Xuli Wang

Targeted systemic delivery of a therapeutic siRNA with a multifunctional carrier controls tumor proliferation in mice

In this study, novel peptide-targeted delivery systems were developed for systemic and targeted delivery of therapeutic siRNA based on a multifunctional carrier, (1-aminoethyl)iminobis[N-(oleicylcysteinylhistinyl-1-aminoethyl)propionamide] (EHCO), which showed pH-sensitive amphiphilic cell membrane disruption. EHCO formed stable nanoparticles with siRNA. Targeted siRNA delivery systems were readily formed by surface modification of the nanoparticles. PEGylation of the siRNA/EHCO nanoparticles significantly reduced nonspecific cell uptake. The incorporation of a bombesin peptide or RGD peptide via a PEG spacer resulted in receptor-mediated cellular uptake and high gene silencing efficiency in U87 cells. Fluorescence confocal microscopic studies demonstrated that EHCO/siRNA nanoparticles and PEG modified EHCO/siRNA nanoparticles were able to facilitate endosomal escape of the siRNA delivery systems. Systemic administration of a therapeutic anti-HIF-1alpha siRNA with the peptide-targeted delivery systems resulted in significant tumor growth inhibition than a nontargeted delivery system or free siRNA via intravenous injection in nude mice bearing human glioma U87 xenografts. The results indicate a great promise of the multifunctional carrier EHCO for systemic and targeted delivery of therapeutic siRNA to treat human diseases with RNAi.

A peptide-targeted delivery system with pH-sensitive amphiphilic cell membrane disruption for efficient receptor-mediated siRNA delivery.

The efficient delivery of therapeutic siRNA into cells of interest is a critical challenge to broad application of RNAi. In this study, we developed a peptide-targeted delivery system for highly efficient receptor-mediated cellular siRNA delivery. The targeted delivery system was readily prepared by in situ functionalization of a polymerizable pH-sensitive amphiphilic surfactant, N-(1-aminoethyl)iminobis[N-(oleicyl-cysteinyl-histinyl-1-aminoethyl)propionamide] (EHCO) and self-assembly with siRNA. The intrinsic pH-sensitive amphiphilicity of EHCO at pH 5-6 was able to induce cell membrane disruption at endosomal pH and facilitate endosomal escape of the siRNA nanoparticles after internalization. The siRNA/EHCO nanoparticles and PEGylated siRNA/EHCO nanoparticles were not cytotoxic as compared to PEI/siRNA or TransFast/siRNA nanoparticles. siRNA/EHCO nanoparticles resulted in higher siRNA delivery efficiency than PEI and TransFast. The PEGylation of the siRNA/EHCO nanoparticles significantly reduced non-specific cell uptake. The incorporation of a bombesin peptide via a PEG spacer resulted in specific cellular uptake and high gene silencing efficiency in CHO-d1EGFP cells with overexpression of bombesin receptors. Receptor-mediated endocytosis and pH-sensitive amphiphilic endosomal escape are the advantageous features of the targeted siRNA delivery system for highly efficient cell-specific siRNA delivery. This novel targeted delivery system holds a great promise for systemic and targeted delivery of therapeutic siRNA.

In vivo evaluation of a PAMAM-cystamine-(Gd-DO3A) conjugate as a biodegradable macromolecular MRI contrast agent

