Discovery of mitochondrion-targeting copper(II)-plumbagin and -bipyridine complexes as chemodynamic therapy agents with enhanced antitumor activity.

Four copper(II)-plumbagin and -bipyridine complexes (Cu1-Cu4) were synthesized as chemodynamic therapy agents with enhanced antitumor activity. As lipophilic and positively charged compounds, Cu1-Cu4 were preferentially accumulated in mitochondria and activated the mitochondrial apoptosis pathway. Mechanistic studies showed that Cu1-Cu4 reacted with GSH to reduce Cu2+ ions to Cu+ ions, catalyzed the formation of toxic hydroxyl radicals (˙OH) from hydrogen peroxide (H2O2) through a Fenton-like reaction, induced mitochondrial dysfunction, and activated caspase-9/3, which eventually led to apoptosis. Cu1-Cu4 arrested HeLa cells in the S phase and eventually killed cancer cells. Cu2 showed a favorable pharmacokinetic profile in mice. Moreover, Cu2 effectively inhibited the growth of HeLa xenografts in nude mice and showed low toxicity in vivo.

Development and preliminary validation of Cancer-related Psychological Flexibility Questionnaire.

The Cancer-related Psychological Flexibility Questionnaire (CPFQ) was developed and validated for assessing cancer patients' psychological flexibility, including attitudes and behavior toward cancer. In a systematic process, the CPFQ identified four factors through principal component analysis and confirmatory factor analysis: Cancer Acceptance, Cancer Avoidance, Activity Engagement, and Valued Action. The results of this study reveal that the CPFQ has a clear factor structure and good psychometric properties. The specific nature of cancer and the need for a specific measure of cancer patient psychological flexibility make this questionnaire valuable for research on psychological flexibility in cancer patients.

Clinical significance of circulating tumor cells in predicating the outcomes of patients with colorectal cancer.

BACKGROUND: Relapse and metastasis of patients with Colorectal Cancer (CRC) is the major obstacle to the long-term life of patients. Its mechanisms remain defined. METHODS: A total of 48 CRC patients were enrolled and 68 samples were obtained from the peripheral blood of patients before or after treatments in this study. Twenty non-cancer patients were also detected as a negative control. Circulating Tumor Cells (CTCs), including Epithelial CTCs (eCTCs), Mesenchymal (MCTCs), and epithelial/mesenchymal mixed phenotypes (mixed CTCs), were identified by CanPatrolTM CTC enrichment and RNA in situ hybridization. The relationship between CTCs number and Progression-Free Survival (PFS) or Overall Survival (OS) was evaluated. RESULTS: Thirty-four of 48 patients (70.8%) were found to have positive CTCs. Total CTCs and MCTCs in the post-treatment had a significant correlation PFS and OS. When total CTCs or MCTCs in 5 mL blood of patients were more than 6 CTCs or 5 MCTCs, PFS of the patients was significantly shorter (p < 0.05) than that in patients with less than 6 CTCs or 5 MCTCs. The patients with > 5 CTCs count changes were found to exhibit poor PFS and OS rates (p < 0.05). CONCLUSION: Total CTCs and MCTCs number detection in patients with colorectal cancer was very useful biomarker for predicting the prognosis of patients. Higher CTCs or MCTCs had poorer PFS and OS rates.

Clinical significance of circulating tumor cells in predicating the outcomes of patients with colorectal cancer

Abstract Background: Relapse and metastasis of patients with Colorectal Cancer (CRC) is the major obstacle to the long-term life of patients. Its mechanisms remain defined. Methods: A total of 48 CRC patients were enrolled and 68 samples were obtained from the peripheral blood of patients before or after treatments in this study. Twenty non-cancer patients were also detected as a negative control. Circulating Tumor Cells (CTCs), including Epithelial CTCs (eCTCs), Mesenchymal (MCTCs), and epithelial/ mesenchymal mixed phenotypes (mixed CTCs), were identified by CanPatrolTM CTC enrichment and RNA in situ hybridization. The relationship between CTCs number and Progression-Free Survival (PFS) or Overall Survival (OS) was evaluated. Results: Thirty-four of 48 patients (70.8%) were found to have positive CTCs. Total CTCs and MCTCs in the post-treatment had a significant correlation PFS and OS. When total CTCs or MCTCs in 5 mL blood of patients were more than 6 CTCs or 5 MCTCs, PFS of the patients was significantly shorter (p < 0.05) than that in patients with less than 6 CTCs or 5 MCTCs. The patients with > 5 CTCs count changes were found to exhibit poor PFS and OS rates (p < 0.05). Conclusion: Total CTCs and MCTCs number detection in patients with colorectal cancer was very useful biomarker for predicting the prognosis of patients. Higher CTCs or MCTCs had poorer PFS and OS rates.

