Yajun Duan

Polyamidoamine dendrimers with a modified Pentaerythritol core having high efficiency and low cytotoxicity as gene carriers.

Polyamidoamine (PAMAM) dendrimers represent one of the most efficient polymeric gene carriers. This study describes a new family of PAMAM dendrimers that can be synthesized using a Pentaerythritol derivative (PD) as a core that possesses 12 branches. This new approach in the synthesis of divergent dendrimers provided a rapid increase in the number of branches, which made it easier to obtain dendrimers with high generation and large enough molecular size. The PD dendrimers of generations 3-5 synthesized in this study could efficiently condense DNA into nanoscale complexes with slightly positive charges. Their transfection efficiency was evaluated in different cell lines. These PD dendrimers were found to show higher transfection efficiency, but much lower cytotoxicity, than the commercial nonviral gene carriers polyethyleneimine (PEI), polylysine (PLL), and PAMAM dendrimers with an ethylenediamine core (generations 5 and 7). The results indicate that, with high transfection efficiency and low cytotoxicity, the PD dendrimers hold promise as novel nonviral gene carriers.

Novel peptide–dendrimer conjugates as drug carriers for targeting nonsmall cell lung cancer

Phage display technology has been demonstrated to be a powerful tool for screening useful ligands that are capable of specifically binding to biomarkers on the surface of tumor cells. The ligands found by this technique, such as peptides, have been successfully applied in the fields of early cancer diagnostics and chemotherapy. In this study, a novel nonsmall cell lung cancer-targeting peptide (LCTP, sequence RCPLSHSLICY) was screened in vivo using a Ph.D.-C7C™ phage display library. In order to develop a universal tumor-targeting drug carrier, the LCTP and fluorescence-labeled molecule (FITC) were conjugated to an acetylated polyamidoamine (PAMAM) dendrimer of generation 4 (G4) to form a PAMAM–Ac–FITC–LCTP conjugate. The performance of the conjugate was first tested in vitro. In vitro results of cell experiments analyzed by flow cytometry and inverted fluorescence microscopy indicated that PAMAM–Ac–FITC–LCTP was enriched more in NCI-H460 cells than in 293T cells, and cellular uptake was both time- and dose-dependent. The tissue distribution of the conjugate in athymic mice with lung cancer xenografts was also investigated to test the targeting efficiency of PAMAM–Ac–FITC–LCTP in vivo. The results showed that LCTP can effectively facilitate the targeting of PAMAM–Ac–FITC–LCTP to nonsmall cell lung cancer cells and tumors. These results suggest that the LCTP-conjugated PAMAM dendrimer might be a promising drug carrier for targeted cancer diagnosis and treatment.

Low Oxygen Tension and Synthetic Nanogratings Improve the Uniformity and Stemness of Human Mesenchymal Stem Cell Layer

A free-standing, robust cell sheet comprising aligned human mesenchymal stem cells (hMSCs) offers many interesting opportunities for tissue reconstruction. As a first step toward this goal, a confluent, uniform hMSC layer with a high degree of alignment and stemness maintenance needs to be created. Hypothesizing that topographical cue and a physiologically relevant low-oxygen condition could promote the formation of such an hMSC layer, we studied the culture of hMSCs on synthetic nanogratings (350 nm width and 700 nm pitch) and either under 2 or 20% O(2). Culturing hMSCs on the nanogratings highly aligned the cells, but it tended to create patchy layers and accentuate the hMSC differentiation. The 2% O(2) improved the alignment and uniformity of hMSCs, and reduced their differentiation. Over a 14-day culture period, hMSCs in 2% O(2) showed uniform connexon distribution, secreted abundant extracellular matrix (ECM) proteins, and displayed a high progenicity. After 21-day culture on nanogratings, hMSCs exposed to 2% O(2) maintained a higher viability and differentiation capacity. This study established that a 2% O(2) culture condition could restrict the differentiation of hMSCs cultured on nanopatterns, thereby setting the foundation to fabricate a uniformly aligned hMSC sheet for different regenerative medicine applications.

