Prodrug-Like, PEGylated Protein Toxin Trichosanthin for Reversal of Chemoresistance.

Multidrug resistance (MDR) is a main obstacle in cancer chemotherapy. The MDR mechanisms involve P-glycoprotein (P-gp) overexpression, abnormality of apoptosis-related protein, and altered expression of drug-targeting proteins. Therapeutic proteins are emerging as candidates for overcoming cancer MDR because of not only their large molecular size that potentially circumvents the P-gp-mediated drug efflux but also their distinctive bioactivity distinguished from small-molecular drugs. Herein we report trichosanthin, a plant protein toxin, possesses synergistic effect with paclitaxel (PTX) in the PTX-resistance A549/T nonsmall cell lung cancer (NSCLC) cells, by reversing PTX-caused caspase 9 phosphorylation and inducing caspase 3-dependent apoptosis. Moreover, via intein-mediated site-specific protein ligation, a matrix metalloproteinase (MMP)-activatable cell-penetrating trichosanthin delivery system was constructed by modification of a cell-penetrating peptide and MMP-2-sensitive PEGylation to overcome the limitation of in vivo application of trichosanthin, by improving the short half-life and poor tumor targeting, as well as immunogenicity. In a mouse model bearing A549/T tumor, the MMP-activatable trichosanthin was further tested for its application for MDR reversal in combination with PTX liposomes. The delivery system showed synergy effect with PTX-loaded liposome in treating MDR cancer in vivo.

Targeting lipid metabolism to overcome EMT-associated drug resistance via integrin ß3/FAK pathway and tumor-associated macrophage repolarization using legumain-activatable delivery.

Epithelial-mesenchymal transition (EMT) is closely associated with the development of drug resistance. Lipid metabolism plays an important role in EMT. This work was to study the cholesterol-lowering drug simvastatin for reversing EMT-associated resistance to chemotherapy via lipid metabolism. METHODS: The combination of simvastatin and paclitaxel was used to overcome the EMT-associated drug resistance. For dual-action on both cancer cells and tumor-associated macrophages (TAM), the tumor microenvironment-activatable multifunctional liposomes were developed for drug codelivery. The liposomes were modified with a hairpin-structured, activatable cell-penetrating peptide that is specifically responsive to the tumor-associated protease legumain. RESULTS: It was revealed simvastatin can disrupt lipid rafts (cholesterol-rich domains) and suppress integrin-ß3 and focal adhesion formation, thus inhibiting FAK signaling pathway and re-sensitizing the drug-resistant cancer cells to paclitaxel. Furthermore, simvastatin was able to re-polarize tumor-associated macrophages (TAM), promoting M2-to-M1 phenotype switch via cholesterol-associated LXR/ABCA1 regulation. The repolarization increased TNF-α, but attenuated TGF-ß, which, in turn, remodeled the tumor microenvironment and suppressed EMT. The liposomal formulation achieved enhanced treatment efficacy. CONCLUSION: This study provides a promising simvastatin-based nanomedicine strategy targeting cholesterol metabolism to reverse EMT and repolarize TAM to treat drug-resistant cancer. The elucidation of the molecular pathways (cholesterol/lipid raft/integrin ß3/FAK and cholesterol-associated LXR/ABCA1 regulation) for anti-EMT and the new application of simvastatin should be of clinical significance.

Targeting death receptors for drug-resistant cancer therapy: Codelivery of pTRAIL and monensin using dual-targeting and stimuli-responsive self-assembling nanocomposites.

Chemoresistance remains a formidable hurdle against cancer therapy. Seeking for novel therapy strategies is an urgent need for those who no longer benefit from chemotherapy. Chemoresistance is usually associated with the dysfunction of intrinsic apoptosis. Targeting extrinsic apoptosis via TRAIL signaling and the death receptors could be a potential solution to treat chemoresistant cancer. A highly biocompatible nano system for codelivery of the TRAIL DNA and the death receptor sensitizer monensin was developed, in which low-molecular-weight PEI (LMW-PEI) was crosslinked by the sulfhydryl cyclodextrin via disulfide bonds, and then bound with DNA, thus forming the bioreducible polyplex cores. In addition, the cyclodextrin also functioned as a carrier for the hydrophobic monensin via host-guest inclusion. Poly-γ-glutamic acid (γ-PGA) was used to modify the polyplex core via charge interaction. The γ-PGA corona can specifically bind with the tumor-associated gamma-glutamyl transpeptidase (GGT) overexpressed on the tumor cells, and achieve tumor-targeting delivery. Moreover, the tumor-homing peptide RGD-modified γ-PGA was also prepared as the surface coating materials for further improving gene delivery efficiency. This gene delivery system was characterized by the dual ligand-targeting, dual stimuli-responsive features. The ligands of RGD and γ-PGA can target the tumor-associated receptors (i.e., integrin and GGT). The conformation of γ-PGA is pH-sensitive, and the tumor acidic micro environments could trigger the detachment of surface-coating γ-PGA. The disulfide crosslinking LMW-PEI is redox-sensitive, and its fast disassembling in the tumor cells could favor the efficient gene delivery. The anti-tumor efficacy was demonstrated both in vitro and in vivo. Moreover, MYC-mediated synthetic lethality could be an important mechanism for overcoming the drug resistance. An important finding of our studies is the demonstration of the in vivo treatment efficacy of TRAIL/monensin, thus providing a potential novel therapeutic strategy for overcoming drug-resistant cancer.

