Stable Loading and Delivery of Melittin with Lipid-Coated Polymeric Nanoparticles for Effective Tumor Therapy with Negligible Systemic Toxicity.

Melittin is a potential anticancer candidate with remarkable antitumor activity and ability to overcome tumor drug resistance. However, the clinical applications of melittin are largely restricted by its severe hemolytic activity and nonspecific cytotoxicity after systemic administration. Here, a biocompatible and stable melittin-loaded lipid-coated polymeric nanoparticle (MpG@LPN) formulation that contains a melittin/poly-γ-glutamic acid nanoparticle inner core, a lipid membrane middle layer, and a polyethylene glycol (PEG) and PEG-targeting molecule outer shell was designed. The formulations were prepared by applying a self-assembly procedure based on intermolecular interactions, including electrostatic attraction and hydrophobic effect. The core-shell MpG@LPN presented high efficiency for melittin encapsulation and high stability in physiological conditions. Hemolysis and cell proliferation assays showed that the PEG-modified MpG@LPN had almost no hemolytic activity and nonspecific cytotoxicity even at high concentrations. The modification of targeting molecules on the MpG@LPNs allowed for the selective binding with target tumor cells and cytolytic activity via apoptosis induction. In vivo experiments revealed that MpG@LPNs can remarkably inhibit the growth of tumors without the occurrence of hemolysis and tissue toxicity. Results suggested that the developed MpG@LPN with a core-shell structure can effectively address the main obstacles of melittin in clinical applications and has great potential in cancer treatment.

Self-Assembled Nanoparticles Prepared from Low-Molecular-Weight PEI and Low-Generation PAMAM for EGFRvIII-Chimeric Antigen Receptor Gene Loading and T-Cell Transient Modification.

BACKGROUND: The complex preparation procedures and severe toxicities are two major obstacles facing the wide use of chimeric antigen receptor-modified T (CAR-T) cells in clinical cancer immunotherapy. The nanotechnology-based T cell temporary CAR modification may be a potential approach to solve these problems and make the CAR-T cell-based tumor therapy feasible and broadly applicable. METHODS: A series of plasmid DNA-loaded self-assembled nanoparticles (pDNA@SNPsx/y) prepared from adamantane-grafted polyamidoamine (Ad-PAMAM) dendrimers of different generations (G1 or G5) and cyclodextrin-grafted branched polyethylenimine (CD-PEI) of different molecular weights (800, 2000, or 25,000 Da) were characterized and evaluated. The detailed physicochemical properties, cellular interaction, and cytotoxicity of selected pDNA@SNPG1/800 were systematically investigated. Thereafter, the epidermal growth factor receptor variant III (EGFRvIII) CAR-expression plasmid vector (pEGFRvIII-CAR) was constructed and encapsulated into SNPG1/800. The resulting pEGFRvIII-CAR@SNPG1/800 was used for Jurkat cell transient transfection, and the EGFRvIII-CAR expressed in transfected cells was measured by flow cytometry and Western blot. Finally, the response of EGFRvIII CAR-positive Jurkat T cell to target tumor cell was evaluated. RESULTS: The pDNA@SNPG1/800 showed the highest efficacy in Jurkat cell gene transfection and exhibited low cytotoxicity. pEGFRvIII-CAR@SNPG1/800 can efficiently deliver pEGFRvIII-CAR into Jurkat T cells, thereby resulting in transient EGFRvIII-CAR expression in transfected cells. EGFRvIII-CAR that is present on the cell membrane enabled Jurkat T cells to recognize and bind specifically with EGFRvIII-positive tumor cells. CONCLUSION: These results indicated that pEGFRvIII-CAR@SNPG1/800 can effectively achieve T-cell transient CAR modification, thereby demonstrating considerable potential in CAR-T cancer therapy.

Solubilization and delivery of Ursolic-acid for modulating tumor microenvironment and regulatory T cell activities in cancer immunotherapy.

Ursolic acid (UA) is a potent triterpenoid compound found in plants and fruits with activities modulating key cell signaling pathways involving STATs, NF-κB, and TRAIL. But it's highly hydrophobic and very poorly soluble in nature. It had been prepared as nanocrystals, solid dispersion and loaded in nanoparticles but the achieved systemic exposure and circulation half-life were not ideal. We reported the development of UA-liposomes made by HPßCD assisted active loading. Compared to lipid suspensions of UA (Lipid-UA) with similar lipid composition, the novel process enabled the formation of UA-Ca crystalline structures inside the liposomes and therefore sustained release of UA in vivo. While the UA-liposomes were not generally toxic towards 4T1 triple negative breast cancer cells, they could effectively modulate CD4+CD25+Foxp3+ T cells from 4T1 tumor bearing mouse by inhibiting STAT5 phosphorylation and IL-10 secretion. In vivo administration of UA-liposomes at 10 mg/kg dose led to reduced numbers of myeloid derived suppressor cells (MDSCs) and regulatory T cells (Tregs) residing in tumor tissues. These changes signified the correction of the tumor mediated immune-suppressive microenvironment. The UA-liposomes treatment alone was already effective in deterring tumor growth. Such a formulation may be highly promising as an immunotherapy agent and be combined with chemotherapeutics or targeted drugs.

