Intracellular Delivery of Antisense DNA and siRNA with Amino Groups Masked with Disulfide Units.

Efficient methods for delivery of antisense DNA or small interfering RNA (siRNA) are highly needed. Cationic materials, which are conventionally used for anionic oligonucleotide delivery, have several drawbacks, including aggregate formation, cytotoxicity and a low endosome escape efficiency. In this report a bio-reactive mask (i.e., disulfide unit) for cationic amino groups was introduced, and the mask was designed such that it was removed at the target cell surface. Insolubility and severe cellular toxicity caused by exposed cationic groups are avoided when using the mask. Moreover, the disulfide unit used to mask the cationic group enabled direct delivery of oligonucleotides to the cell cytosol. The molecular design reported is a promising approach for therapeutic applications.

Click Chemistry as a Tool for Cell Engineering and Drug Delivery.

Click chemistry has great potential for use in binding between nucleic acids, lipids, proteins, and other molecules, and has been used in many research fields because of its beneficial characteristics, including high yield, high specificity, and simplicity. The recent development of copper-free and less cytotoxic click chemistry reactions has allowed for the application of click chemistry to the field of medicine. Moreover, metabolic glycoengineering allows for the direct modification of living cells with substrates for click chemistry either in vitro or in vivo. As such, click chemistry has become a powerful tool for cell transplantation and drug delivery. In this review, we describe some applications of click chemistry for cell engineering in cell transplantation and for drug delivery in the diagnosis and treatment of diseases.

Efficient delivery of mesenchymal stem/stromal cells to injured liver by surface PEGylation.

BACKGROUND: Mesenchymal stem/stromal cells (MSCs) have been used in clinical trials for various diseases. These have certain notable functions such as homing to inflammation sites, tissue repair, and immune regulation. In many pre-clinical studies, MSCs administered into peripheral veins demonstrated effective therapeutic outcomes. However, most of the intravenously administered MSCs were entrapped in the lung, and homing to target sites was less than 1%. This occurred mainly because of the adhesion of MSCs to vascular endothelial cells in the lung. To prevent this adhesion, we modified the surface of MSCs with polyethylene glycol (PEG; a biocompatible polymer) using the avidin-biotin complex (ABC) method. METHODS: The surface of MSCs was modified with PEG using the ABC method. Then, the cell adhesion to mouse aortic endothelial cells and the tissue distribution of PEG-modified MSCs were evaluated. Moreover, the homing to the injured liver and therapeutic effect of PEG-modified MSCs were evaluated using carbon tetrachloride-induced acute liver failure model mice. RESULTS: The PEG modification significantly suppressed the adhesion of MSCs to cultured mouse aortic endothelial cells as well as the entrapment of MSCs in the lungs after intravenous injection in mice. PEG-modified MSCs efficiently homed to the injured liver of carbon tetrachloride-induced acute liver failure model mice. More importantly, the cells significantly suppressed serum transaminase levels and leukocyte infiltration into the injured liver. CONCLUSION: These results indicate that PEG modification to the surface of MSCs can suppress the lung entrapment of intravenously administered MSCs and improve their homing to the injured liver.

Intravenous injection of mesenchymal stem cell spheroids improves the pulmonary delivery and prolongs in vivo survival.

BACKGROUND: Because of the excellent therapeutic potential, mesenchymal stem cells (MSCs) have been used as cell therapeutics for various diseases. However, the survival rate and duration of MSCs after transplantation are extremely low and short, respectively. To solve these problems, in this study, we prepared multicellular spheroids of MSCs and investigated their survival and function after intravenous injection in mice. METHODS AND RESULTS: The murine adipose-derived MSC line m17.ASC was cultured in agarose-based microwell plates to obtain size-controlled m17.ASC spheroids of an average diameter and cell number of approximately 170 µm and 1100 cells/spheroid, respectively. The intravenously injected m17.ASC spheroids mainly accumulated in the lung and showed a higher survival rate than suspended m17.ASC cells during the experimental period of 7 days. m17.ASC spheroids efficiently reduced the lipopolysaccharide-induced increase in plasma concentrations of interleukin-6 and tumor necrosis factor-α. CONCLUSIONS: These results indicate that spheroid formation improved the pulmonary delivery and survival of MSCs, as well as their therapeutic potential against inflammatory pulmonary diseases.

Mesenchymal stem/stromal cells as next-generation drug delivery vehicles for cancer therapeutics.

INTRODUCTION: Drug delivery to solid tumors remains a significant therapeutic challenge. Mesenchymal stem/stromal cells (MSCs) home to tumor tissues and can be employed as tumor targeted drug/gene delivery vehicles. Reportedly, therapeutic gene- or anti-cancer drug-loaded MSCs have shown remarkable anti-tumor effects in preclinical studies, and some clinical trials for assessing therapeutic MSCs in patients with cancer have been registered. AREAS COVERED: In the present review, we first discuss the source and interdonor heterogeneity of MSCs, their tumor-homing mechanism, and the route of MSC administration in MSC-based cancer therapy. We then summarize the therapeutic applications of MSCs as a drug delivery vehicle for therapeutic genes or anti-cancer drugs and the drug delivery mechanism from drug-loaded MSCs to cancer cells. EXPERT OPINION: Although numerous preclinical studies have revealed significant anti-tumor effects, several clinical trials assessing MSC-based cancer gene therapy have failed to demonstrate corroborative results, documenting limited therapeutic effects. Notably, a successful clinical outcome with MSC-based cancer therapy would require the interdonor heterogeneity of administered MSCs to be resolved, along with improved tumor-homing efficiency and optimized drug delivery efficiency from MSCs to cancer cells.

Development of nanoparticles derived from corn as mass producible bionanoparticles with anticancer activity.

