Redox stimulus disulfide conjugated polyethyleneimine as a shuttle for gene transfer.

Redox-responsive cationic polymers have gained considerable attention in gene delivery due to low cytotoxicity and spatio-temporal release of DNA into the cells. Here, we reported the synthesis of reducible disulfide conjugated polyethyleneimine (1.8 kDa) (denoted as SS-PEI) and its application to transfer pEGFP-ZNF580 plasmid (pZNF580) into EA.hy926 cell. This reducible SS-PEI polymer was prepared by one-step polycondensation reaction of low molecular weight PEI with bis-(p-nitrophenyl)-3,3'-dithiodipropionate. The SS-PEI successfully condensed pZNF580 into nano-sized complexes (170 ± 1.5 nm to 255 ± 1.6 nm) with zeta potentials of 3 ± 0.4 mV to 17 ± 0.9 mV. The complexes could be triggered to release pZNF580 when exposed to the reducing environment of 5 mM dithiothreitol. Besides, the SS-PEI exhibited low cytotoxicity. In vitro transfection results showed that SS-PEI exhibited good transfection efficiency comparable to PEI25kDa. Thus, the SS-PEI could act as an reducible gene carrier with good transfection efficiency and low cytotoxicity.

Multifunctional Gene Carriers Labeled by Perylene Diimide Derivative as Fluorescent Probe for Tracking Gene Delivery.

Multifunctional carriers with both gene transfection property and fluorescent tracking function have attracted significant attention in recent years. Herein, a kind of perylene diimide derivative (PDI-C10C8) is conjugated onto the polyethylenimine-g-poly(lactide-co-glycolide)-g-polyethylenimine (PLGA-PEI) polymer to obtain fluorescent multifunctional polymer and micelles (abbreviated as MP). Then, the REDV-G-TAT-G-NLS (TP-G) peptide sequence is grafted onto this MP to obtain multifunctional micelles labeled by perylene diimide derivative (MP-TP-G). These micelles exhibit enhanced photobleaching stability compared with the reference Cy5-labeled micelles, and the fluorescent images of cellular uptake show bright red emission without any background noise. Confocal laser scanning microscope (CLSM) experiments show that gene complexes can deliver gene into nucleus. MP-TP-G carriers do not enter into the cell nucleus, which proves that the nuclear localization signal sequence may not exert its nucleus accumulation ability via conjugating to the amphiphilic polymers. The high transfection efficiency and the enhanced photobleaching stability, combined with the ability to monitor the detailed process of cellular uptake and gene delivery, make these multifunctional micelles have great potential application for endothelialization of artificial blood vessels and gene delivery process study.

Novel Zinc Finger Transcription Factor ZFP580 Facilitates All-Trans Retinoic Acid -Induced Vascular Smooth Muscle Cells Differentiation by Rarα-Mediated PI3K/Akt and ERK Signaling.

BACKGROUND/AIMS: Phenotypic switching of vascular smooth muscle cells (VSMC) plays a vital role in the development of vascular diseases. All-trans retinoic acid (ATRA) is known to regulate VSMC phenotypes. However, the underlying mechanisms remain completely unknown. Here, we have investigated the probable roles and underlying mechanisms of the novel C2H2 zinc finger transcription factor ZFP580 on ATRA-induced VSMC differentiation. METHODS: VSMCs were isolated, cultured, and identified. VSMCs were infected with an adenovirus encoding ZFP580 or Ad-siRNA to silence ZFP580. The expression levels of ZFP580, SMα-actin, SM22α, SMemb, RARα, RARß, and RARγ were assayed by Q-PCR and western blot. A rat carotid artery injury model and morphometric analysis of intimal thickening were also used in this study. RESULTS: ATRA caused a significant reduction of VSMC proliferation and migration in a doseand time-dependent manner. Moreover, it promoted VSMC differentiation by enhancing expression of differentiation markers and reducing expression of dedifferentiation markers. This ATRA activity was accompanied by up-regulation of ZFP580, with concomitant increases in RARα expression. In contrast, silencing of the RARα gene or inhibiting RARα with its antagonist Ro41-5253 abrogated the ATRA-induced ZFP580 expression. Furthermore, ATRA binding to RARα induced ZFP580 expression via the PI3K/Akt and ERK pathways. Adenovirusmediated overexpression of ZFP580 promoted VSMC differentiation by enhancing expression of SM22α and SMα-actin and reducing expression of SMemb. In contrast, silencing ZFP580 dramatically reduced the expression of differentiation markers and increased expression of dedifferentiation markers. The classic rat carotid artery balloon injury model demonstrated that ZFP580 inhibited proliferation and intimal hyperplasia in vivo. CONCLUSION: The novel zinc finger transcription factor ZFP580 facilitates ATRA-induced VSMC differentiation by the RARα-mediated PI3K/Akt and ERK signaling pathways. This might represent a novel mechanism of regulation of ZFP580 by ATRA and RARα, which is critical for understanding the biological functions of retinoids during VSMC phenotypic modulation.