Peroxisome Proliferator-activated Receptor γ Activation by Ligands and Dephosphorylation Induces Proprotein Convertase Subtilisin Kexin Type 9 and Low Density Lipoprotein Receptor Expression

Background: PCSK9 regulates cholesterol homeostasis by enhancing the LDLR protein degradation. The effects of PPARγ on PCSK9 and LDLR expression remain unknown. Results: PPARγ activation by ligands or dephosphorylation induces PCSK9 and LDLR expression and cholesterol metabolism. Conclusion: PPARγ is an important transcriptional factor in regulating PCSK9 and LDLR expression. Significance: We define a new signaling pathway that regulates PCSK9 and LDLR expression. Proprotein convertase subtilisin kexin type 9 (PCSK9) plays an important role in cholesterol homeostasis by enhancing the degradation of LDL receptor (LDLR) protein. Peroxisome proliferator-activated receptor γ (PPARγ) has been shown to be atheroprotective. PPARγ can be activated by ligands and/or dephosphorylation with ERK1/2 inhibitors. The effect of PPARγ on PCSK9 and LDLR expression remains unknown. In this study, we investigated the effects of PPARγ on PCSK9 and LDLR expression. At the cellular levels, PPARγ ligands induced PCSK9 mRNA and protein expression in HepG2 cells. PCSK9 expression was induced by inhibition of ERK1/2 activity but inhibited by ERK1/2 activation. The mutagenic study and promoter activity assay suggested that the induction of PCSK9 expression by ERK1/2 inhibitors was tightly linked to PPARγ dephosphorylation. However, PPARγ activation by ligands or ERK1/2 inhibitors induced hepatic LDLR expression. The promoter assay indicated that the induction of LDLR expression by PPARγ was sterol regulatory element-dependent because PPARγ enhanced sterol regulatory element-binding protein 2 (SREBP2) processing. In vivo, administration of pioglitazone or U0126 alone increased PCSK9 expression in mouse liver but had little effect on PCSK9 secretion. However, the co-treatment of pioglitazone and U0126 enhanced both PCSK9 expression and secretion. Similar to in vitro, the increased PCSK9 expression by pioglitazone and/or U0126 did not result in decreased LDLR expression and function. In contrast, pioglitazone and/or U0126 increased LDLR protein expression and membrane translocation, SREBP2 processing, and CYP7A1 expression in the liver, which led to decreased total and LDL cholesterol levels in serum. Our results indicate that although PPARγ activation increased PCSK9 expression, PPARγ activation induced LDLR and CYP7A1 expression that enhanced LDL cholesterol metabolism.

Co-electrospun fibrous scaffold-adsorbed DNA for substrate-mediated gene delivery.

Incorporation of gene into electrospun nanofibers for localized gene transfection of target cells represents a robust platform for tissue regeneration. In this study, a new two-step approach was explored to immobilize DNA onto electrospun nanofibers for effective gene delivery, that is, nonviral gene vector of polyethylene glycol (PEG)-modified polyethylenimine (PEI) was incorporated into scaffolds by electrospinning and then target DNA was adsorbed onto the electrospun nanofibers via electrostatic interaction between DNA and PEI-PEG. PEI-PEG/DNA particles formed from the released DNA, and PEI-PEG had a uniform particle size of approximately 200 nm. This nanofiber-based gene delivery system exhibited high transfection efficiency, in which >65% of human embryonic kidney 293 cells and >40% of mesenchymal stem cells were transfected with green fluorescent protein gene. Compared with PEI, PEG modification of PEI had improved the biocompatibility and further increased the transfection efficiency. These results suggest that the combination of nonviral gene carrier with electrospun nanofibers could be used for localized gene delivery, which has multifold potential applications in tissue engineering or as an in vivo substrate for tissue regeneration.