Mouse strain specificity of DAAO inhibitors-mediated antinociception.

D-Amino acid oxidase (DAAO) specifically catalyzes the oxidative deamination of neutral and polar D-amino acids and finally yields byproducts of hydrogen peroxide. Our previous work demonstrated that the spinal astroglial DAAO/hydrogen peroxide (H2 O2 ) pathway was involved in the process of pain and morphine antinociceptive tolerance. This study aimed to report mouse strain specificity of DAAO inhibitors on antinociception and explore its possible mechanism. DAAO inhibitors benzoic acid, CBIO, and SUN significantly inhibited formalin-induced tonic pain in Balb/c and Swiss mice, but had no antinociceptive effect in C57 mice. In contrast, morphine and gabapentin inhibited formalin-induced tonic pain by the same degrees among Swiss, Balb/c and C57 mice. Therefore, mouse strain difference in antinociceptive effects was DAAO inhibitors specific. In addition, intrathecal injection of D-serine greatly increased spinal H2 O2 levels by 80.0% and 56.9% in Swiss and Balb/c mice respectively, but reduced spinal H2 O2 levels by 29.0% in C57 mice. However, there was no remarkable difference in spinal DAAO activities among Swiss, Balb/c and C57 mice. The spinal expression of glutathione (GSH) and glutathione peroxidase (GPx) activity in C57 mice were significantly higher than Swiss and Balb/c mice. Furthermore, the specific GPx inhibitor D-penicillamine distinctly restored SUN antinociception in C57 mice. Our results reported that DAAO inhibitors produced antinociception in a strain-dependent manner in mice and the strain specificity might be associated with the difference in spinal GSH and GPx activity.

FDA Approval Summary: Selumetinib for Plexiform Neurofibroma.

On April 10, 2020, the FDA approved selumetinib (KOSELUGO, AstraZeneca) for the treatment of pediatric patients 2 years of age and older with neurofibromatosis type 1 who have symptomatic, inoperable plexiform neurofibromas. Approval was based on demonstration of a durable overall response rate per Response Evaluation in Neurofibromatosis and Schwannomatosis criteria and supported by observed clinical improvements in plexiform neurofibroma-related symptoms and functional impairments in 50 pediatric patients with inoperable plexiform neurofibromas in a single-arm, multicenter trial. The overall reponse rate per NCI investigator assessment was 66% (95% confidence interval, 51-79) with at least 12 months of follow-up. The median duration of response was not reached, and 82% of responding patients experienced duration of response ≥12 months. Clinical outcome assessment endpoints provided supportive efficacy data. Risks of selumetinib are consistent with MAPK (MEK) inhibitor class effects, including ocular, cardiac, musculoskeletal, gastrointestinal, and dermatologic toxicities. Safety was assessed across a pooled database of 74 pediatric patients with plexiform neurofibromas and supported by adult and pediatric selumetinib clinical trial data in cancer indications. The benefit-risk assessment for selumetinib in patients with inoperable plexiform neurofibromas was considered favorable.

Chinese Version of the Psychological Inflexibility in Pain Scale for Cancer Patients Reporting Chronic Pain.