Intein-mediated site-specific synthesis of tumor-targeting protein delivery system: Turning PEG dilemma into prodrug-like feature.

Poor tumor-targeted and cytoplasmic delivery is a bottleneck for protein toxin-based cancer therapy. Ideally, a protein toxin drug should remain stealthy in circulation for prolonged half-life and reduced side toxicity, but turn activated at tumor. PEGylation is a solution to achieve the first goal, but creates a hurdle for the second because PEG rejects interaction between the drugs and tumor cells therein. Such PEG dilemma is an unsolved problem in protein delivery. Herein proposed is a concept of turning PEG dilemma into prodrug-like feature. A site-selectively PEGylated, gelatinase-triggered cell-penetrating trichosanthin protein delivery system is developed with three specific aims. The first is to develop an intein-based ligation method for achieving site-specific modification of protein toxins. The second is to develop a prodrug feature that renders protein toxins remaining stealthy in blood for reduced side toxicity and improved EPR effect. The third is to develop a gelatinase activatable cell-penetration strategy for enhanced tumor targeting and cytoplasmic delivery. Of note, site-specific modification is a big challenge in protein drug research, especially for such a complicated, multifunctional protein delivery system. We successfully develop a protocol for constructing a macromolecular prodrug system with intein-mediated ligation synthesis. With an on-column process of purification and intein-mediated cleavage, the site-specific PEGylation then can be readily achieved by conjugation with the activated C-terminus, thus constructing a PEG-capped, cell-penetrating trichosanthin system with a gelatinase-cleavable linker that enables tumor-specific activation of cytoplasmic delivery. It provides a promising method to address the PEG dilemma for enhanced protein drug delivery, and importantly, a facile protocol for site-specific modification of such a class of protein drugs for improving their druggability and industrial translation.

Dual-Targeting Magnetic PLGA Nanoparticles for Codelivery of Paclitaxel and Curcumin for Brain Tumor Therapy.

Chemotherapy is one of the most important strategies for glioma treatment. However, the "impermeability" of the blood-brain barrier (BBB) impedes most chemotherapeutics from entering the brain, thereby rendering very few drugs suitable for glioma therapy, letting alone application of a combination of chemotherapeutics. Thereby, there is a pressing need to overcome the obstacles. A dual-targeting strategy was developed by a combination of magnetic guidance and transferrin receptor-binding peptide T7-mediated active targeting delivery. The T7-modified magnetic PLGA nanoparticle (NP) system was prepared with co-encapsulation of the hydrophobic magnetic nanoparticles and a combination of drugs (i.e., paclitaxel and curcumin) based on a "one-pot" process. The combined drugs yielded synergistic effects on inhibition of tumor growth via the mechanisms of apoptosis induction and cell cycle arrest, displaying significantly increased efficacy relative to the single use of each drug. Dual-targeting effects yielded a >10-fold increase in cellular uptake studies and a >5-fold enhancement in brain delivery compared to the nontargeting NPs. For the in vivo studies with an orthotopic glioma model, efficient brain accumulation was observed by using fluorescence imaging, synchrotron radiation X-ray imaging, and MRI. Furthermore, the antiglioma treatment efficacy of the delivery system was evaluated. With application of a magnetic field, this system exhibited enhanced treatment efficiency and reduced adverse effects. All mice bearing orthotopic glioma survived, compared to a 62.5% survival rate for the combination group receiving free drugs. This dual-targeting, co-delivery strategy provides a potential method for improving brain drug delivery and antiglioma treatment efficacy.