Targeted cellular delivery of robust enzyme nanoparticles for the treatment of drug-induced hepatotoxicity and liver injury.

Direct delivery of proteins into cells has been considered an effective approach for treating the protein-related diseases. However, clinical use of proteins has still been limited due to their instability in the blood and poor membrane permeability. To achieve an efficient cellular delivery of the protein to target cells via a systemic administration, a multifunctional carrier system having desirable stability both in the blood stream and the cells, specific cell-targeting property and endosomal escape functions may be required. In this study, we prepared a catalytic nanoparticle containing an active enzyme by cross-tethering multiple superoxide dismutase (SOD) molecules with catechol-derivatized hyaluronic acid (HA). The permeable shell of hydrophilic HA chains effectively protects the enzyme from degradation in the blood after intravenous administration and provides an additional function for targeting hepatocytes expressing HA receptor (CD44). The structure and catalytic activity of the enzyme molecules in the nanoparticle were not significantly compromised in the nanoparticle. In addition, ultra-small calcium phosphate nanoparticles (USCaP, 2-5 nm) were crystalized and decorated on the surface of the nanoparticle for the efficient endosomal escape after cellular uptake. The SOD-containing nanoparticle fortified with USCaP was used for the treatment of acetaminophen (APAP)-induced fulminant hepatotoxicity and liver injury. The nanoparticle achieved the efficient hepatic cellular delivery of SOD via a systemic administration and resulted in efficient removal of reactive oxygen species (ROS) in the liver and remarkable improvement of APAP-induced hepatotoxicity and liver injury in animals. STATEMENT OF SIGNIFICANCE: Despite the enormous therapeutic potential, the intracellular delivery of proteins has been limited due to their poor membrane permeability and stability. In this study, we demonstrated an active enzyme-containing nanoparticle functionalized by hyaluronic acid and ultra-small size calcium phosphate nanoparticles (2-5 nm) for targeted cellular delivery of superoxide dismutase (SOD). The nanoparticle was designed to integrate all the essential functions, including serum stability, target specificity, and endosomal escape capability, for a systemic delivery of a therapeutic protein to the cells of the liver tissue. The intravenous administration of the nanoparticle efficiently removes reactive oxygen species (ROS) in the liver and remarkably improves the drug-induced hepatotoxicity and the progress of fulminant liver injury in an acetaminophen-overdose animal model.

Enhanced Cancer Vaccination by In Situ Nanomicelle-Generating Dissolving Microneedles.

Efficient delivery of tumor antigens and immunostimulatory adjuvants into lymph nodes is crucial for the maturation and activation of antigen-presenting cells (APCs), which subsequently induce adaptive antitumor immunity. A dissolving microneedle (MN) has been considered as an attractive method for transcutaneous immunization due to its superior ability to deliver vaccines through the stratum corneum in a minimally invasive manner. However, because dissolving MNs are mostly prepared using water-soluble sugars or polymers for their rapid dissolution in intradermal fluid after administration, they are often difficult to formulate with poorly water-soluble vaccine components. Here, we develop amphiphilic triblock copolymer-based dissolving MNs in situ that generate nanomicelles (NMCs) upon their dissolution after cutaneous application, which facilitate the efficient encapsulation of poorly water-soluble Toll-like receptor 7/8 agonist (R848) and the delivery of hydrophilic antigens. The sizes of NMCs range from 30 to 40 nm, which is suitable for the efficient delivery of R848 and antigens to lymph nodes and promotion of cellular uptake by APCs, minimizing systemic exposure of the R848. Application of MNs containing tumor model antigen (OVA) and R848 to the skin of EG7-OVA tumor-bearing mice induced a significant level of antigen-specific humoral and cellular immunity, resulting in significant antitumor activity.

Self-assembled PEGylated albumin nanoparticles (SPAN) as a platform for cancer chemotherapy and imaging.

