Yingzhi Chen

Recent progress in microRNA delivery for cancer therapy by non-viral synthetic vectors☆

MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression. Because of significant changes in their expression in cancer, miRNAs are believed to be key factors in cancer genetics and to have potential as anticancer drugs. However, the delivery of miRNAs is limited by many barriers, such as low cellular uptake, immunogenicity, renal clearance, degradation by nucleases, elimination by phagocytic immune cells, poor endosomal release, and untoward side effects. Nonviral delivery systems have been developed to overcome these obstacles. In this review, we provide insights into the development of non-viral synthetic miRNA vectors and the promise of miRNA-based anticancer therapies, including therapeutic applications of miRNAs, challenges of vector design to overcome the delivery obstacles, and the development of miRNA delivery systems for cancer therapy. Additionally, we highlight some representative examples that give a glimpse into the current trends into the design and application of efficient synthetic systems for miRNA delivery. Overall, a better understanding of the rational design of miRNA delivery systems will promote their translation into effective clinical treatments.

Green synthesis of hyaluronic acid-based silver nanoparticles and their enhanced delivery to CD44+ cancer cells

The potent antitumor activities of silver nanoparticles (AgNP) have attracted great attention. However, the application of AgNP is restricted by its non-specific delivery and poor cellular uptake. We developed a novel hyaluronic acid (HA)-based strategy for the green synthesis of AgNP, in which HA was used as the reducing agent and stabilizer. More importantly, HA is a ligand of CD44, and the HA-modified AgNP can target CD44 receptors that are overexpressed in many types of cancer cells. The CD44-dependent endocytosis can significantly increase the intracellular delivery of HA-AgNP, compared to the non-modified one. The antitumor efficacy was significantly improved by HA modification. Furthermore, we found that multiple mechanisms were involved for the enhanced anticancer activities of HA-AgNP, including the decline of mitochondrial membrane potential, cell-cycle arrest, apoptosis, and autophagy. The HA-based strategy for green synthesis and CD44-targeting delivery provided a promising solution for AgNP-mediated cancer treatment.

Hydrothermal Synthesis and Biocompatibility Study of Highly Crystalline Carbonated Hydroxyapatite Nanorods

Highly crystalline carbonated hydroxyapatite (CHA) nanorods with different carbonate contents were synthesized by a novel hydrothermal method. The crystallinity and chemical structure of synthesized nanorods were studied by Fourier transform infrared spectroscopy (FTIR), X-ray photo-electronic spectroscopy (XPS), X-ray diffraction (XRD), Raman spectroscopy, and transmission electron microscopy (TEM). The biocompatibility of synthesized CHA nanorods was evaluated by cell viability and alkaline phosphatase (ALP) activity of MG-63 cell line. The biocompatibility evaluation results show that these CHA nanorods are biologically active apatites and potentially promising bone-substitute biomaterials for orthopedic application.

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.

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.

Co-Delivery of Trichosanthin and Albendazole by Nano-Self-Assembly for Overcoming Tumor Multidrug-Resistance and Metastasis

Multidrug resistance (MDR) and metastasis are the major obstacles in cancer chemotherapy. Nanotechnology-based combination therapy is a useful strategy. Recently, the combination of biologics and small drugs has attracted much attention in cancer therapy. Yet, the treatment outcomes are often compromised by the different pharmacokinetic profiles of the co-administered drugs thus leading to inconsistent drug uptake and suboptimal drug combination at the tumor sites. Nanotechnology-based co-delivery offers a promising method to address this problem, which is well demonstrated in the use of small drug combinations. However, co-delivery of the drugs bearing different physicochemical properties (e.g., proteins and small drugs) remains a formidable challenge. Herein, we developed a self-assembled nanosystem for co-delivery of trichosanthin (TCS) protein and albendazole (ABZ) as a combination therapy for overcoming MDR and metastasis. TCS is a ribosome-inactivating protein with high antitumor activity. However, the druggability of TCS is poor due to its short half-life, lack of tumor-specific action, and low cell uptake. ABZ is a clinically used antihelmintic drug, which can also inhibit tubulin polymerization and thus serve as a potential antitumor drug. In our work, ABZ was encapsulated in the albumin-coated silver nanoparticles (termed ABZ@BSA/Ag NP). The thus-formed NPs were negatively charged and could tightly bind with the cationic TCS that was modified with a cell-penetrating peptide (CPP) low-molecular-weight protamine (termed rTL). Via the stable charge interaction, the nanosystem (rTL/ABZ@BSA/Ag NP) was self-assembled, and featured by the TCS corona. The co-delivery system efficiently inhibited the proliferation of the drug-resistant tumor cells (A549/T and HCT8/ADR) by impairing the cytoskeleton, arresting the cell cycle, and enhancing apoptosis. In addition, the migration and invasion of tumor cells were inhibited presumably due to the impeded cytoskeleton functions. The anti-MDR effect was further confirmed by the in vivo studies with the subcutaneous A549/T tumor mouse model. More importantly, the co-delivery system was demonstrated to be able to inhibit metastasis. The co-delivery system of TCS/ABZ provided a potential strategy for both overcoming drug resistance and inhibiting tumor metastasis.