BACKGROUND: Cancer-related chronic pain is reported by many patients during treatment. There are very few Chinese tools for measuring psychological inflexibility caused by cancer pain, particularly with regard to psychological processes that might influence pain severity and function disorder during cancer treatment. OBJECTIVE: To culturally adapt the Psychological Inflexibility in Pain Scale (PIPS) to Chinese cancer patients experiencing chronic pain, including the determination of psychometric properties of the translated PIPS. METHODS: This cross-sectional study included 2 phases: (1) translation and cultural adaptation and (2) determination of psychometric properties of the translated PIPS. In total, 389 cancer patients with several types of cancer experiencing chronic pain enrolled from May to September 2018 at a tertiary cancer hospital in Yuelu District of Hunan Province, China. RESULTS: The Chinese PIPS version was semantically equivalent to the original. It had a 2-factor structure with satisfactory content validity (content validity index = 0.78-1.00), convergent and discriminant validity (composite reliability and average variance extracted at 0.41-0.89, P < .001), criterion-related validity (r = 0.54 and 0.41, P < .001), Cronbach's α coefficients (α = .87), and test-retest reliability (0.9 ≤ r ≤ 0.98). CONCLUSIONS: The Chinese PIPS version has been culturally adapted and has strong psychometric properties. The scale is a psychometrically sound assessment of psychological inflexibility that can be used for future studies of pain and pain management for cancer patients. IMPLICATIONS FOR PRACTICE: The study provides a vital tool for the psychological management of cancer patients with chronic pain.

Six1 is negatively correlated with poor prognosis and reduces 5-fluorouracil sensitivity via attenuating the stemness of hepatocellular carcinoma cells.

The promoting roles of transcriptional factor six1 have been shown in various tumors, such as breast cancer and colorectal Cancer. However, its roles in hepatocellular carcinoma (HCC) cell stemness and chemotherapeutic sensitivity are never been revealed. In the present study, we showed that six1 expression was negatively correlated the overall survival of HCC patients and significantly increased in HCC tissues. Analysis on normal hepatic cells and HCC cells obtained the consistent result. Functional experiments revealed that six1 knockdown enhanced 5-fluorouracil (5-FU) sensitivity and reduced the stemness of HCC cells. Additionally, six1 knockdown partially reversed 5-FU resistance and attenuated the stemness in 5-FU-resistant HCC cells. Furthermore, we demonstrated that six1 directly bound to sox2 (a stemness master regulator) promoter, enhanced its transcription and expression. Overexpression of sox2 rescued the inhibitory effects of six1 knockdown on the stemness and 5-FU sensitivity of HCC cells. Thus, our work identified a novel six1/sox2 axis in regulating the stemness of HCC cells.

[Status of antibiotic use in hospitalized children with community-acquired pneumonia in multiple regions of China].

OBJECTIVE: To investigate the use of antibiotics in children with community-acquired pneumonia (CAP) in multiple regions of China, and to provide a reference for CAP standard treatment and rational antibiotic use in children. METHODS: The medical data of 1 383 children with CAP who were hospitalized in the department of pediatrics in 10 grade A tertiary hospitals from 9 cities between April 14, 2014 and January 1, 2016 were reviewed, to analyze the status of antibiotic use in hospitalized children in North China, Northeast China, East China, and South China. RESULTS: The overall rate of antibiotic use in children with CAP was 89.08%, with 88.7% in North China, 95.5% in Northeast China, 83.3% in East China, and 86.6% in South China. The main types of antibiotics used were cephalosporins, macrolides, compound preparations of ß-lactam antibiotics, polyphosphoric broad-spectrum antibiotics and other ß-lactam antibiotics. The selection of antibiotics was generally rational, but antibiotics were still used in some patients with viral infection alone or a combined use of ≥2 kinds of antibiotics were noted in some patients with infection caused by one kind of pathogen. Irrational antibiotic use was observed in 131 children (10.63%). CONCLUSIONS: There are high rates of antibiotic use and irrational use of antibiotics among children with CAP. Standard management of antibiotic use in children with CAP should be strengthened.

Neuroprotective effects of genistein on SH-SY5Y cells overexpressing A53T mutant α-synuclein.

Genistein, a potent antioxidant compound, protects dopaminergic neurons in a mouse model of Parkinson's disease. However, the mechanism underlying this action remains unknown. This study investigated human SH-SY5Y cells overexpressing the A53T mutant of α-synuclein. Four groups of cells were assayed: a control group (without any treatment), a genistein group (incubated with 20 µM genistein), a rotenone group (treated with 50 µM rotenone), and a rotenone + genistein group (incubated with 20 µM genistein and then treated with 50 µM rotenone). A lactate dehydrogenase release test confirmed the protective effect of genistein, and genistein remarkably reversed mitochondrial oxidative injury caused by rotenone. Western blot assays showed that BCL-2 and Beclin 1 levels were markedly higher in the genistein group than in the rotenone group. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling revealed that genistein inhibited rotenone-induced apoptosis in SH-SY5Y cells. Compared with the control group, the expression of NFE2L2 and HMOX1 was significantly increased in the genistein + rotenone group. However, after treatment with estrogen receptor and NFE2L2 channel blockers (ICI-182780 and ML385, respectively), genistein could not elevate NFE2L2 and HMOX1 expression. ICI-182780 effectively prevented genistein-mediated phosphorylation of NFE2L2 and remarkably suppressed phosphorylation of AKT, a protein downstream of the estrogen receptor. These findings confirm that genistein has neuroprotective effects in a cell model of Parkinson's disease. Genistein can reduce oxidative stress damage and cell apoptosis by activating estrogen receptors and NFE2L2 channels.