Potential Signaling Pathways Involved in the Clinical Application of Oxymatrine.

Oxymatrine, an alkaloid component extracted from the roots of Sophora species, has been shown to have antiinflammatory, antifibrosis, and antitumor effects and the ability to protect against myocardial damage, etc. The potential signaling pathways involved in the clinical application of oxymatrine might include the TGF-ß/Smad, toll-like receptor 4/nuclear factor kappa-light-chain-enhancer of activated B cells, toll-like receptor9/TRAF6, Janus kinase/signal transduction and activator of transcription, phosphatidylinositol-3 kinase/Akt, delta-opioid receptor-arrestinl-Bcl-2, CD40, epidermal growth factor receptor, nuclear factor erythroid-2-related factor 2/heme oxygenase-1 signaling pathways, and dimethylarginine dimethylaminohydrolase/asymmetric dimethylarginine metabolism pathway. In this review, we summarize the recent investigations of the signaling pathways related to oxymatrine to provide clues and references for further studies on its clinical application. Copyright © 2016 John Wiley & Sons, Ltd.

Liver Stiffness Measurement is Useful in Predicting Type 2 Diabetes Mellitus Among Nonalcohol Fatty Liver Disease Patients.

Background: Type 2 diabetes mellitus (T2DM) and non-alcoholic fatty liver disease (NAFLD) are closely related conditions. Aim: This study investigated a group of individuals with NAFLD to evaluate if liver fibrosis, identified by FibroScan, correlated with T2DM. Methods: 154 NAFLD patients obtained FibroScan, liver ultrasonography (US), and a thorough assessment of clinical implications and chemical biomarkers. Results: In comparison to the NAFLD without T2DM group, the hemoglobin A1c(HBA1c)(mmol/mol%), homeostasis model of assessment for insulin resistance index (HOMA-IR), gamma-glutamyl transferase (GGT), fibrosis indices, and liver stiffness measurement (LSM) values were all considerably higher in the NAFLD with T2DM group. Patients with NAFLD and T2DM had considerably lower serum uric acid(SUA) levels than those with NAFLD alone.Those with severe fibrosis (79.3%, 23/29) in the NAFLD group showed a greater frequency of T2DM than those with mild fibrosis (45.6%, 21/46) or no fibrosis (27.85%, 22/79) (P=0.000). LSM value and elements of the metabolic syndrome (MetS) were independent risk factors for incident T2DM among NAFLD patients (OR=1.466, 95% CI [1.139-1.888], P=0.003; and OR=0.273, 95% CI [0.081-0.916], P=0.036). Conclusion: FibroScan can identify significant fibrosis, which is independently linked to a higher prevalence of T2DM. As a result, it is crucial to make use of this technology to predict T2DM in NAFLD patients.

Octreotide for primary moderate to severe acute pancreatitis: a meta-analysis.

BACKGROUND/AIMS: To systematically evaluate the effect and safety of octreotide on primary moderate to severe acute pancreatitis. METHODOLOGY: The Cochrane Central Register of Controlled Trials (CENTRAL) in The Cochrane Library, PubMed, EMBASE, Science Citation Index Expanded (SCI-E), and Chinese Biomedicine Database (CBM) were searched in September 2011. Major outcomes contained mortality, incidence rate of complications, rate of surgical intervention, and length of hospital stay. RESULTS: Eleven randomized clinical trials with 720 participants were included and evaluated, only two of which had a high study quality and were combined in meta-analysis. The pool estimate of RR of mortality was 0.88 (95% CI: 0.53, 1.45) and that of incidence rate of complication was 1.08 (95% CI: 0.94, 1.26), both of which had no significant difference. The other two outcomes could not be combined for lack of enough data. CONCLUSIONS: Present evidence does not approve octreotide's benefit in the major outcomes of moderate to severe acute pancreatitis and further randomized controlled trials with high quality and large sample size are required.