Multi-functional self-assembled nanoparticles for pVEGF-shRNA loading and anti-tumor targeted therapy.

Although RNA interference (RNAi) technology shows great potential in cancer treatment, the tumor target delivery and sufficient cytosolic transport of RNAi agents are still the main obstacles for its clinical applications. Herein, we report a functional supramolecular self-assembled nanoparticle vector for RNAi agent loading and tumor target therapy. Molecular block adamantane-grafted poly(ethylene glycol) (Ad-PEG) was modified with epidermal growth factor receptor (EGFR)-specific binding ligand GE11 or pH-sensitive fusogenic peptide GALA and then used for self-assembly with cyclodextrin-grafted branched polyethylenimine (CD-PEI), adamantane-grafted polyamidoamine dendrimer (Ad-PAMAM), and plasmid DNA containing a small hairpin RNA expression cassette against vascular endothelial growth factor (VEGF) into functional DNA-loaded supramolecular nanoparticles (GE11&GALA-pshVEGF@SNPs) based on molecular recognition and charge interaction. These functional peptides facilitated the target cell binding, internalization, and endosomal escape of GE11&GALA-pshVEGF@SNPs, resulting in increased reporter gene expression and efficient targeted gene silencing. The systemic delivery of the GE11&GALA-pshVEGF@SNPs can efficiently downregulate the intratumoral VEGF protein levels, reduce blood vessel formation, and significantly inhibit A549 xenograft tumor growth. These results reveal the potential of these multifunctional self-assembled nanoparticles as a nucleic acid drug delivery system for the treatment of lung cancer.

Gene/paclitaxel co-delivering nanocarriers prepared by framework-induced self-assembly for the inhibition of highly drug-resistant tumors.

While drug resistance has been recognized as the main cause of unsuccessful chemotherapy, the efficient inhibition of highly drug-resistant tumors still remains a significant challenge, especially for in vivo treatments. Drug resistance has been associated with the high expression of the multi-drug resistance gene 1 (MDR1), which can encode an efflux transporter known as P-glycoprotein (P-gp) that is located in the cellular membrane. Therefore, the combined delivery of MDR1-inhibited genes and chemotherapeutic drugs is anticipated to enable the effective inhibition of drug-resistant tumors. Herein, highly paclitaxel (PTX)-resistant ovarian (OV) cancer with a drug resistance index reaching up to ~ 60 was chosen to evaluate the performance of an efficient gene/drug co-delivery nanocarrier. Inspired by the self-assembly that occurs in cells and exosomes, we designed a biomimetic lipid/dextran hybrid nanocarrier with a diameter of ~ 100 nm to enhance the endocytosis and the efficiency of drug/gene release within the cells. This nanocarrier was fabricated via the frame-guided self-assembly of lipid amphiphiles on the surfaces of redox-cleavable dextran-based nanogels. The anionic MDR1-siRNA and the hydrophobic drug PTX were respectively loaded into the cationic lipid shell and the hydrophobic internal core of the hybrid nanocarriers. MDR1-siRNA can knock down MDR1, promoting the accumulation of PTX in cells, and thus is expected to achieve an efficient inhibitory effect against highly PTX-resistant cancer cells. Both in vitro and in vivo studies revealed that this dual-delivery system significantly enhanced the therapeutic effect in comparison with that provided by a PTX-only system. Thus, the construction of gene/chemo co-delivered lipid/dextran nanocarriers provides a new strategy to inhibit highly drug-resistant tumors both in vitro and in vivo. In addition, this work will contribute toward the development of urgently needed tumor nanotherapy that is able to overcome drug resistance while also offering an unmatched range of effective therapeutic nanocarriers. STATEMENT OF SIGNIFICANCE: The biomimetic lipid/dextran hybrid nanocarrier with a diameter of ~ 100 nm, which was fabricated via the frame-guided self-assembly of lipid amphiphiles onto the surface of redox-cleavable dextran-based nanogels, provides a model carrier to co-deliver MDR1-siRNA and PTX.  The MDR1-siRNA/PTX co-loaded biomimetic lipid/dextran hybrid nanocarriers demonstrate good capability in overcoming the PTX-resistance in highly chemo-resistant human ovarian (OV) cancer cells both in vitro and in vivo.