Xian-Rong Qi

Tumor-targeting dual peptides-modified cationic liposomes for delivery of siRNA and docetaxel to gliomas.

Combinations of drugs promoting anti-angiogenesis and apoptosis effects are meaningful for cancer therapy. In the present study, dual peptides-modified liposomes were designed by attaching two receptor-specific peptides, specifically low-density lipoprotein receptor-related protein receptor (Angiopep-2) and neuropilin-1 receptor (tLyP-1) for brain tumor targeting and tumor penetration. Vascular endothelial growth factor (VEGF) siRNA and chemotherapeutic docetaxel (DTX) were chosen as the two payloads because VEGF is closely associated with angiogenesis, and DTX can kill tumor cells efficiently. Binding to glioma cells, co-delivery of siRNA and DTX in human glioblastoma cells (U87 MG) and murine brain microvascular endothelial cells (BMVEC), VEGF gene silencing, antiproliferation and anti-tumor effects of the dual peptides-modified liposomes were evaluated in vitro and in vivo. The dual peptides-modified liposomes persisted the binding ability to glioma cells, enhanced the internalization via specific receptor mediated endocytosis and tissue penetration, thus the dual peptides-modified liposomes loading VEGF siRNA and DTX possessed stimulative gene silencing and antiproliferation activity compared with non-modified and single peptide-modified liposomes. The co-delivery research revealed different intracellular behavior of hydrophilic large molecular and lipophilic small molecule, the former involves endocytosis and subsequent escape of endosome/lysosomes, while the latter experiences passive diffusion of lipophilic small drugs after its release. Furthermore, the dual peptides-modified liposomes showed superiority in anti-tumor efficacy, combination of anti-angiogenesis by VEGF siRNA and apoptosis effects by DTX, after both intratumor and system application against mice with U87 MG tumors, and the treatment did not activate system-associated toxicity or the innate immune response. Combination with the dual peptides-guided tumor homing and penetration, the dual peptides-modified liposomes provide a strategy for effective targeting delivery of siRNA and DTX into the glioma cell and inhibition of tumor growth in a synergistic manner.

Chemotherapeutic drug delivery to cancer cells using a combination of folate targeting and tumor microenvironment-sensitive polypeptides

Chemotherapeutic agents often cause severe side effects because they produce a similar cytotoxicity in both cancerous and healthy cells. In this study, a rational strategy was implemented to take advantage of a combination of both tumor microenvironment-sensitive polypeptides (TMSP) and folate to create a more selective and efficient drug delivery system to target cancer cells. TMSP and folate were conjugated to the distal ends of DSPE-PEG2000-maleimide and DSPE-PEG5000-amine to create DSPE-PEG2000-TMSP and DSPE-PEG5000-folate, respectively, which were incorporated onto the surface of a docetaxel-loaded nanostructured lipid carrier (F/TMSP-DTX-NLC). TMSP are comprised of polycationic cell-penetrating peptides (CPP) and polyanionic inhibitory peptides, which are coupled via a proteinase-sensitive cleavable linker. The linker can be cleaved in the presence of matrix metalloprotease-2 and -9 (MMP-2/9). TMSP provides the ability to enhance specific cancer cellular uptake after selectively unmasking polyanionic inhibitory peptides in MMP-2/9 protease-oversecretion tumor tissue, whereas in circulation, the penetration is shielded. The folate moiety binds selectively to folate receptor-positive tumors. The cleaved dual-modified nanocarriers are then taken up by the tumor cells via both receptor-mediated endocytosis and CPP penetrating action to overcome the higher interstitial pressure in the tumor. The nanocarrier system demonstrated a small size, high encapsulation efficiency (>95%), sustained release and targeted delivery. The strong cellular uptake and cytotoxic activity of dual-modified F/TMSP-DTX-NLC in KB, HT-1080, MCF-7 and A549 cells verified the correlation with folate receptor expression and MMP-2/9 secretion. The remarkable penetration into KB and HT-1080 multicellular tumor spheroids confirmed that the temporary mask of the polyanionic inhibitory peptide in TMSP does not disturb the penetration ability of CPP in the tumor microenvironment with abundant proteases. Furthermore, the active targeting and triggered activation exhibited higher antitumor efficacy and lower systemic toxicity with the KB tumor model in nude mice compared to the nonmodified DTX-NLC and Taxotere(®). These results suggested that the application of combined TMSP and folate modifications may be an approach in the selectively targeted delivery of anticancer drugs with low systemic toxicity.