Paclitaxel (PTX) is used as a major antitumor agent for the treatment of recurrent and metastatic breast cancer. For the clinical application of PTX, it needs to be dissolved in an oil/detergent-based solvent due to its poor solubility in an aqueous medium. However, the formulation often causes undesirable complications including hypersensitivity reactions and limited tumor distribution, resulting in a lower dose-dependent antitumor effect. Herein, we introduce a facile and oil-free method to prepare albumin-based PTX nanoparticles for efficient systemic cancer therapy using a conjugate of human serum albumin (HSA) and poly(ethyleneglycol) (PEG). PTX were efficiently incorporated in the self-assembled HSA-PEG nanoparticles (HSA-PEG/PTX) using a simple film casting and re-hydration procedure without additional processes such as application of high pressure/shear or chemical crosslinking. The spherical HSA-PEG nanoparticle with a hydrodynamic diameter of ca. 280 nm mediates efficient cellular delivery, leading to comparable or even higher cytotoxicity in various breast cancer cells than that of the commercially available Abraxane®. When systemically administered in a mouse xenograft model for human breast cancer, the HSA-PEG-based nanoparticle formulation exhibited an extended systemic circulation for more than 96 h and enhanced intratumoral accumulation, resulting in a remarkable anticancer effect and prolonged survival of the animals.

Inhibition of Tumor Growth via Systemic siRNA Delivery Using Reducible Bile Acid-Conjugated Polyethylenimine.

RNA interference (RNAi), mediated by small interfering RNA (siRNA), has been considered as a potential therapeutic agent for cancer owing to its ability to suppress target genes in a sequence-specific manner. In this study, a conjugate of the low molecular weight (MW) polyethylenimine (PEI) (MW 1800) and deoxycholic acid (DA) was further modified with 4-fluorothiophenol (FTP) (TP-DA-PEI) to achieve systemic siRNA delivery. The thiophenol group would be involved with disulfide bonds between the polymer chains and siRNA modified with free thiols (thiol-siRNA) to form and π⁻π interactions between the pendent aromatic groups and coprostane ring of the bile acid. The TP-DA-PEI conjugates could generate stable nanoparticles with thiol-siRNA. The TP-DA-PEI conjugate not only achieved enhanced intracellular uptake, serum stability, and transfection efficiency, but also showed high accumulation of TP-DA-PEI/thiol-siRNA polyplexes and significant tumor growth inhibition effect in tumor-bearing mice after systemic administration.

Microneedle arrays coated with charge reversal pH-sensitive copolymers improve antigen presenting cells-homing DNA vaccine delivery and immune responses.

Successful delivery of a DNA vaccine to antigen-presenting cells and their subsequent stimulation of CD4+ and CD8+ T cell immunity remains an inefficient process. In general, the delivery of prophylactic vaccines is mainly mired by low transfection efficacy, poor immunogenicity, and safety issues from the materials employed. Currently, several strategies have been exploited to improve immunogenicity, but an effective strategy for safe and pain-free delivery of DNA vaccines is complicated. Herein, we report the rapid delivery of polyplex-based DNA vaccines using microneedle arrays coated with a polyelectrolyte multilayer assembly of charge reversal pH-responsive copolymer and heparin. The charge reversal pH-responsive copolymer, composed of oligo(sulfamethazine)-b-poly(ethylene glycol)-b-poly(amino urethane) (OSM-b-PEG-b-PAEU), was used as a triggering layer in the polyelectrolyte multilayer assembly on microneedles. Charge reversal characteristics of this copolymer, that is, the OSM-b-PEG-b-PAEU copolymer exhibit, positive charge at low pH (pH4.03) and becoming negative charge when exposed to physiological pH conditions (pH7.4), allowing the facile assembly and disassembly of polyelectrolyte multilayers. The electrostatic repulsion between heparin and OSM-b-PEG-b-PAEU charge reversal copolymer triggered the release of DNA vaccines. DNA vaccines laden on microneedles are effectively transfected into RAW 264.7 macrophage cells in vitro. Vaccination of BALB/c mice by DNA vaccine-loaded microneedle arrays coated with a polyelectrolyte multilayer generated antigen-specific robust immune responses. These findings provide potential strategy of charge reversal pH-responsive copolymers coated microneedles for DNA vaccine delivery.