Glioma Dual-Targeting Nanohybrid Protein Toxin Constructed by Intein-Mediated Site-Specific Ligation for Multistage Booster Delivery

Malignant glioma is one of the most untreatable cancers because of the formidable blood-brain barrier (BBB), through which few therapeutics can penetrate and reach the tumors. Biologics have been booming in cancer therapy in the past two decades, but their application in brain tumor has long been ignored due to the impermeable nature of BBB against effective delivery of biologics. Indeed, it is a long unsolved problem for brain delivery of macromolecular drugs, which becomes the Holy Grail in medical and pharmaceutical sciences. Even assisting by targeting ligands, protein brain delivery still remains challenging because of the synthesis difficulties of ligand-modified proteins. Herein, we propose a rocket-like, multistage booster delivery system of a protein toxin, trichosanthin (TCS), for antiglioma treatment. TCS is a ribosome-inactivating protein with the potent activity against various solid tumors but lack of specific action and cell penetration ability. To overcome the challenge of its poor druggability and site-specific modification, intein-mediated ligation was applied, by which a gelatinase-cleavable peptide and cell-penetrating peptide (CPP)-fused recombinant TCS toxin can be site-specifically conjugated to lactoferrin (LF), thus constructing a BBB-penetrating, gelatinase-activatable cell-penetrating nanohybrid TCS toxin. This nanohybrid TCS system is featured by the multistage booster strategy for glioma dual-targeting delivery. First, LF can target to the BBB-overexpressing low-density lipoprotein receptor-related protein-1 (LRP-1), and assist with BBB penetration. Second, once reaching the tumor site, the gelatinase-cleavable peptide acts as a separator responsive to the glioma-associated matrix metalloproteinases (MMPs), thus releasing to the CPP-fused toxin. Third, CPP mediates intratumoral and intracellular penetration of TCS toxin, thereby enhancing its antitumor activity. The BBB penetration and MMP-2-activability of this delivery system were demonstrated. The antiglioma activity was evaluated in the subcutaneous and orthotopic animal models. Our work provides a useful protocol for improving the druggability of such class of protein toxins and promoting their in-vivo application for targeted cancer therapy.

Poly-γ-glutamic acid-based GGT-targeting and surface camouflage strategy for improving cervical cancer gene therapy

Polycation-based delivery presents a major method for non-viral gene therapy. However, the disadvantages of cationic vectors are their tendencies to be captured and eliminated by the reticuloendothelial system due to their excessive positive charges and nonspecific interaction with normal cells that leads to adverse effects. PEGylation was applied to solve these major problems. Yet, PEG chains can severely compromise cellular uptake and yield unsatisfying efficiency resulting in a so-called PEG dilemma. We developed a γ-PGA-based GGT-targeting and surface camouflage strategy by constructing a ternary complex system via a layer-by-layer self-assembly method. The biodegradable polyanion γ-PGA could protect the PEI/pDNA complexes from interaction with the body fluid components; however, in endosome, the polyanion facilitated the intracellular release of PEI/pDNA. The γ-PGA/PEI/pDNA nanoparticles possessed a markedly improved serum-tolerant capability. More importantly, γ-PGA interacts with the tumor-associated γ-glutamyl transpeptidase (GGT) that can mediate endocytosis of the nanoparticles. With pTRAIL as the therapeutic gene, the γ-PGA/PEI/pTRAIL nanoparticles effectively inhibited tumor cell proliferation by inducing cell apoptosis and arresting cell cycles. The in vivo results displayed effective suppression of tumor growth, and high treatment efficacy in the mice bearing cervical tumor. The γ-PGA-based GGT-targeting and surface camouflage strategy is a potential method for improved gene delivery and cancer therapy.

Biomimetic albumin-modified gold nanorods for photothermo-chemotherapy and macrophage polarization modulation

Nanotechnology-based photothermal therapy has attracted great attention in the past decade. Nevertheless, photothermal therapy has some inherent drawbacks, such as the uneven heat production and limited laser penetration, often leading to insufficient treatment outcomes. Here, we developed a combination strategy to improve cancer therapy. The biomimetic albumin-modified gold nanorods (AuNRs) were prepared with incorporation of paclitaxel (PTX). This therapeutic system was characterized by several features. First, the albumin modification enhanced the biocompatibility and colloidal stability. Second, the surface-coated albumin promoted cellular uptake via the albumin-binding protein pathway. Third, PTX was incorporated via hydrophobic interaction between PTX and the albumin lipophilic domain. Fourth, the system can be used for combined photothermo-chemotherapy for yielding synergistic effects. The antitumor activity of the system was evaluated both in vitro and in vivo using the HCT116 colon cancer cell and tumor model. The combination therapy was found with an enhanced treatment efficiency and no obvious side effect. Most importantly, the thermal effect was also discovered with the ability to modulate the tumor microenvironments and suppress the macrophages polarization towards the M2 pro-tumor phenotype. It could be a mechanism for photothermal immunotherapy. The combination strategy and the system provide a potential method for cancer therapy.

CPP-mediated Protein Delivery in a Noncovalent Form: Proof-of-Concept for Percutaneous and Intranasal Delivery

Macromolecular drugs (e.g., proteins and nucleic acids) are highly environmentally liable and unstable, and their administration is strictly limited to injection. Moreover, a vast majority of macromolecules are cell membrane- impermeable, and it is a critical issue to enhance the cellular uptake efficiency for improving the treatment outcomes. Cell-penetrating peptide (CPP)-assisted strategy is promising for effective macromolecular delivery. As a case in point, CPP-mediated protein delivery has been considered as a revolutionary breakthrough. With aid of CPP, virtually all pro- teins can become cell-permeable. Generally, CPP-protein delivery works in a covalent delivery pattern, by which CPP and its cargo are linked via covalent bond. Recently, noncovalent delivery has also attracted attention for its potential application for protein delivery. In the presented work, the noncovalent pattern was demonstrated for its feasibi lity in percutaneous and nose-to-brain delivery with TAT/GFP as model drug, in comparison with the covalent method. Noncovalent CPP/protein delivery and its noninvasive application may provide a facile method for protein therapy.