pH-responsive complexes using prefunctionalized polymers for synchronous delivery of doxorubicin and siRNA to cancer cells

A nanocarrier delivery system that can simultaneously deliver a chemotherapeutic drug and siRNA to the tumor is emerging as a promising treatment strategy for cancer treatment. In this study, a multifunctional PHD/PPF/siRNA complexes was developed by one-step assembly of prefunctionalized polymers: PEI-HZ-DOX (PHD) and PEI-PEG-Folate (PPF) with siRNA. The PHD, a conjugate of PEI (polyethylenimine) with doxorubicin (DOX) via a pH-responsive hydrazone linkage, enables pH-controlled drug release. The PPF, a tumor-targeting folate ligand conjugated to PEI using polyethyleneglycol (PEG) as a linker, enables immune evasion and cell-specific targeting. The prefunctionalized PHD and PPF as well as the self-assembly complexes reveals advantage on safety in further application for siRNA delivery. By exploiting distinct triple ratios of PHD, PPF and siRNA during nanocomplexes formulation, the folate surface density, DOX loading amount and siRNA complexation can be precisely and reproducibly changed. The studies showed that the complexes was capable of delivering siRNA and DOX to cancerous cells and release synchronously in cell by acid-triggered manner, i.e. hydrazone bond cleavage and endosome/lysosome escape using flow cytometry and confocal laser scanning microscopy analysis. The results highlight the potential for therapeutic gene silencing in vitro and in vivo using RT-PCR and non-invasive in vivo imaging systems. The PHD/PPF/siRNA complexes can increase DOX and siRNA accumulation in cancerous cells and decrease the nonspecific distribution in normal tissues by the combination of EPR effect of nanocarriers, pH-triggered drug release, folate-mediated targeted delivery, and synergistic action of DOX and siRNA.

Superparamagnetic iron oxide nanoparticles stabilized by alginate: Pharmacokinetics, tissue distribution, and applications in detecting liver cancers

The objectives of this study were to describe the pharmacokinetics and tissue distribution of superparamagnetic iron oxide nanoparticle (SPIO) stabilized with alginate (SPIO-alginate), and investigate its potential in detecting liver cancers as a newly developed magnetic resonance (MR) contrast agent. Pharmacokinetics and tissue distribution of SPIO-alginate were investigated in Sprague-Dawley rats. The results showed that SPIO-alginate was eliminated rapidly from serum with the half-life of 0.27 h at 109.5 micromol Fe/kg and accumulated dominantly in liver and spleen with a total percentage of more than 90% of dose after intravenous injection. The studies of pharmacokinetics and distribution of SPIO-alginate in rats indicated the MR contrast agent, based on SPIO, mainly accumulating in targeting organs that contain phagocytosing cells, i.e. liver and spleen. The efficacies in detecting hepatocellular carcinoma (HCC) of rat with primary liver cancer and xenograft liver cancers of rabbit were investigated before and after injection of SPIO-alginate. The signal intensity of liver parenchyma in rabbit with VX2 tumor after injection of SPIO-alginate was reduced sharply resulting in a significant contrast between liver parenchyma and tumor. Detection of the HCC in rat model was also demonstrated. The present study provides evidence that SPIO-alginate might have the ability to improve the detection of liver tumors as an MR contrast agent, and the efficacy is associated with the SPIO specifically located in Kupffer cells in hepatic sinusoid.

PSA-responsive and PSMA-mediated multifunctional liposomes for targeted therapy of prostate cancer