Target-specific delivery of siRNA by stabilized calcium phosphate nanoparticles using dopa-hyaluronic acid conjugate.

Low cytotoxicity and high cellular gene delivery capability are among the most important prerequisites for the selection of a non-viral carrier. Although calcium phosphate (CAP) nanoparticles have been long used for animal cell transfection, its rapid and uncontrollable crystal growth and lack of tissue specificity are among the most challenging problems that limit its use in the clinic. In this study, we report the development of CAP nanoparticles stabilized by a conjugate of the mussel-inspired adhesive molecule, 3,4-dihydroxy-l-phenylalanine (dopa), and a nontoxic hydrophilic natural polymer, hyaluronic acid (HA), for targeted siRNA delivery to tumors. CAP/siRNA/dopa-HA can form compact nanoparticles that effectively protect siRNA from enzymatic degradation despite the structural drawbacks of siRNA, such as low charge density and short and rigid structure. In addition, stabilized CAP nanoparticles were able to maintain their colloidal stability in a physiological salt condition for over a week. The superior ability of CAP/siRNA/dopa-HA to maintain the integrity of encapsulated siRNA and the stability in solution of the nanoparticles allow this formulation to achieve improved intratumoral accumulation of siRNA and a high level of target gene silencing in solid tumors after systemic administration. Considering its biocompatibility, transfection efficacy, and tumor targeting capability, this stabilized calcium phosphate nanoparticle-based gene delivery platform should be considered a promising candidate carrier for systemic siRNA delivery and targeted cancer therapy.

Polyplex-releasing microneedles for enhanced cutaneous delivery of DNA vaccine.

Microneedle (MN)-based DNA vaccines have many advantages over conventional vaccines administered by hypodermic needles. However, an efficient strategy for delivering DNA vaccines to intradermal cells has not yet been established. Here, we report a new approach for delivering polyplex-based DNA vaccines using MN arrays coated with a pH-responsive polyelectrolyte multilayer assembly (PMA). This approach enabled rapid release of polyplex upon application to the skin. In addition to the polyplex-releasing MNs, we attempted to further maximize the vaccination by developing a polymeric carrier that targeted resident antigen presenting cells (APCs) rich in the intradermal area, as well as a DNA vaccine encoding a secretable fusion protein containing amyloid beta monomer (Aß1-42), an antigenic determinant. The resulting vaccination system was able to successfully induce a robust humoral immune response compared to conventional subcutaneous injection with hypodermal needles. In addition, antigen challenge after immunization elicited an immediate and strong recall immune response due to immunogenic memory. These results suggest the potential utility of MN-based polyplex delivery systems for enhanced DNA vaccination.

Enhanced transfection by antioxidative polymeric gene carrier that reduces polyplex-mediated cellular oxidative stress.

PURPOSE: To test the hypothesis in which polyplex-induced oxidative stress may affect overall transfection efficiency, an antioxidative transfection system minimizing cellular oxidative stress was designed for enhanced transfection. METHODS: An amphiphilic copolymer (PEI-PLGA) was synthesized and used as a micelle-type gene carrier containing hydrophobic antioxidant, α-tocopherol. Cellular oxidative stress and the change of mitochondrial membrane potential after transfection was measured by using a fluorescent probe (H2DCFDA) and lipophilic cationic probe (JC-1), respectively. Transfection efficiency was determined by measuring a reporter gene (luciferase) expression level. RESULTS: The initial transfection study with conventional PEI/plasmid DNA polyplex showed significant generation of reactive oxygen species (ROS). The PEI-PLGA copolymer successfully carried out the simultaneous delivery of α-tocopherol and plasmid DNA (PEI-PLGA/Toco/pDNA polyplex) into cells, resulting in a significant reduction in cellular ROS generation after transfection and helped to maintain the mitochondrial membrane potential (ΔΨ). In addition, the transfection efficiency was dramatically increased using the antioxidative transfection system. CONCLUSIONS: This work showed that oxidative stress would be one of the important factors that should be considered in designing non-viral gene carriers and suggested a possible way to reduce the carrier-mediated oxidative stress, which consequently leads to enhanced transfection.