Association of LPP and TAGAP Polymorphisms with Celiac Disease Risk: A Meta-Analysis.

Background: Lipoma preferred partner (LPP) and T-cell activation Rho GTPase activating protein (TAGAP) polymorphisms might influence the susceptibility to celiac disease. Therefore, we performed a meta-analysis by identifying relevant studies to estimate the risks of these polymorphisms on celiac disease. Methods: The PubMed, Web of Science and Embase databases were searched (up to October 2016) for LPP rs1464510 and TAGAP rs1738074 polymorphisms. Results: This meta-analysis included the same 7 studies for LPP rs1464510 and TAGAP rs1738074. The minor risk A allele at both rs1464510 and rs1738074 carried risks (odds ratios) of 1.26 (95% CI: 1.22-1.30) and 1.17 (95% CI: 1.14-1.21), respectively, which contributed to increased risks in all celiac disease patients by 10.72% and 6.59%, respectively. The estimated lambdas were 0.512 and 0.496, respectively, suggesting that a co-dominant model would be suitable for both gene effects. Conclusions: This meta-analysis provides robust estimates that polymorphisms in LPP and TAGAP genes are potential risk factors for celiac disease in European and American. Prospective studies and more genome-wide association studies (GWAS) are needed to confirm these findings, and some corresponding molecular biology experiments should be carried out to clarify the pathogenic mechanisms of celiac disease.

EphA2 targeting peptide tethered bioreducible poly(cystamine bisacrylamide-diamino hexane) for the delivery of therapeutic pCMV-RAE-1γ to pancreatic islets.

The pathogenesis of type-1 diabetes is complicated, and a clear, single mechanism has yet to be identified. Reports have indicated that the activating receptor NKG2D plays an important role in the development of disease. Exploiting a natural phenomenon observed in tumors, plasmid DNA encoding for a soluble ligand to NKG2D (sRAE-1γ) was isolated and engineered into a plasmid expression system. A polymeric gene delivery system was developed to deliver the soluble RAE-1 plasmid locally to the pancreatic islets for the prevention of type-1 diabetes. The bioreducible cationic polymer poly(cystamine bisacrylamide-diamino hexane) (p(CBA-DAH)) was modified with poly(ethylene glycol) (PEG) and the targeting peptide CHVLWSTRC, known to target the EphA2 and EphA4 receptors. The PEG serves to improve stability and tissue selectivity, while the peptide will target EphA2 and A4, overexpressed in the pancreatic microvasculature. The targeting polymer Eph-PEG-p(CBA-DAH) shows selective uptake by the target cell line, indicative of the targeting properties that will be seen in systemic administration. Using the delivery system, the therapeutic plasmid can be delivered to the pancreas, reduce interactions between the beta-cells and infiltrating NKG2D positive lymphocytes, and effectively protect beta-cells from autoimmune destruction and prevent type 1 diabetes.

[Effect of xuezhlkang capsule in intervening different Chinese medical syndrome patterns of non-alcoholic fatty liver disease complicated with carotid atherosclerosis].