In the hormone-refractory stage of prostate cancer (PC), the expression of prostate-specific antigen (PSA) and prostate-specific membrane antigen (PSMA) often remains highly active. Accumulating studies have demonstrated that these two proteins are attractive targets for specific delivery of functional molecules to advanced PC, not merely as potential sensitive markers for PC detection. In this study, we constructed a dual-modified liposome that incorporated PSA-responsive and PSMA-mediated liposomes and potentially offers double selectivity for PC. The folate moiety binds quickly to PSMA-positive tumors, and the PSA-responsive moiety is cleaved by PSA that was enriched in tumor tissues. The activated liposomes (folate and cell-penetrating peptides dual-modifications) are subsequently taken up by the tumor cells via polyarginines penetrating effects and receptor-mediated endocytosis. To corroborate these assumptions, a series of experiments were conducted, including PSA-responsive peptide hydrolysis kinetics, cellular uptake, internalization mechanism and escape from endosomes in PC-3 and/or 22Rv1 cells, biodistribution and antitumor activity of siRNA-loaded liposomes after systemic administration, gene silencing and cell apoptosis in vitro and in vivo. The results reveal that multivalent interactions play a key role in enhancing PC cell recognition and uptake while reducing nonspecific uptake. The dual-modified liposomes carrying small interfering RNA (siRNA) have significant advantages over the control liposomes, including single-modified (folate, CPP, PSA-responsive only) and non-modified liposomes. The dual-modified liposomes elevated cellular uptake, downregulated expression of polo-like kinase 1 (PLK-1) and augmented cell apoptosis in prostate tumor cells. The entry of the dual-modified liposomes into 22Rv1 cells occurred via multiple endocytic pathways, including clathrin-mediated endocytosis and macropinocytosis, followed by an effective endosomal escape of the entrapped siRNA into the cytoplasm. In vivo studies conducted on a 22Rv1 xenograft murine model demonstrated that the dual-modified liposomes demonstrated the maximized accumulation, retention and knockdown of PLK-1 in tumor cells, as well as the strongest inhibition of tumor growth and induction of tumor cell apoptosis. In terms of targeting capacity and therapeutic potency, the combination of a PSA-responsive and PSMA-mediated liposome presents a promising platform for therapy and diagnosis of PSMA/PSA-positive PC.

Enhancing cellular uptake of activable cell-penetrating peptide–doxorubicin conjugate by enzymatic cleavage

The use of activable cell-penetrating peptides (ACPPs) as molecular imaging probes is a promising new approach for the visualization of enzymes. The cell-penetrating function of a polycationic cell-penetrating peptide (CPP) is efficiently blocked by intramolecular electrostatic interactions with a polyanionic peptide. Proteolysis of a proteinase-sensitive substrate present between the CPP and polyanionic peptide affords dissociation of both domains and enables the activated CPP to enter cells. This ACPP strategy could also be used to modify antitumor agents for tumor-targeting therapy. Here, we aimed to develop a conjugate of ACPP with antitumor drug doxorubicin (DOX) sensitive to matrix metalloproteinase-2 and -9 (MMP-2/9) for tumor-targeting therapy purposes. The ACPP-DOX conjugate was successfully synthesized. Enzymatic cleavage of ACPP-DOX conjugate by matrix metalloproteinase (MMP)-2/9 indicated that the activation of ACPP-DOX occurred in an enzyme concentration–dependent manner. Flow cytometry and laser confocal microscope studies revealed that the cellular uptake of ACPP-DOX was enhanced after enzymatic-triggered activation and was higher in HT-1080 cells (overexpressed MMPs) than in MCF-7 cells (under-expressed MMPs). The antiproliferative assay showed that ACPP had little toxicity and that ACPP-DOX effectively inhibited HT-1080 cell proliferation. These experiments revealed that the ACPP-DOX conjugate could be triggered by MMP-2/9, which enabled the activated CPP-DOX to enter cells. ACPP-DOX conjugate may be a potential prodrug delivery system used to carry antitumor drugs for MMP-related tumor therapy.

Enhanced brain distribution and pharmacodynamics of rivastigmine by liposomes following intranasal administration.

Alzheimers disease (AD) is a common progressive neurodegenerative disorder associated with cholinergic neurons degeneration. The blood-brain barrier (BBB) not only provides protection for the brain but also hinders the treatment and diagnosis of this neurological disease, because the drugs must cross BBB to reach the lesions. The present work was aimed at formulating rivastigmine liposomes (Lp) and cell-penetrating peptide (CPP) modified liposomes (CPP-Lp) to improve rivastigmine distribution in brain and proceed to enhance pharmacodynamics by intranasal (IN) administration and minimize side effects. The results revealed that Lp especially the CPP-Lp can enhance the permeability across the BBB by murine brain microvascular endothelial cells model in vitro. IN administration of rivastigmine solution and rivastigmine liposomes demonstrated the capacity to improve rivastigmine distribution and adequate retention in CNS regions especially in hippocampus and cortex, which were the regions most affected by AD, than that of IV administration. Importantly, the lagging but intense inhibition of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) activities were relative to the extended release, absorption and retention. In addition, there was very mild nasal toxicity of liposomal formulations. The data suggest that rivastigmine liposomes especially CPP-Lp improve the brain delivery and enhance pharmacodynamics which respect to BBB penetration and nasal olfactory pathway into brain after IN administration, and simultaneously decrease the hepatic first pass metabolism and gastrointestinal adverse effects.