Macromolecular Gd(III) chelates are superior magnetic resonance imaging (MRI) contrast agents for blood pool and tumor imaging. However, their clinical development is limited by the safety concerns related to the slow excretion and long-term gadolinium tissue accumulation. A generation 6 PAMAM Gd(III) chelate conjugate with a cleavable disulfide spacer, PAMAM-G6-cystamine-(Gd-DO3A), was prepared as a biodegradable macromolecular MRI contrast agent with rapid excretion from the body. T1 and T2 relaxivities of the contrast agent were 11.6 and 13.3 mM −1sec−1 at 3T, respectively. Blood pool and tumor contrast enhancement of the agent were evaluated in female nude mice bearing MDA-MB-231 human breast carcinoma xenografts with a nondegradable conjugate PAMAM-G6-(Gd-DO3A) as a control. PAMAM-G6-cystamine-(Gd-DO3A) resulted in significant contrast enhancement in the blood for about 5 mins, and Gd-DO3A was released from the conjugate and rapidly excreted via renal filtration after the disulfide spacer was cleaved. The nondegradable control had much longer blood circulation and excreted more slowly from the body. PAMAM-G6-cystamine-(Gd-DO3A) also resulted in more prominent tumor contrast enhancement than the control. However, PAMAM-G6-cystamine-(Gd-DO3A) demonstrated high toxicity due to the intrinsic toxicity of PAMAM dendrimers. In conclusion, although PAMAM-G6-cystamine-(Gd-DO3A) showed some advantages compared with the nondegradable control, PAMAM dendrimers are not suitable carriers for biodegradable macromolecular MRI contrast agents, due to their high toxicity.

Graphene-based anticancer nanosystem and its biosafety evaluation using a zebrafish model.

In this paper, a facile strategy to develop graphene-based delivery nanosystems for effective drug loading and sustained drug release was proposed and validated. Specifically, biocompatible naphthalene-terminated PEG (NP) and anticancer drugs (curcumin or doxorubicin (DOX)) were simultaneously integrated onto oxidized graphene (GO), leading to self-assembled, nanosized complexes. It was found that the oxidation degree of GO had a significant impact on the drug-loading efficiency and the structural stability of nanosystems. Interestingly, the nanoassemblies resulted in more effective cellular entry of DOX in comparison with free DOX or DOX-loaded PEG-polyester micelles at equivalent DOX dose, as demonstrated by confocal microscopy studies. Moreover, the nanoassemblies not only exhibited a sustained drug release pattern without an initial burst release, but also significantly improved the stability of formulations which were resistant to drug leaking even in the presence of strong surfactants such as aromatic sodium benzenesulfonate (SBen) and aliphatic sodium dodecylsulfonate (SDS). In addition, the nanoassemblies without DOX loading showed negligible in vitro cytotoxicity, whereas DOX-loaded counterparts led to considerable toxicity against HeLa cells. The DOX-mediated cytotoxicity of the graphene-based formulation was around 20 folds lower than that of free DOX, most likely due to the slow DOX release from complexes. A zebrafish model was established to assess the in vivo safety profile of curcumin-loaded nanosystems. The results showed they were able to excrete from the zebrafish body rapidly and had nearly no influence on the zebrafish upgrowth. Those encouraging results may prompt the advance of graphene-based nanotherapeutics for biomedical applications.

Synthesis of poly[(5-benzyloxy-trimethylene carbonate)-co-(5,5-dimethyl-trimethylene carbonate)] catalyzed by immobilized lipase on silica particles with different size

Abstract Porcine pancreas lipase (PPL) immobilized on silica particles with different size were prepared and employed successfully for ring-opening co-polymerization of 5-benzyloxy-trimethylene carbonate (BTMC) with 5,5-dimethyl-trimethylene carbonate (DTC) for the first time. Three kinds of silica particles with different sizes (150–250, 75–150 and 1 μm) were selected as carriers for enzyme immobilization. The structure of copolymers were confirmed by 1 H and 13 C NMR which showed no decarboxylation occurrence during the polymerization. The ( M n ) of poly(BTMC- co -DTC) decreased rapidly with the increasing of immobilized PPL concentration. The carrier size of immobilized PPL affected both the catalytic activity and the polymer yield. The highest molecular weight ( M n =26,400) of poly(BTMC- co -DTC) was obtained at around 0.1% concentration of immobilized PPL on silica particles with size of 75–150 μm.

