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.

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 polyarginine's 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.

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.

Comparative studies of polyethylenimine-doxorubicin conjugates with pH-sensitive and pH-insensitive linkers.

Doxorubicin (DOX) was conjugated to polyethylenimine (PEI) in several synthetic ways and created PEI-succinic anhydride (SUC)-DOX, PEI-N-succinimidyl 3-maleimidopropionate (SMP)-DOX and PEI-3-maleimidopropionic acid hydrazide (MPH)-DOX conjugates using pH-insensitive linkage (amide bond) or pH-sensitive linkage (hydrazone bond). The effects of the conjugates on anticancer therapy against human breast cancer cells were investigated for in vitro release under different pH, cellular uptake, intracellular localization and antiproliferative activity. DOX release from PEI-MPH-DOX formed via hydrazone bonds occurred in an acid-triggered manner and increased with decreasing pH. But PEI-SUC-DOX and PEI-SMP-DOX formed via amide bonds were quite stable and released negligible DOX under both neutral and acidic conditions. The results from flow cytometry and confocal laser scanning microscopy analysis showed that conjugates uptakes were PEI-SUC-DOX > PEI-SMP-DOX > PEI-MPH-DOX, which was consistent with the primary amine content and more efficient than free DOX due to the intense nonspecific interaction between the positively charged conjugates and the negatively charged cell surface proteoglycans. PEI-MPH-DOX showed higher antiproliferative activity than that of PEI-SUC-DOX and PEI-SMP-DOX. The antiproliferative activity of PEI-MPH-DOX was achieved through the endocytosis, nuclear entry of released DOX and interaction with the DNA. However, PEI-SUC-DOX and PEI-SMP-DOX showed more endocytosis but less nuclear entry leading to lower antiproliferative activity. According to the measurements, we determined that enhanced endocytosis and pH-sensitive drug release were the critical processes in determining anticancer therapy of drug-polymer conjugate. The PEI-MPH-DOX conjugate could be sifted to use as a prodrug for cancer therapy and also as a novel delivery carrier to codelivery DOX and gene in further research.

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 drug's 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.