Remote loading of diclofenac, insulin and fluorescein isothiocyanate labeled insulin into liposomes by pH and acetate gradient methods.

Remote loading of the model drugs diclofenac, insulin and fluorescein isothiocyanate labeled insulin (FITC-insulin) into liposomes by formation of transmembrane gradients were examined. A trapping efficiency of almost 100% was obtained for liposomal diclofenac, by the calcium acetate gradient method, whereas liposomes prepared by the conventional reverse-phase evaporation vesicle method had 1-8% trapping efficiencies. Soybean-derived sterol was a better stabilizer of the dipalmitoylphosphatidylcholine bilayer membrane than cholesterol, as shown from trapping efficiencies and drug release. The pH gradient method resulted in a 5-50% of FITC-insulin liposomal trapping efficiency, while insulin could not be loaded by this method. Liposomes released calcein in response to insulin, showing insulin interacts with the liposomal membrane in the presence of a transmembrane gradient. The present work has demonstrated a remote loading method for weak acids such as diclofenac into liposomes by the acetate gradient method. From the result of remote loading of FITC-insulin into liposomes by the pH gradient method, this method may be available for the preparation of liposomal peptides.

Enhanced antitumor efficacy and decreased toxicity by self-associated docetaxel in phospholipid-based micelles

To overcome the poor aqueous solubility of docetaxel (DTX) and the side effects of the emulsifier in the marketed formulation, we have developed a DTX-loaded micelle using a nontoxic and biodegradable amphiphilic diblock copolymer, methoxy polyethylene glycol-distearoylphosphatidylethanolamine (mPEG(2000)-DSPE). The prepared micelles exhibited a core-shell structure, and DTX was successfully encapsulated in the core with an encapsulation efficiency of 97.31 ± 2.95% and a drug loading efficiency of 3.14 ± 0.13%. The micelles were spherical with a hydrodynamic diameter of approximately 20 nm, which could meet the requirement for in vivo administration, and were expected to enhance the drugs antitumor efficacy and to decrease its toxicity. To evaluate the DTX-loaded micelles, we chose a well marketed formulation, Taxotere(®), as the control. The prepared DTX micelle had a similar antiproliferative effect to Taxotere(®) in vitro but a significantly better antitumor efficacy than Taxotere(®) in vivo, which may be caused by passive targeting of the tumor by the micelles. In addition, the safety evaluation revealed that the DTX micelle was a qualified drug for use in vivo. Based on the experimental results, we propose that mPEG(2000)-DSPE micelle is a potent carrier for DTX.

Development of small interfering RNA delivery system using PEI-PEG-APRPG polymer for antiangiogenic vascular endothelial growth factor tumor-targeted therapy

Background Small interfering RNA (siRNA) can silence target genes in the cytoplasm and be a major tool in gene therapy. Vascular endothelial growth factor (VEGF), a potent regulator of angiogenesis, is overexpressed in most tumors and is closely associated with tumor growth and metastasis. It has been shown that inhibition of VEGF expression by siRNA is an effective and useful method for antiangiogenic tumor therapy. Methods In the present study, we synthesized a targeted delivery system of PEI-PEG-APRPG incorporating angiogenic vessel-homing Ala-Pro-Arg-Pro-Gly (APRPG) peptide into cationic polyethylenimine (PEI) via a hydrophilic poly(ethylene glycol) (PEG) spacer. Results PEI-PEG-APRPG effectively condensed siRNA into 20–50 nm nanoparticles with a positive surface charge using a suitable N/P ratio. The siRNA/PEI-PEG-APRPG complex effectively enhanced the stability of siRNA in RNase A, and improved the proliferation-inhibiting ability and transfection efficiency of siRNA in vitro and tumor accumulation in vivo. In addition, the siRNA/PEI-PEG-APRPG complex exhibited high efficiency as antitumor therapy with regard to tumor growth, microvessel density, and VEGF protein and mRNA levels. Conclusion These findings suggest that PEI-PEG-APRPG effectively delivers siRNA to tumors overexpressing VEGF and thereby inhibits tumor growth.