FIB-4 is closer to FibroScan screen results to detecting advanced liver fibrosis and maybe facilitates NAFLD warning.

To assess the relationship between clinical biochemical characteristics and steatosis or fibrosis by Fibroscan in non-alcoholic fatty liver disease (NAFLD) patients in order to seek the simple effective screening method closed to the results of the fibroScan measurement. A cross-sectional study was conducted on 188 patients with NAFLD who underwent FibroScan examinations. Demographic data and clinical biochemical characteristics were collected and analyzed. The result showed elevated serum uric acid (SUA) (P = .023, odds ratio [OR = 1.005, 95% CI (1.001-1.009) and metabolic syndrome (MetS) (P = .000, OR = 4.549, 95%CI (1.974-10.484) were associated with severe steatosis (controlled attenuation parameter, CAP ≥ 300 dB/m). The magnitude of liver stiffness measured using FibroScan was positively correlated with aspartate transaminase/alanine aminotransferase (AST/ALT) ratio (R = 0.419, P = .000), AST to platelet ratio index (APRI) score (R = 0.309, P = .000), and Fibrosis-4 score (FIB-4) (R = 0.507, P = .000). The areas under the receiver operating curve (ROC) of AST/ALT, APRI, and FIB-4 for mild or severe fibrosis were 0.563, 0.696, and 0.728, respectively, and those for advanced fibrosis were 0.648, 0.750, and 0.821, respectively. The FIB-4 index cutoff value was 1.65 with a sensitivity of 68.3% and specificity of 89.8% during the diagnosis of advanced fibrosis. MetS and elevated SUA are associated with severe steatosis according to the CAP value screen, whereas FIB-4, as the fibrosis score method, is closer to the liver stiffness measurement results from FibroScan, which may facilitate early warning of NAFLD in the community or in remote areas.

Clinical Classification of Obesity and Implications for Metabolic Dysfunction-Associated Fatty Liver Disease and Treatment.

Obesity,and metabolic dysfunction-associated fatty liver disease (MAFLD) have reached epidemic proportions globally. Obesity and MAFLD frequently coexist and act synergistically to increase the risk of adverse clinical outcomes (both hepatic and extrahepatic). Type 2 diabetes mellitus (T2DM) is the most important risk factor for rapid progression of steatohepatitis and advanced fibrosis. Conversely, the later stages of MAFLD are associated with an increased risk of T2DM incident. According to the proposed criteria, MAFLD is diagnosed in patients with liver steatosis and in at least one in three: overweight or obese, T2DM, or signs of metabolic dysregulation if they are of normal weight. However, the clinical classification and correlation between obesity and MAFLD is more complex than expected. In addition, treatment for obesity and MAFLD are associated with a reduced risk of T2DM, suggesting that liver-based treatments could reduce the risk of developing T2DM. This review describes the clinical classification of obesity and MAFLD, discusses the clinical features of various types of obesity and MAFLD, emphasizes the role of visceral obesity and insulin resistance (IR) in the development of MAFLD,and summarizes the existing treatments for obesity and MAFLD that reduce the risk of developing T2DM.

"Intercellular Mass Transport" Mimic Enables ASO Entry Completely into the Cell Nucleus for Enhanced Ischemia Therapy.