Self-Assembled Vehicle Construction via Boronic Acid Coupling and Host–Guest Interaction for Serum-Tolerant DNA Transport and pH-Responsive Drug Delivery

By exploiting boronic acid coupling and host-guest chemistry, a pH-responsive drug/gene co-delivery nanoplatform is designed for cancer treatments with the excellently serum-tolerant transfection activity and the capability to load and release hydrophobic drugs in an acidity-accelerated manner. Via boronate linkage, γ-CD is allowed to spontaneously attach onto phenylboronic-acid-modified oligoethylenimine (PEI1.8K-PB2.9 ) at neutral condition. The formed vehicle/DNA nanoformulation is thus surrounded densely by γ-CD moieties to biomimic the carbohydrate-rich cell surface, providing a novel approach to overcome serum-susceptible drawbacks frequently associated with synthetic gene carriers. PEI1.8K-PB2.9 -γ-CD conjugates demonstrate significantly improved cell-biocompatibility and transfection activity over PEI1.8K-PB2.9 . Noticeably, serum-associated inhibition effect is negligible for PEI1.8K-PB2.9 -γ-CD-mediated transfection whereas marked transfection reduction occurs for PEI25K and PEI1.8K-PB2.9 upon serum exposure. Consequently, PEI1.8K-PB2.9 -γ-CDs afford much higher transfection efficiency, that is, 25-fold higher luciferase expression over PEI25K in presence of 30% serum. An anticancer drug of doxorubicin (DOX) is shown to be readily accommodated into the nanoformulation via host-guest chemistry and intracellularly co-delivered together with plasmid DNA. Due to the acidity-labile feature of boronate linkage, DOX/γ-CD inclusion complexes would be mostly detached from the nanoformulation triggered by acidity, leading to faster drug release. Furthermore, drug inclusion does not alter the serum-compatible transfection efficiency of PEI1.8K-PB2.9 -γ-CD.

A boronate-linked linear-hyperbranched polymeric nanovehicle for pH-dependent tumor-targeted drug delivery.

Advanced drug delivery systems, which possess post-functionalization feasibility to achieve targetability and traceability, favorable pharmacokinetics with dynamic but controllable stability, and preferable tumor accumulation with prolonged drug residence in disease sites, represent ideal nanomedicine paradigm for tumor therapy. To address this challenge, here we reported a dynamic module-assembly strategy based on reversible boronic acid/1,3-diol bioorthogonality. As a prototype, metastable hybrid nanoself-assembly between hydrophobic hyperbranched diol-enriched polycarbonate (HP-OH) and hydrophilic linear PEG terminated with phenylboronic acid (mPEG-PBA) is demonstrated in vitro and in vivo. The nanoconstruction maintained excellent stability with little leakage of loaded drugs under the simulated physiological conditions. Such a stable nanostructure enabled the effective in vivo tumor accumulation in tumor site as revealed by NIR imaging technique. More importantly, this nanoconstruction presented a pH-labile destruction profile in response to acidic microenvironment and simultaneously the fast liberation of loaded drugs. Accordingly at the cellular level, the intracellular structural dissociation was also proved in terms of the strong acidity in late endosome/lysosome, thus favoring the prolonged retention of remaining drug-loaded HP-OH aggregates within tumor cells. Hence, our delicate design open up a dynamical module-assembly path to develop site and time dual-controlled nanotherapeutics for tumor chemotherapy, allowing enhanced tumor selectivity through prolonged retention of delivery system in tumor cells followed by a timely drug release pattern.

Structural effect on degradability and in vivo contrast enhancement of polydisulfide Gd(III) complexes as biodegradable macromolecular MRI contrast agents

The structural effect of biodegradable macromolecular magnetic resonance imaging (MRI) contrast agents, polydisulfide gadolinium (Gd)(III) chelates, on their in vitro degradability, and cardiovascular and tumor imaging were evaluated in mice. Polydisulfide Gd(III) chelates, Gd-DTPA cystamine copolymers (GDCC), Gd-DTPA l-cystine copolymers (GDCP), Gd-DTPA d-cystine copolymers (dGDCP) and Gd-DTPA glutathione (oxidized) copolymers (GDGP), with different sizes and narrow molecular weight distribution were prepared and evaluated both in vitro and in vivo in mice bearing MDA-MB-231 tumor xenografts. GDGP with large steric hindrance around the disulfide bonds had greater T(1) and T(2) relaxivities than GDCC, GDCP and dGDCP. The degradability of the polydisulfide by the endogenous thiols decreased with increasing steric effects around the disulfide bonds in the order of GDCC>GDCP, dGDCP>GDGP. The size and degradability of the contrast agents had a significant impact on vascular contrast enhancement kinetics. The agents with a large size and low degradability resulted in more prolonged vascular enhancement than the agents with a small size and high degradability. It seems that the size and degradability of the agents did not significantly affect tumor enhancement. All agents resulted in significant contrast enhancement in tumor tissue. This study has demonstrated that the vascular enhancement kinetics of the polydisulfide MRI contrast agents can be controlled by their sizes and structures. The polydisulfide Gd(III) chelates are promising biodegradable macromolecular MRI contrast agents for magnetic resonance angiography and cancer imaging.