OBJECTIVE: To observe the intervening effects of Xuezhikang Capsule (XZK) on levels of blood lipid and other related indices in patients with different Chinese medical syndrome patterns of non-alcoholic fatty liver disease complicated carotid atherosclerosis (NAFLD-CAS), and to seek out the most appropriate pattern to indicate XZK for making guidance of its utilization. METHODS: Chinese medical syndrome in 74 patients of NAFLD-CAS were classified into 4 patterns, 34 of Pi-deficiency phlegm-dampness pattern (A), 24 of dampness-heat accumulation pattern (B), 12 of phlegm-stasis intertwined pattern (C), and 4 of Gan-Shen yin-deficiency pattern (D). Excepting those of pattern D were excluded due to too small samples, all patients were treated with XZK for 3 months. Blood levels of blood lipids, including triglyceride (TG), total cholesterol (TC), and low-density lipoprotein cholesterol (LDL-C), as well as high-sensitivity C-reactive protein (hs-CRP), and tumor necrosis factor alpha (TNF-alpha) were detected and compared before and after treatment. RESULTS: The effective rate of XZK on patients of the three patterns, in A-C order, was 97.06%, 91.67%, 91.67%, respectively, with the optimal overall efficacy showed on pattern A. All the indices detected significantly decreased after treatment in all three patterns (P < 0.01), among them, excepting the difference of TG level between groups showed no significance (P > 0.05), the decrements of others were more significant in pattern A than in other two patterns (P < 0.05 or P < 0.01). CONCLUSION: XZK could reduce the levels of blood lipids, hs-CRP and TNF-alpha in NAFLD-CAS patients, and the Pi-deficiency phlegm-dampness syndrome pattern was the optimal indication of XZK treatment.

Bioreducible polymer-transfected skeletal myoblasts for VEGF delivery to acutely ischemic myocardium.

Implantation of skeletal myoblasts to the heart has been investigated as a means to regenerate and protect the myocardium from damage after myocardial infarction. While several animal studies utilizing skeletal myoblasts have reported positive findings, results from clinical studies have been mixed. In this study we utilize a newly developed bioreducible polymer system to transfect skeletal myoblasts with a plasmid encoding vascular endothelial growth factor (VEGF) prior to implantation into acutely ischemic myocardium. VEGF has been demonstrated to promote revascularization of the myocardium following myocardial infarction. We report that implanting VEGF expressing skeletal myoblasts into acutely ischemic myocardium produces superior results compared to implantation of untransfected skeletal myoblasts. Skeletal myoblasts expressing secreted VEGF were able to restore cardiac function to non-diseased levels as measured by ejection fraction, to limit remodeling of the heart chamber as measured by end systolic and diastolic volumes, and to prevent myocardial wall thinning. Additionally, arteriole and capillary formation, retention of viable cardiomyocytes, and prevention of apoptosis was significantly improved by VEGF expressing skeletal myoblasts compared to untransfected myoblasts. This work demonstrates the feasibility of using bioreducible cationic polymers to create engineered skeletal myoblasts to treat acutely ischemic myocardium.

Reductive degradation behavior of bioreducible poly(disulfide amine) for enhancing SiRNA efficiency.

Bioreducible cationic polymer poly(CBA-DAH) containing repeated disulfide linkages on the polymer backbone was synthesized through Michael-type polyadditions of CBA to DAH monomers. Poly(CBA-DAH) could spontaneously form nanoscale polyelectrolyte complexes through electrostatic interactions with siRNA in an aqueous phase. These nanoparticles were rapidly degraded under the reductive cytoplasmic environment with subsequently releasing the siRNA cargo into the cytoplasm where RNAi takes place, as a result of the breakdown of disulfide bonds in the polymers. The reductive degradation behavior of the poly(CBA-DAH)/siRNA polyplexes is more likely to increase RNAi activity with enhancing the cytoplasmic localization of siRNA molecules. Poly(CBA-DAH) may have great potential as a gene carrier especially for therapeutic applications of siRNAs owing to the reductive degradation characteristics.

Polymer transfected primary myoblasts mediated efficient gene expression and angiogenic proliferation.