Currently, the development of a new therapeutic technology is focused on antisense oligonucleotides (ASOs), where ASOs are used to complementarily pair with DNA, messenger RNA, or long noncoding RNA (lncRNA) to regulate the cell behavior by inhibiting the target gene expression. However, the targeted regulation toward nuclear genes still faces great challenges in ASO delivery for clinical applications, i.e., two essential criteria (high nuclear entry and delivery vehicle safety/simplification) generally compromise each other and are not simultaneously satisfactory. Herein, for the first time, inspired by "intercellular-mass-transport", we report an important discovery that the cell membrane of endothelial cells (ECs) serving as the biointerface enables ASOs to rapidly and completely enter the EC nucleus. Thereby, we innovatively fabricate a nanosystem only by sequential self-assembly of natural/off-the-shelf biomaterials to well overcome the above-mentioned contradiction. The efficacy is strikingly superior to that of the previous delivery vehicles. Furthermore, our technology is applied to successfully silence lncRNA MEG3 in the EC nucleus, significantly augmenting EC morphogenesis. More importantly, this nanosystem is applicable for in vivo intramuscular injection to enhance the therapeutic outcome in a critical limb ischemia mouse model. This work brings a new hope for the technological innovation of ASO nuclear delivery and opens a new avenue to explore natural/off-the-shelf materials for cargo delivery into subcellular compartments.

Exploring the links between gut microbiome changes and irritable bowel syndrome in Han populations in the Tibetan Plateau. / è¥¿è—é«˜åŽŸæ±‰æ—äººç¾¤çš„è‚ é“å¾®ç”Ÿç‰©å˜åŒ–ä¸Žè‚ æ˜“æ¿€ç»¼åˆå¾ä¹‹é—´çš„å ³ç³»ç ”ç©¶.

The gut microbiome shows changes under a plateau environment, while the disbalance of intestinal microbiota plays an important role in the pathogenesis of irritable bowel syndrome (IBS); however, the relationship between the two remains unexplored. In this work, we followed up a healthy cohort for up to a year before and after living in a plateau environment and performed 16S ribosomal RNA (rRNA) sequencing analysis of their fecal samples. Through evaluating the participants' clinical symptoms, combined with an IBS questionnaire, we screened the IBS sub-population in our cohort. The sequencing results showed that a high-altitude environment could lead to changes in the diversity and composition of gut flora. In addition, we found that the longer the time volunteers spent in the plateau environment, the more similar their gut microbiota composition and abundance became compared to those before entering the plateau, and IBS symptoms were significantly alleviated. Therefore, we speculated that the plateau may be a special environment that induces IBS. The taxonomic units g_Alistipes, g_Oscillospira, and s_Ruminococcus_torques, which had been proved to play important roles in IBS pathogenesis, were also abundant in the IBS cohort at high altitudes. Overall, the disbalance of gut microbiota induced by the plateau environment contributed to the high frequency of IBS and the psychosocial abnormalities associated with IBS. Our results prompt further research to elucidate the relevant mechanism.

Superlow Dosage of Intrinsically Bioactive Zinc Metal-Organic Frameworks to Modulate Endothelial Cell Morphogenesis and Significantly Rescue Ischemic Disease.

Despite long-term efforts for ischemia therapy, proangiogenic drugs hardly satisfy therapy/safety/cost/mass production multiple evaluations and meanwhile with a desire to minimize dosages, thereby clinical applications have been severely hampered. Recently, metal ion-based therapy has emerged as an effective strategy. Herein, intrinsically bioactive Zn metal-organic frameworks (MOFs) were explored by bridging the dual superiorities of proangiogenic Zn2+ and facile/cost-effective/scalable MOFs. Zn-MOFs could enhance the morphogenesis of vascular endothelial cells (ECs) via the PI3K/Akt/eNOS pathway. However, high dosage is inevitable and Zn-MOFs suffer from insolubility and low stability, which lead to the bioaccumulation of Zn-MOFs and seriously potential toxicity risks. To alleviate this, it is required to decrease the dosage, but this can be entrapped into the dosage/therapy/safety contradiction and disappointing therapy effect. To address these challenges, the bioavailability of Zn-MOFs is urgent to improve for the minimization of dosage and significant therapy/safety. The mitochondrial respiratory chain is Zn2+ active, which inspired us to codecorate EC-targeted and mitochondria-localizing-sequence peptides onto Zn-MOF surfaces. Interestingly, after codecoration, a 100-fold reduced dosage acquired equally powerful vascularization, and the superlow dosage significantly rescued ischemia (4.4 µg kg-1, about one order of magnitude lower than the published minimal value). Additionally, no obvious muscle injury was found after treatment. Potential toxicity risks were alleviated, benefiting from the superlow dosage. This advanced drug simultaneously satisfied comprehensive evaluations and dosage minimization. This work utilizes engineering thought to rationally design "all-around" bioactive MOFs and is expected to be applied for ischemia treatment.

