characterized size distribution and surface charge of 6 PNLGs after complexation with either nucleic acid or Ad. Among all 6 PNLGs, the 5 amine group PNLG showed the strongest efficacy in delivering nucleic acid as well as Ad vectors. Interestingly, cellular uptake results showed higher uptake ability in Ad complexed with 2 amine group PNLG than Ad/5 amine group PNLG, suggesting that the size of Ad/PNLGs is more essential than the surface charge for cellular uptake in polymers with charges greater than 30 mV. Moreover, the endosome escape ability of Ad/PNLGs increased depending on the number of amine groups, but decreased by PEG size. Cancer cell killing efficacy and immune response studies of oncolytic Ad/PNLGs showed 5 amine group PNLG to be a more effective and safe carrier for delivering Ad. Overall, these studies provide new insights into the functional mechanism of polymer-based approaches to either nucleic acid or Ad/nanocomplex. Furthermore, the identified ideal biocompatible PNLG polymer formulation (5 amine/2k PEG for nucleic acid, 5 amine/5k PEG for Ad) demonstrated high transduction efficiency as well as therapeutic value (efficacy and safety) and thus has strong potential for in vivo therapeutic use in the future

Characteristics of the out-of-hospital cardiac arrest case group and age-, sex-, and urbanization level-matched control group.

Characteristics of the out-of-hospital cardiac arrest case group and age-, sex-, and urbanization level-matched control group.</p

Interaction analysis for OHCA incidence of insomnia according to depression.

Interaction analysis for OHCA incidence of insomnia according to depression.</p

Characteristics of the study population according to the depression.

Characteristics of the study population according to the depression.</p

Multivariable conditional logistic regression analysis of insomnia and depression for out-of-hospital cardiac arrest.

Multivariable conditional logistic regression analysis of insomnia and depression for out-of-hospital cardiac arrest.</p

Characteristics of the study population according to the insomnia.

Characteristics of the study population according to the insomnia.</p

Safety Profiles and Antitumor Efficacy of Oncolytic Adenovirus Coated with Bioreducible Polymer in the Treatment of a CAR Negative Tumor Model

Adenovirus (Ad) vectors show promise as cancer gene therapy delivery vehicles, but immunogenic safety concerns and coxsackie and adenovirus receptor (CAR)-dependency have limited their use. Alternately, biocompatible and bioreducible nonviral vectors, including arginine-grafted cationic polymers, have been shown to deliver nucleic acids through a cell penetration peptide (CPP) and protein transduction domain (PTD) effect. We utilized the advantages of both viral and nonviral vectors to develop a hybrid gene delivery vehicle by coating Ad with mPEG-PEI-<i>g</i>-Arg-S-S-Arg-<b>g</b>-PEI-mPEG (Ad/PPSA). Characterization of Ad/PPSA particle size and zeta potential showed an overall size and cationic charge increase in a polymer concentration-dependent manner. Ad/PPSA also showed a marked transduction efficiency increase in both CAR-negative and -positive cells compared to naked Ad. Competition assays demonstrated that Ad/PPSA produced higher transgene expression levels than naked Ad and achieved CAR-independent transduction. Oncolytic Ad (DWP418)/PPSA was able to overcome the nonspecificity of polymer-only therapies by demonstrating cancer-specific killing effects. Furthermore, the DWP418/PPSA nanocomplex elicited a 2.24-fold greater antitumor efficacy than naked Ad in vivo. This was supported by immunohistochemical confirmation of Ad E1As accumulation in MCF7 xenografted tumors. Lastly, intravenous injection of DWP418/PPSA elicited less innate immune response compared to naked Ad, evaluated by interleukin-6 cytokine release into the serum. The increased antitumor effect and improved vector targeting to both CAR-negative and -positive cells make DWP418/PPSA a promising tool for cancer gene therapy

Factorial Design Based Multivariate Modeling and Optimization of Tunable Bioresponsive Arginine Grafted Poly(cystaminebis(acrylamide)-diaminohexane) Polymeric Matrix Based Nanocarriers

Desired characteristics of nanocarriers are crucial to explore its therapeutic potential. This investigation aimed to develop tunable bioresponsive newly synthesized unique arginine grafted poly­(cystaminebis­(acrylamide)-diaminohexane) [ABP] polymeric matrix based nanocarriers by using L9 Taguchi factorial design, desirability function, and multivariate method. The selected formulation and process parameters were ABP concentration, acetone concentration, the volume ratio of acetone to ABP solution, and drug concentration. The measured nanocarrier characteristics were particle size, polydispersity index, zeta potential, and percentage drug loading. Experimental validation of nanocarrier characteristics computed from initially developed predictive model showed nonsignificant differences (<i>p</i> > 0.05). The multivariate modeling based optimized cationic nanocarrier formulation of <100 nm loaded with hydrophilic acetaminophen was readapted for a hydrophobic etoposide loading without significant changes (<i>p</i> > 0.05) except for improved loading percentage. This is the first study focusing on ABP polymeric matrix based nanocarrier development. Nanocarrier particle size was stable in PBS 7.4 for 48 h. The increase of zeta potential at lower pH 6.4, compared to the physiological pH, showed possible endosomal escape capability. The glutathione triggered release at the physiological conditions indicated the competence of cytosolic targeting delivery of the loaded drug from bioresponsive nanocarriers. In conclusion, this unique systematic approach provides rational evaluation and prediction of a tunable bioresponsive ABP based matrix nanocarrier, which was built on selected limited number of smart experimentation