Recent studies showed that plant-derived nanoparticles (NPs) can be easily produced in high yields and have potential applications as therapeutic agents or delivery carriers for bioactive molecules. In this study, we selected corn as it is inexpensive to grow and mass-produced globally. Super sweet corn was homogenized in water to obtain corn juice, which was then centrifuged, filtered through a 0.45-µm-pore size syringe filter, and ultracentrifuged to obtain NPs derived from corn, or corn-derived NPs (cNPs). cNPs obtained were approximately 80 nm in diameter and negatively charged (- 17 mV). cNPs were taken up by various types of cells, including colon26 tumor cells and RAW264.7 macrophage-like cells, with selective reduction of the proliferation of colon26 cells. Moreover, cNPs induced tumor necrosis factor-α release from RAW264.7 cells. cNPs and RAW264.7 in combination significantly suppressed the proliferation of colon26/fluc cells. Daily intratumoral injections of cNPs significantly suppressed the growth of subcutaneous colon26 tumors in mice, with no significant body weight loss. These results indicate excellent anti-tumor activity of cNPs.

Anticancer drug-loaded mesenchymal stem cells for targeted cancer therapy.

Mesenchymal stem cells (MSCs) have a tumor-homing ability-they accumulate inside tumors after systemic injection, and may thus be useful as carriers for tumor-targeting therapy. To use MSCs effectively as an anti-cancer therapy, they must first be functionalized with a large amount of anti-cancer drugs without causing any significant changes to their tumor-tropism. In the present study, we attempted to modify the cell surface of MSCs with doxorubicin-loaded liposomes (DOX-Lips), using the avidin-biotin complex method, and evaluated delivery efficiency and anti-tumor efficacy of DOX-Lip-modified MSCs. The amount of DOX in DOX-Lip-modified C3H10T1/2 cells, a murine mesenchymal stem cell line, was approximately 21.5 pg per cell, with no significant changes to the tumor-tropism of C3H10T1/2 cells. Notably, DOX-Lip-modified C3H10T1/2 cells significantly suppressed the proliferation of firefly luciferase-expressing murine colon adenocarcinoma colon26/fluc cells, compared to DOX-Lips alone. Fluorescent DOX accumulated at the cell contact surface and inside green fluorescence protein-expressing colon26 (colon26/GFP) in co-cultures of DOX-Lip-modified C3H10T1/2 and colon26/GFP cells. This localized distribution was not observed when only DOX-Lips was added to colon26/GFP cells. These results suggest that DOX-Lips are efficiently delivered from DOX-Lip-modified C3H10T1/2 cells to the neighboring colon26 cells. Furthermore, DOX-Lip-modified C3H10T1/2 cells suppressed tumor growth in subcutaneous tumor-bearing mice, and in a lung metastasis mouse model. Taken together, these results indicate that the intercellular delivery of DOX may be enhanced using DOX-Lip-modified MSCs as an efficient carrier system for targeted tumor therapy.

Multifunctionalization of Cells with a Self-Assembling Molecule to Enhance Cell Engraftment.

Cell-based therapy is a promising approach to restoring lost functions to compromised organs. However, the issue of inefficient cell engraftment remains to be resolved. Herein, we take a chemical approach to facilitate cell engraftment by using self-assembling molecules which modify two cellular traits: cell survival and invasiveness. In this system, the self-assembling molecule induces syndecan-4 clusters on the cellular surface, leading to enhanced cell viability. Further integration with Halo-tag technology provided this self-assembly structure with matrix metalloproteinase-2 to functionalize cells with cell-invasion activity. In vivo experiments showed that the pretreated cells were able to survive injection and then penetrate and engraft into the host tissue, demonstrating that the system enhances cell engraftment. Therefore, cell-surface modification via an alliance between self-assembling molecules and ligation technologies may prove to be a promising method for cell engraftment.

Stable Surface Modification of Mesenchymal Stem Cells Using the Avidin-Biotin Complex Technique.

Mesenchymal stem cells (MSCs) hold promise in cell-based therapies because of their strong tissue repair ability and immunosuppressive effects; however, the therapeutic efficacy of transplanted MSCs is limited due to low survival rates and short-term functioning after transplantation. While the functionalization of MSCs is an ideal way to solve these problems, conventional cell functionalization methods have disadvantages such as cell damage, changes in cellular characteristics, and short-term modification. This unit describes a technique for MSC functionalization by surface modification via the avidin-biotin complex (ABC). This technique provides long-term modification MSC surfaces with biotinylated compounds. This easy method of MSC functionalization will support effective MSC-based therapy. © 2018 by John Wiley & Sons, Inc.

Long-term drug modification to the surface of mesenchymal stem cells by the avidin-biotin complex method.

Mesenchymal stem cells (MSCs) have various functions, making a significant contribution to tissue repair. On the other hand, the viability and function of MSCs are not lasting after an in vivo transplant, and the therapeutic effects of MSCs are limited. Although various chemical modification methods have been applied to MSCs to improve their viability and function, most of conventional drug modification methods are short-term and unstable and cause cytotoxicity. In this study, we developed a method for long-term drug modification to C3H10T1/2 cells, murine mesenchymal stem cells, without any damage, using the avidin-biotin complex method (ABC method). The modification of NanoLuc luciferase (Nluc), a reporter protein, to C3H10T1/2 cells by the ABC method lasted for at least 14 days in vitro without major effects on the cellular characteristics (cell viability, cell proliferation, migration ability, and differentiation ability). Moreover, in vivo, the surface Nluc modification to C3H10T1/2 cells by the ABC method lasted for at least 7 days. Therefore, these results indicate that the ABC method may be useful for long-term surface modification of drugs and for effective MSC-based therapy.