RNA-seq Analysis Reveals Potential Molecular Mechanisms of ZNF580/ZFP580 Promoting Neuronal Survival and Inhibiting Apoptosis after Hypoxic-ischemic Brain damage.

Neonatal hypoxic-ischemic brain damage (HIBD) is one of the main causes of neonatal acute death and chronic nervous system impairment, but still lacks effective treatments. ZNF580/ZFP580, reported in our previous studies, may be a newly identified member of the Krüppel-like factor (KLF) family, and has anti-apoptotic effects during ischemic myocardial injury. In the present study, we showed that the expression levels of both ZFP580/ZNF580 mRNA and protein increased significantly in neonatal HIBD rats and oxygen-glucose deprivation (OGD) SH-SY5Y cell models. ZNF580 overexpression promoted neuron survival and suppressed neuron apoptosis after OGD in neuron-like SH-SY5Y cells, while interference with ZNF580 resulted in the opposite results. RNA-seq analysis identified 248 differentially-expressed genes (DEGs) between ZNF580 overexpression SH-SY5Y cells and interference-expressed SH-SY5Y cells. Gene Ontology functional enrichment analysis showed that these DEGs played significant roles in the growth, development, and regeneration of axons, DNA biosynthetic processes, DNA replication, and apoptosis. Kyoto Encyclopedia of Genes and Genomes enrichment analysis indicated that these DEGs were found in some pathways, including ferroptosis, glutamatergic synapses, protein processing in the endoplasmic reticulum, estrogen signaling pathways, the TGF-beta signaling pathway, and the longevity regulating pathway. The qRT-PCR validation results were consistent with RNA-seq results, which showed that HSPA5, IGFBP3, NTN4, and KLF9 increased in ZNF580-overexpressed SH-SY5Y cells and decreased in interference-expressed SH-SY5Y cells, when compared with normal cells. Together, the results suggested that ZNF580 targeted these genes to inhibit neuronal apoptosis.

A two-pronged approach to regulate the behaviors of ECs and SMCs by the dual targeting-nanoparticles.

Inhibiting vascular restenosis remains a tricky challenge for the postoperative development of cardiovascular interventional therapy. The ideal approaches should activate endothelial cells (ECs) and restrain smooth muscle cells (SMCs), however, they are commonly contradictory. Herein, a strategy was developed for synchronizing ECs promotion and SMCs inhibition by codelivery DNA and siRNA for combination therapy. Thus, an easy and efficient strategy integrated dual-superiorities of precise targeting and dual therapeutic genes to precisely regulate the behaviors of ECs and SMCs. The ECs-targeting REDV peptide and SMCs-targeting VAPG peptide grafted anionic polymers were used to surface-functionalize the delivery nanoplatforms for vascular endothelial growth factor (VEGF) plasmids and ERK2 siRNA delivery, respectively. The dual targeting-nanoparticles were prepared by physical mixing method, and their outstanding advantages were confirmed by the co-culture experiments. In comparison with single targeting-nanoparticles and dual non-targeting-nanoparticles, the dual targeting-nanoparticles simultaneously enhanced ECs proliferation/migration and restrained SMCs proliferation/migration. Moreover, the dual targeting-nanoparticles group manifested the highest VEGF expression in ECs and the lowest ERK2 expression in SMCs. In summary, the two-pronged strategy with dual targeting-nanoparticles provides a valuable cornerstone for synchronizing ECs promotion and SMCs inhibition.

Value of anti-p53 antibody as a biomarker for hepatocellular carcinoma: Evidence from a meta-analysis.