Inhibition of ERK1/2 and Activation of LXR Synergistically Reduce Atherosclerotic Lesions in ApoE-Deficient Mice

Objective— Activation of liver X receptor (LXR) inhibits atherosclerosis but induces hypertriglyceridemia. In vitro, it has been shown that mitogen-activated protein kinase kinase 1/2 (MEK1/2) inhibitor synergizes LXR ligand–induced macrophage ABCA1 expression and cholesterol efflux. In this study, we determined whether MEK1/2 (U0126) and LXR ligand (T0901317) can have a synergistic effect on the reduction of atherosclerosis while eliminating LXR ligand–induced fatty livers and hypertriglyceridemia. We also set out to identify the cellular mechanisms of the actions. Approach and Results— Wild-type mice were used to determine the effect of U0126 on a high-fat diet or high-fat diet plus T0901317-induced transient dyslipidemia and liver injury. ApoE deficient (apoE−/−) mice or mice with advanced lesions were used to determine the effect of the combination of T0901317 and U0126 on atherosclerosis and hypertriglyceridemia. We found that U0126 protected animals against T0901317-induced transient or long-term hepatic lipid accumulation, liver injury, and hypertriglyceridemia. Meanwhile, the combination of T0901317 and U0126 inhibited the development of atherosclerosis in a synergistic manner and reduced advanced lesions. Mechanistically, in addition to synergistic induction of macrophage ABCA1 expression, the combination of U0126 and T0901317 maintained arterial wall integrity, inhibited macrophage accumulation in aortas and formation of macrophages/foam cells, and activated reverse cholesterol transport. The inhibition of T0901317-induced lipid accumulation by the combined U0126 might be attributed to inactivation of lipogenesis and activation of lipolysis/fatty acid oxidation pathways. Conclusions— Our study suggests that the combination of mitogen-activated protein kinase kinase 1/2 inhibitor and LXR ligand can function as a novel therapy to synergistically reduce atherosclerosis while eliminating LXR-induced deleterious effects.

Inhibition of Glutathione Production Induces Macrophage CD36 Expression and Enhances Cellular-oxidized Low Density Lipoprotein (oxLDL) Uptake

Background: The GSH-dependent antioxidant system reduces atherosclerosis. Results: Inhibition of GSH production by BSO enhanced CD36 translational efficiency to induce CD36 protein expression and lipid accumulation that was blocked by antioxidant (enzyme). Conclusion: Alterations of cellular GSH and GSH/GSSG status regulate macrophage CD36 expression and cellular oxLDL uptake. Significance: Our study demonstrates an important anti-atherogenic function of the GSH-dependent antioxidant system. The glutathione (GSH)-dependent antioxidant system has been demonstrated to inhibit atherosclerosis. Macrophage CD36 uptakes oxidized low density lipoprotein (oxLDL) thereby facilitating foam cell formation and development of atherosclerosis. It remains unknown if GSH can influence macrophage CD36 expression and cellular oxLDL uptake directly. Herein we report that treatment of macrophages with l-buthionine-S,R-sulfoximine (BSO) decreased cellular GSH production and ratios of GSH to glutathione disulfide (GSH/GSSG) while increasing production of reactive oxygen species. Associated with decreased GSH levels, macrophage CD36 expression was increased, which resulted in enhanced cellular oxLDL uptake. In contrast, N-acetyl cysteine and antioxidant enzyme (catalase or superoxide dismutase) blocked BSO-induced CD36 expression as well as oxLDL uptake. In vivo, administration of mice with BSO increased CD36 expression in peritoneal macrophages and kidneys. BSO had no effect on CD36 mRNA expression and promoter activity but still induced CD36 protein expression in macrophages lacking peroxisome proliferator-activated receptor γ expression, suggesting it induced CD36 expression at the translational level. Indeed, we determined that BSO enhanced CD36 translational efficiency. Taken together, our study demonstrates that cellular GSH levels and GSH/GSSG status can regulate macrophage CD36 expression and cellular oxLDL uptake and demonstrate an important anti-atherogenic function of the GSH-dependent antioxidant system by providing a novel molecular mechanism.

A tumor targeted gene vector modified with G250 monoclonal antibody for gene therapy.