FGF19/FGFR4 signaling contributes to the resistance of hepatocellular carcinoma to sorafenib

BackgroundSorafenib, a multi-kinase inhibitor, is used as a standard therapy for advanced hepatocellular carcinoma (HCC). However, complete remission has not been achieved and the molecular basis of HCC resistance to sorafenib remains largely unknown. Previous studies have shown that fibroblast growth factor 19 (FGF19) expression correlates with tumor progression and poor prognosis of HCC. Here, we demonstrate the novel role of FGF19 in HCC resistance to sorafenib therapy.MethodsFGF19 Knockdown cells were achieved by lentiviral-mediated interference, and FGFR4 knockout cells were achieved by CRISPR-Cas9. Protein levels of FGF19, FGFR4 and c-PARP in various HCC cell lines were measured by Western blotting analysis. Cell viability was determined by MTS assay, apoptosis was determined by DAPI nuclear staining and Western blot of c-PRAP, and ROS generation was determined by DCFH-DA staining and electrochemical biosensor.ResultsWe showed that FGF19, when overexpressed, inhibited the effect of sorafenib on ROS generation and apoptosis in HCC. In contrast, loss of FGF19 or its receptor FGFR4 led to a remarkable increase in sorafenib-induced ROS generation and apoptosis. In addition, knockdown of FGF19 in sorafenib-resistant HCC cells significantly enhanced the sensitivity to sorafenib. Importantly, targeting FGF19/FGFR4 axis by ponatinib, a third-generation inhibitor of chronic myeloid leukemia, overcomes HCC resistance of sorafenib by enhancing ROS-associated apoptosis in sorafenib-treated HCC.ConclusionOur results provide the first evidence that inhibition of FGF19/FGFR4 signaling significantly overcomes sorafenib resistance in HCC. Co-treatment of ponatinib and sorafinib may represent an effective therapeutic approach for eradicating HCC.

New amphiphilic carriers forming pH-sensitive nanoparticles for nucleic acid delivery.

Amphiphilic lipids are promising for efficient intracellular delivery of nucleic acids. In this study, two new amphiphilic carriers, EKHCO and EHHKCO, were designed and synthesized as multifunctional carriers for efficient intracellular delivery of nucleic acids. The critical micelle concentrations of EKHCO and EHHKCO were 9.50 and 6.87 microM, respectively. Dynamic light scattering showed that the surfactants complexed with plasmid DNA and siRNA to form stable nanoparticles at the concentrations below their critical micelle concentrations. The nanoparticles of the surfactants with pDNA and siRNA exhibited pH-sensitive hemolysis against rat red blood cells when the pH decreased from 7.4 to 5.5, the endosomal-lysosomal pH. The nanoparticles of EHHKCO showed more concentration-dependent pH sensitivity than those of EKHCO. The EHHKCO and EKHCO nanoparticles of both pNDA and siRNA exhibited low cytotoxicity of at physiological pH. Both EKHCO and EHHKCO resulted in high intracellular uptake of pDNA and siRNA. EKHCO and EHHKCO resulted in relatively lower luciferase expression efficiency in U87 cells than DOTAP but produced a much higher percentage of GFP expression in the transfected cells than DOTAP. Both EKHCO and EHHKCO mediated much higher gene silencing efficiency of luciferase and green fluorescence protein (GFP) than DOTAP. The surfactants were more effective for intracellular siRNA delivery than intracellular delivery of plasmid DNA. The pH-sensitive amphiphilic carriers are promising multifunctional carriers for intracellular delivery of nucleic acids.