INTRODUCTION: The incidence of hepatocellular carcinoma (HCC) ranks sixth in the world, but its mortality is the third highest due to the lack of early diagnostic markers. Nowadays, the increase of autoantibody levels has been found in many cancers, and many studies have begun to pay attention to the detection of anti-p53 antibodies in HCC. The purpose of this study is to quantitatively and comprehensively analyze the potential diagnostic value of anti-p53 autoantibodies in HCC METHODS:: English articles up to November 2019 were collected. The overall sensitivity and specificity were calculated. Besides, the positive likelihood ratio, negative likelihood ratio (NLR), diagnostic odds ratio (DOR), and summary receiver operating characteristic curves of the overall diagnostic accuracy of anti-p53 antibody were calculated by STATA software. Finally, according to the heterogeneity of the results, the subgroup analysis, and the publication bias were performed. RESULTS: A total of 16 eligible studies were incorporated into this meta-analysis, including 1323 patients with HCC and 1896 control. The pooled sensitivity was 0.28(0.17-0.41) and specificity was 0.98 (0.95-0.99). The pooled DOR was 10.44 (6.31-17.29) and the pooled NLR was 0.74 (0.63-0.86). The area under ROC curve of symmetrical ROC was 0.840. CONCLUSIONS: The anti-p53 antibody has a high specificity for HCC, but the low sensitivity is not perfect and would limit the clinical application. The anti-p53 antibody would help rule out HCC but not help rule in HCC for early diagnosis. Whether combined as a diagnostic panel with other biomarkers or laboratory tests may prove useful requires further study.

Unexpected Amplification of Synergistic Gene Expression to Boom Vascular Flow in Advantageous Dual-Gene Co-expression Plasmid Delivery Systems over Physically Mixed Strategy.

Gene therapy exerts powerful potential in the treatment of various diseases, such as overexpressing pro-angiogenic gene to accelerate angiogenesis and restore vascular flow of ischemic tissue. Tremendous efforts have been invested in developing gene carriers for high transfection efficiency, while little research has been devoted to synergistically expressing functional proteins via optimizing therapeutic genes. Actually, the amplified gene expression is the ultimate goal of gene delivery. Dual-gene co-delivery and coordinate expression become a "breach" of strengthened gene expression. Herein, we explored the synergistic effects on gene expression and pro-angiogenesis by two typical dual-gene delivery strategies to determine which one is more efficient. The physical mixing method used ZNF580 and VEGF165 plasmids with a 1/1 weight ratio (p1:1), and the other strategy involved chemically inserting ZNF580 and VEGF165 genes into one plasmid as a dual-gene co-expression plasmid (pZNF-VEGF). p1:1 and pZNF-VEGF were loaded by REDV-TAT-NLS-H12 carrier, a promising peptide carrier, to form corresponding dual-gene delivery systems. Both systems exhibited approximately similar size and zeta potential, guaranteeing almost the same cellular uptake. We comprehensively evaluated two delivery systems through gene expression at mRNA and protein levels and angiogenesis-related activities in vitro and in vivo. Interestingly, the pZNF-VEGF group showed a remarkably amplified synergistic effect in the expression of ZNF580 and VEGF165 genes in comparison with the p1:1 group. More importantly, the unexpected amplified synergistic effect of dual-gene co-expression plasmid was further verified for proliferation, migration, and angiogenesis in vitro and in vivo. Accordingly, we believed that the co-delivery of dual genes via constructing co-expression plasmids offers a better option for gene therapy, which can more effectively enhance the synergistic expression of target genes than the physical mixing method.

Cascaded bio-responsive delivery of eNOS gene and ZNF580 gene to collaboratively treat hindlimb ischemia via pro-angiogenesis and anti-inflammation.

Gene therapy is a promising strategy for treating ischemic disease by solving the dual dilemma of ischemia and inflammation. However, its development remains limited by inefficient gene transfection. Hence, we propose a "dual genes + all-adaptive carrier" idea. We have innovatively co-delivered eNOS gene and the ZNF580 gene encoding its transcription factor to enhance the efficiency of eNOS expression. The overexpressed ZNF580 protein significantly promotes angiogenesis via regulating the transcription of multiple genes. This implies a potential synergistic effect of eNOS and ZNF580 genes in anti-ischemic therapy. Additionally, we have designed an all-adaptive gene carrier with cascaded bio-responsive functions based on the characteristic bio-signals of the ischemic site (including extracellular excessive matrix metalloproteinase-2, the endo/lysosomal pH gradient and high cytoplasmic glutathione level). This carrier can sequentially overcome transfection bottlenecks and achieve high transfection. Excitingly, this cascaded bio-responsive delivery strategy remarkably enhanced blood perfusion, accelerated angiogenesis and alleviated inflammation in critical limb ischemia (CLI) mice, which was attributed to the combined effects of pro-angiogenic ZNF580 expression and synergistically produced eNOS expression. Thereby, we believe that the co-delivery of eNOS and ZNF580 genes assisted by a cascaded bio-responsive carrier is a powerful strategy to treat CLI.