G250 is a tumor associated antigen that is found on > 90% of renal cell carcinoma (RCC). In order to develop a highly targeting gene vector for RCC gene therapy, G250 monoclonal antibody was prepared, purified and characterized. The antibody was chemically bound to Polyethylenimine (PEI) to form the IgG-PEI conjugate. The conjugate is capable of forming DNA complexes in the size of nano meters and with a narrow size distribution. The targeting effect and transfection efficiency were tested on five cell lines, ketr 3, Hela, ACHN, HepG2, and smooth muscle cells. The transfection was quantitatively determined by fluorescence activated cell sorting (FACS) and luciferase assay. The FACS results show that for G250 positive cells ketr 3 and Hela, the transfection efficiency of IgG-PEI are 2-fold higher than that of PEI. But for G250 negative cells, antibody modification has no effect on transfection. The expression of luciferase in ketr 3 cells which is expressed as enzyme activity is 15-fold and 61-fold higher than that in ACHN and SMC, respectively. In the presence of free antibody, the targeting effect of IgG-PEI is impaired and the transfection efficiency is normalized. It indicates that G250 antibody is an ideal targeting ligand for delivery of genes into RCC. Application of this IgG-PEI conjugate in RCC gene therapy will be of great interest.

Activation of liver X receptor induces macrophage interleukin-5 expression

Background: LXR inhibits the development of atherosclerosis. It remains unknown whether LXR regulates IL-5 expression, an atheroprotective cytokine, in macrophages. Results: LXR induces macrophage IL-5 expression in an LXRE-dependent manner. It also induces IL-5 expression in aortic root area of LDLR−/− mice. Conclusion: Macrophage IL-5 is a target gene for LXR activation. Significance: The increased IL-5 expression can be related to LXR-induced anti-atherosclerosis. IL-5 stimulates production of T15/EO6 IgM antibodies that can block the uptake of oxidized low density lipoprotein by macrophages, whereas a deficiency in macrophage IL-5 expression accelerates development of atherosclerosis. Liver X receptors (LXRs) are ligand-activated transcription factors that can induce macrophage ABCA1 expression and cholesterol efflux, thereby inhibiting the development of atherosclerosis. However, it remains unknown whether additional mechanisms, such as the regulation of macrophage IL-5 expression, are related to the anti-atherogenic properties of LXR. We initially defined IL-5 expression in macrophages where the LXR ligand (T0901317) induced macrophage IL-5 protein expression and secretion. The overexpression of LXR increased, whereas its knockdown inhibited IL-5 expression. Furthermore, we found that LXR activation increased IL-5 transcripts, promoter activity, formation of an LXR·LXR-responsive element complex, and IL-5 protein stability. In vivo, we found that T0901317 increased IL-5 and total IgM levels in plasma and IL-5 expression in multiple tissues in wild type mice. In LDL receptor knock-out (LDLR−/−) mice, T0901317 increased IL-5 expression in the aortic root area. Taken together, our studies demonstrate that macrophage IL-5 is a target gene for LXR activation, and the induction of macrophage IL-5 expression can be related to LXR-inhibited atherosclerosis.

Poly(ethylene glycol)-Grafted Polyethylenimine Modified with G250 Monoclonal Antibody for Tumor Gene Therapy

To improve the biocompatibility of a gene vector and to avoid its being eliminated by the immune system, polyethylenimine (PEI) was modified with poly(ethylene glycol) (PEG) before G250 monoclonal antibody (mAb) conjugation. G250-PEI-PEG was capable of forming complexes with DNA in the right size distribution, and the G250 mAb modification significantly improved PEI transfection of G250-positive cells. The highest transfection efficiency was seen in HeLa cells as determined by flow cytometry after transfection with the gene encoding green fluorescent protein: 2-fold higher compared with the transfection of HepG2 cells. Blocking the surface antigen on the cell membrane of HeLa cells by incubation with free G250 mAb, or by downregulating G250 expression by small interfering RNA transfection, resulted in a remarkable decrease in transfection efficiency. These data indicate the targeting effect of G250 antibody modification. The presence of serum decreased transfection efficiency in a concentration-dependent manner. However, the transfection of HeLa cells with G250-PEI-PEG remained significant in the presence of 30% serum. In an in vivo study, G250-PEI-PEG exhibited high transfection efficiency in tumors. In addition, pathological analysis did not show obvious toxicity caused by the materials used. These suggest that PEG- and G250 mAb-modified PEI could be a useful nonviral gene vector for in vivo study.