This study was designed to assess the in vitro gene expression efficiency and therapeutic effectiveness of polymer mediated transfection of primary myoblasts. Autologous primary myoblast transplantation may improve the function of infarcted myocardium via myogenesis. In addition, primary myoblasts can carry exogenous angiogenic genes that encode angiogenic factors to promote therapeutic angiogenesis. Viral vectors have limited clinical application due to the induction of inflammatory reactions, tumorigenic mutations and genome integration. To overcome these problems, two new biodegradable poly(disulfide amine)s, poly(cystaminebisacryamide-diaminohexane) [poly(CBA-DAH)] and poly(cystaminebisacryamide-diaminohexane-arginine) [poly(CBA-DAH-R)], were synthesized as polymer carriers for gene delivery. In this study, primary myoblasts were isolated and purified from rat skeletal muscles. Based on an optimized polymer mediated transfection procedure using a luciferase assay and confocal microscopy, these two poly(disulfide amine)s induced up to 16-fold higher luciferase expression and much higher green fluorescence protein expression than branched poy(ethylenimine) (bPEI, 25kDa) in primary myoblasts. By flow cytometry, poly(CBA-DAH) and poly(CBA-DAH-R) promote rates of cellular uptake of florescence-labeled polymer/pDNA complexes of 97% and 99%, respectively, which are rates higher than that of bPEI 25kDa (87%). Both poly(disulfide amine)s were much less cytotoxic than bPEI 25kDa. The in vitro time-course and co-culture experiments verified that polymer engineered primary myoblasts have the ability to stimulate endothelial proliferation. These data confirmed that poly(disulfide amine)s are the safe and feasible polymeric gene carriers to transfect VEGF(165) into primary myoblasts. Polymer engineered primary myoblasts have potential for therapeutic application in the treatment of ischemic heart diseases.

A family of bioreducible poly(disulfide amine)s for gene delivery.

A family of bioreducible poly(disulfide amine)s, which differ in the length of polymethylene spacer [-(CH(2))(n)-] in the main chain and the side chain, has been synthesized. These bioreducible poly(disulfide amine)s exhibit local environment specific degradability and are associated with lower cytotoxicity than branched poly(ethylenimine) (bPEI, 25 kDa). These cationic polymers also show higher buffering capacity and protonation degree than bPEI, facilitating the endosomal escape of carried genetic materials. The transfection efficiency of these agents is oligomethylene length dependent. Poly(cystaminebisacrylamide-spermine) [poly(CBA-SP)], poly(cystaminebisacrylamide-bis(3-aminopropyl)-1,3-propanediamine) [poly(CBA-APPD)], and poly(cyxtaminebisacrylamide-bis(3-aminopropyl)-ethylenediamine) [ploy(CBA-APED)] with longer propylene [-(CH(2))(3)-] side spacer, demonstrate higher transfection efficacy than the counterpart poly(cystaminebisacrylamide-bis(2-aminoethyl)-1,3-propanediamine) [poly(CBA-AEPD)] and poly(cystaminebisacrylamide-triethylenetetramine) [poly(CBA-TETA)], which have shorter ethylene [-(CH(2))(2)-] side spacer. The poly(CBA-SP), poly(CBA-APPD), poly(CBA-APED) with the main chain spacer of -(CH(2))(4)-, -(CH(2))(3)-, -(CH(2))(2)- demonstrate similar transfection efficiency, indicating the length of polymer main chain spacer has less influence on transfection efficiency. However, with the same short ethylene [-(CH(2))(2)-] side spacer, poly(CBA-AEPD), with the longer main chain oligomethylene units [-(CH(2))(3)-], showed relatively higher transfection efficiency than poly(CBA-TETA), having shorter main chain oligomethylene units [-(CH(2))(2)-]. Of these polymeric carriers, poly(CBA-SP) demonstrated the highest transfection in the C2C12 cell line, while poly(CBA-APED) showed the highest transfection in the HeLa cell line. All of these agents showed greater transfection activity than commercialized bPEI 25 kDa. The poly(disulfide amine)s are promising safe and efficient non-viral vectors for gene delivery.

Arginine-grafted bioreducible poly(disulfide amine) for gene delivery systems.

Arginine-grafted bioreducible poly(disulfide amine) (ABP) polymer was synthesized for non-viral gene delivery systems. Its Mw was measured to be 4.45x10(3) Da/mole by FPLC-SEC and its PDI value was 1.49. ABP was able to retard pDNA from a weight ratio of 2 but ABP could not retard pDNA even at a weight ratio of 10 in the presence of DTT, showing that it can be biodegraded in reducing environment such as cytoplasm. ABP was examined to form positively charged nano-sized particles (<200 nm) with pDNA. ABP showed no significant cytotoxicity and greatly enhanced transfection efficiency in comparison with unmodified poly(cystaminebisacrylamide-diaminohexane) (poly(CBA-DAH)) and PEI25k in mammalian cells. The transfection efficiency of ABP was not much reduced even in the serum condition. Chloroquine treatment was not found to improve the transfection efficiency of ABP. The cellular uptake pattern of ABP polyplexes was almost similar with poly(CBA-DAH), suggesting that greatly enhanced transfection efficiency of ABP is not induced by its high cellular penetrating ability but may be mediated by other factors such as good nuclear localization ability.