Agmatine-grafted bioreducible poly(l-lysine) for gene delivery with low cytotoxicity and high efficiency.

Bioreducible cationic polymers have gained considerable attention in gene delivery due to their low cytotoxicity and high efficiency. In the present work, we reported a cationic polymer, poly(disulfide-l-lysine)-g-agmatine (denoted as SSL-AG), and evaluated its ability to transfer pEGFP-ZNF580 plasmid (pZNF580) into human umbilical vein endothelial cells (HUVECs). This SSL-AG polymeric carrier efficiently condensed pZNF580 into positively charged particles (<200 nm) through electrostatic interaction. This carrier also exhibited excellent buffering capacity in the physiological environment, good pDNA protection against enzymatic degradation and rapid pDNA release in a highly reducing environment mainly because of the responsive cleavage of disulfide bonds in the polymer backbone. The hemolysis assay and in vitro cytotoxicity assay suggested that the SSL-AG carrier and corresponding gene complexes possessed both good hemocompatibility and great cell viability in HUVECs. The cellular uptake of the SSL-AG/Cy5-oligonucleotide group was 3.6 times that of the poly(l-lysine)/Cy5-oligonucleotide group, and its mean fluorescence intensity value was even higher than that of the PEI 25 kDa/Cy5-oligonucleotide group. Further, the intracellular trafficking results demonstrated that the SSL-AG/Cy5-oligonucleotide complexes exhibited a high nucleus co-localization rate (CLR) value (36.0 ± 2.8%, 3.4 times that of the poly (l-lysine)/Cy5-oligonucleotide group, 1.6 times that of the poly(disulfide-l-lysine)-g-butylenediamine/Cy5-oligonucleotide group) at 24 h, while the endo/lysosomal CLR value was relatively low. This suggested that SSL-AG successfully delivered plasmid into HUVECs with high cellular uptake, rapid endosomal escape and efficient nuclear accumulation owing to the structural advantages of the bioreducible and agmatine groups. In vitro transfection assay also verified the enhanced transfection efficiency in the SSL-AG/pZNF580 group. Furthermore, the results of CCK-8, cell migration and in vitro/vivo angiogenesis assays revealed that pZNF580 delivered by SSL-AG could effectively enhance the proliferation, migration and vascularization of HUVECs. In a word, the SSL-AG polymer has great potential as a safe and efficient gene carrier for gene therapy.

From single to a dual-gene delivery nanosystem: coordinated expression matters for boosting the neovascularization in vivo.

In the past decade, the development of gene carriers has been key in enhancing gene therapy. Gene therapy is associated with not only the delivery process but also gene expression as a prominent role. Herein, for the purpose of achieving a novel breakthrough in gene therapy, we creatively proposed a "strengthened gene expression" idea beyond the range of improving the gene carrier. We constructed three types of gene delivery systems, namely, single-pZNF580 delivery system, single-pVEGF165 delivery system, and dual-gene delivery system. These systems possessed approximate same sizes (∼120 nm) and zeta potentials (∼+20 mV), which indicated negligible differences in their cellular uptake. Interestingly, we found that the gene expression of dual-gene groups significantly increased at the level of both mRNA and protein at least 2 times and 1.5 times as high as single-gene groups, respectively. This "1 + 1 > 2" expression effect benefited from the coordinated expression of the angiogenesis-related genes of ZNF580 and VEGF165. Furthermore, the coordinated effect was also confirmed in HUVEC activities such as an obviously enhanced proliferation and migration of the dual-gene group. Rationally, we further evaluated the effects of coordinated interactions on neovascularization. We observed that the statistic tube number of dual-gene groups was approximately 1.44 times as high as that of single-gene groups. More importantly, this enhanced angiogenesis induced by the coordinated expression was also demonstrated in an in vivo environment. Therefore, we believed that the enhanced gene therapy via the gene expression pathway could provide a creative viewpoint for the design of gene delivery system and therapy.