Cardiomyocyte-targeted siRNA delivery by prostaglandin E(2)-Fas siRNA polyplexes formulated with reducible poly(amido amine) for preventing cardiomyocyte apoptosis.

A cardiomyocyte-targeted Fas siRNA delivery system was developed using prostaglandin E(2) (PGE(2))-modified siRNA polyplexes formed by a reducible poly(amido amine) to inhibit cardiomyocyte apoptosis. PGE(2), which was used as a specific ligand for cardiomyocyte targeting, was conjugated to the terminal-end of the sense siRNA (PGE(2)-siRNA). The reducible cationic copolymer, synthesized via Michael-type polyaddition of 1,6-diaminohexane and cystamine bis-acrylamide (poly(DAH/CBA)), tightly condensed the PGE(2)-siRNA conjugate to form nanosize polyplexes having a diameter of 100-150 nm. The PGE(2)-siRNA/poly(DAH/CBA) polyplexes decomplexed to release PGE(2)-siRNA in a cytosolic reducing environment due to the degradation of the reducible poly(DAH/CBA). The cellular uptake of the PGE(2)-siRNA/poly(DAH/CBA) polyplex was increased in rat cardiomyocytes (H9C2 cells) due to PGE(2) receptor-mediated endocytosis. When H9C2 cells were transfected with siRNA against Fas, a key regulator of ischemia-induced apoptosis, the PGE(2)-Fas siRNA/poly(DAH/CBA) polyplex delivery system led to a significant increase in Fas gene silencing, resulting in inhibition of cardiomyocyte apoptosis. The PGE(2)-Fas siRNA/poly(DAH/CBA) polyplex did not induce interferon-alpha in peripheral blood mononuclear cells. These results suggest that the PGE(2)-Fas siRNA/poly(DAH/CBA) polyplex formulation may be clinically applicable as a cardiomyocyte-targeted Fas siRNA delivery system to inhibit apoptosis in cardiovascular disease.

Novel biodegradable poly(disulfide amine)s for gene delivery with high efficiency and low cytotoxicity.

Novel biodegradable poly(disulfide amine)s with defined structure, high transfection efficiency, and low cytotoxicity were designed and synthesized as nonviral gene delivery carriers. Michael addition between N, N'-cystaminebisacrylamide (CBA) and three N-Boc protected diamines ( N-Boc-1,2-diaminoethane, N-Boc-1,4-diaminobutane, and N-Boc-1,6-diaminohexane) followed by N-Boc deprotection under acidic condition resulted in final cationic polymers with disulfide bonds, tertiary amine groups in main chains, and pendant primary amine groups in side chains. Polymer structures were confirmed by 1H NMR, and their molecular weights were in the range 3.3-4.7 kDa with narrow polydispersity (1.12-1.17) as determined by size exclusion chromatography (SEC). Acid-base titration assay showed that the poly(disulfide amine)s possessed superior buffering capacity to branched PEI 25 kDa in the pH range 7.4-5.1, which may facilitate the escape of DNA from the endosomal compartment. Gel retardation assay demonstrated that significant polyplex dissociation was observed in the presence of 5.0 mM DTT within 1 h, suggesting rapid DNA release in the reduction condition such as cytoplasm due to the cleavage of disulfide bonds. Genetic transfections mediated by these poly(disulfide amine)s were side-chain spacer length dependent. The poly(disulfide amine) with a hexaethylene spacer, poly(CBA-DAH), had comparable transfection efficiency to bPEI 25 kDa in the tested cell lines, i.e., 293T cells, Hela cells, and NIH3T3 cells. This same poly(disulfide amine) mediated 7-fold higher luciferase expression than bPEI 25 kDa in C2C12 cells (mouse myoblast cell line), a cell line difficult to transfect with many cationic polymers. Furthermore, MTT assay indicated that all three poly(disulfide amine)s/pDNA polyplexes were significantly less toxic than bPEI/pDNA complexes.