Multifunctional REDV-G-TAT-G-NLS-Cys peptide sequence conjugated gene carriers to enhance gene transfection efficiency in endothelial cells.

Rapid endothelialization on small diameter artificial blood vessels is an effective strategy to facilitate long-term patency and inhibit thrombosis. The gene delivery can enhance the proliferation and migration of endothelial cells (ECs), which is beneficial for rapid endothelialization. REDV-G-TAT-G-NLS-Cys (abbreviated as TP-G) peptide could weakly condense pEGFP-ZNF580 (pZNF580) and transfect ECs, but its transfection efficiency was still very low because of its low positive charge, low stability and weak endosome escape ability. In order to develop more stable and efficient gene carriers with low cytotoxicity, in the present study, we conjugated different amounts of TP-G peptide onto poly(lactide-co-glycolide)-g-polyethylenimine (PLGA-g-PEI) amphiphilic copolymers via a hetero-poly(ethylene glycol) spacer (OPSS-PEG-NHS). The TP-G peptide and PEI could cooperatively and strongly condense pZNF580. The carrier's cytotoxicity was reduced by the introduction of poly(ethylene glycol) spacer. They condensed pZNF580 to form gene complexes (PPP-TP-G/pZNF580) with suitable size and positive zeta potential for gene delivery. The transfected ECs promoted their migration ability as demonstrated by cell migration assay. The results of cellular uptake and confocal laser scanning microscopy showed significantly high internalization efficiency, endosomal/lysosomal escape and nucleus location of pZNF580 by this multifunctional TP-G peptide sequence conjugated gene delivery system. Furthermore, several inhibitors were used to study the cellular uptake pathways of PPP-TP-G/pZNF580 complexes. The results showed that PPP-TP-G2/Cy5-oligonucleotide complexes exhibited the optimized endocytosis pathways which facilitated for cellular uptake. In conclusion, the multifunctional TP-G peptide conjugated gene carriers could promote the transfection efficiency due to the multifunction of REDV, cell-penetrating peptide and nuclear localization signal in the peptide sequence, which could be a suitable gene carrier for endothelialization.

Co-immobilization of ACH11 antithrombotic peptide and CAG cell-adhesive peptide onto vascular grafts for improved hemocompatibility and endothelialization.

Surface modification by conjugating biomolecules has been widely proved to enhance biocompatibility of small-caliber artificial vascular grafts. In this study, we aimed at developing a multifunctional vascular graft that provides not only good hemocompatibility but also in situ rapid endothelialization. Herein, a vascular graft (inner diameter ∼2 mm) was fabricated by electrospinning with poly(lactic acid-co-caprolactone) and gelatin, and then biofunctionalized with antithrombotic peptide with sequence LTFPRIVFVLG (ACH11) and cell adhesion peptide with sequence CAG through adhesive poly(dopamine) coating. We developed this graft with the synergistic properties of low thrombogenicity and rapid endothelialization. The successful grafting of both CAG and ACH11 peptides was confirmed by Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy. The surface micromorphology of the modified surfaces was observed by field emission scanning electron microscopy. Our results demonstrated that the multifunctional surface suppressed the denaturation of absorbed fibrinogen, hindered coagulation factor Xa activation, and inhibited platelet adhesion and aggregation. Importantly, this modified surface could selectively enhance endothelial cells adhesion, proliferation and release of nitric oxide. Upon in vivo implantation of 6 weeks, the multifunctional vascular graft showed improved patency and superior vascular endothelialization. Overall, the results effectively demonstrated that the co-immobilization of ACH11 and CAG provided a promising method for the improvement of hemocompatibility and endothelialization of vascular grafts. STATEMENT OF SIGNIFICANCE: Electrospun small-caliber vascular grafts are increasingly used to treat cardiovascular diseases. Despite their success related to their good biodegradation and mechanical strength, they have some drawbacks, such as low hemocompatibility and endothelialization. The single-function ligands are insufficient to modify surface with both good hemocompatibility and rapid endothelialization simultaneously. Therefore, we functionalized electrospun vascular graft by novel antithrombotic peptide and cell-adhesive peptide to construct superior anticoagulation and ECs-selective adhesion surface in present study. The multifunctional vascular grafts benefit for high long-term patency and rapid endothelialization.