Cholesterol-Bearing Polysaccharide-Based Nanogels for Development of Novel Immunotherapy and Regenerative Medicine.

Novel functional biomaterials are expected to bring about breakthroughs in developing immunotherapy and regenerative medicine through their application as drug delivery systems and scaffolds. Nanogels are defined as nanoparticles with a particle size of 100 nm or less and as having a gel structure. Nanogels have a three-dimensional network structure of cross-linked polymer chains, which have a high water content, a volume phase transition much faster than that of a macrogel, and a quick response to external stimuli. As it is possible to transmit substances according to the three-dimensional mesh size of the gel, a major feature is that relatively large substances, such as proteins and nucleic acids, can be taken into the gel. Furthermore, by organizing nanogels as a building block, they can be applied as a scaffold material for tissue regeneration. This review provides a brief overview of the current developments in nanogels in general, especially drug delivery, therapeutic applications, and tissue engineering. In particular, polysaccharide-based nanogels are interesting because they have excellent complexation properties and are highly biocompatible.

Three-Dimensional Culture of Cartilage Tissue on Nanogel-Cross-Linked Porous Freeze-Dried Gel Scaffold for Regenerative Cartilage Therapy: A Vibrational Spectroscopy Evaluation.

This study presents a set of vibrational characterizations on a nanogel-cross-linked porous freeze-dried gel (NanoCliP-FD gel) scaffold for tissue engineering and regenerative therapy. This scaffold is designed for the in vitro culture of high-quality cartilage tissue to be then transplanted in vivo to enable recovery from congenital malformations in the maxillofacial area or crippling jaw disease. The three-dimensional scaffold for in-plate culture is designed with interface chemistry capable of stimulating cartilage formation and maintaining its structure through counteracting the dedifferentiation of mesenchymal stem cells (MSCs) during the formation of cartilage tissue. The developed interface chemistry enabled high efficiency in both growth rate and tissue quality, thus satisfying the requirements of large volumes, high matrix quality, and superior mechanical properties needed in cartilage transplants. We characterized the cartilage tissue in vitro grown on a NanoCliP-FD gel scaffold by human periodontal ligament-derived stem cells (a type of MSC) with cartilage grown by the same cells and under the same conditions on a conventional (porous) atelocollagen scaffold. The cartilage tissues produced by the MSCs on different scaffolds were comparatively evaluated by immunohistochemical and spectroscopic analyses. Cartilage differentiation occurred at a higher rate when MSCs were cultured on the NanoCliP-FD gel scaffold compared to the atelocollagen scaffold, and produced a tissue richer in cartilage matrix. In situ spectroscopic analyses revealed the cell/scaffold interactive mechanisms by which the NanoCliP-FD gel scaffold stimulated such increased efficiency in cartilage matrix formation. In addition to demonstrating the high potential of human periodontal ligament-derived stem cell cultures on NanoCliP-FD gel scaffolds in regenerative cartilage therapy, the present study also highlights the novelty of Raman spectroscopy as a non-destructive method for the concurrent evaluation of matrix quality and cell metabolic response. In situ Raman analyses on living cells unveiled for the first time the underlying physiological mechanisms behind such improved chondrocyte performance.

Removal of Perfluorooctanoic Acid from Water Using a Hydrophobic Ionic Liquid Selected Using the Conductor-like Screening Model for Realistic Solvents.

The conductor-like screening model for realistic solvents was used to identify ionic liquids (ILs) to efficiently extract perfluorooctanoic acid (PFOA). The infinite dilution chemical potentials of PFOA in 14 000 ILs were calculated and used as descriptors of the chemical affinities between the ILs and PFOA. Trihexyltetradecylphosphonium pivalate ([P6,6,6,14][Piv]) was found to be a good IL for extracting PFOA because it gave a well-balanced combination of a strong chemical attraction for PFOA and useful physicochemical properties. The results of experiments indicated that [P6,6,6,14][Piv] could remove >99.9% of the PFOA in an aqueous solution. However, problematic emulsification of IL in the aqueous phase occurred at PFOA/IL molar ratios <1.9-2.1, and this limited the PFOA removal rate to 80-91%. The ability of the used IL to extract PFOA was found to be partially regenerated by washing the IL with 1% NaOH, and the IL could be reused to extract PFOA with a removal rate decreased by ∼10% in each cycle.

A solid-in-oil-in-water emulsion: An adjuvant-based immune-carrier enhances vaccine effect.

The biomaterial-based immunoengineering has become one of the most attractive research fields in the last decade. In the present study, a solid-in-oil-in-water (S/O/W) emulsion encapsulating antigen in the oil phase of an oil-in-water (O/W) emulsion was prepared as a novel vaccine carrier consisting of similar materials to the emulsion adjuvant of which the safety, immunogenicity and vaccination efficacy have been already confirmed in human. Direct observation by high-resolution confocal laser scanning microscopy and small angle X-ray scattering analysis showed that the antigens were dispersed inside of the oil phase of the S/O/W emulsion as solid-state particles. The S/O/W emulsion robustly produced antigen-specific antibodies and enhanced the antitumor effects in a therapeutic cancer vaccination compared with free antigens or the O/W emulsion in vivo. This result is in good agreement with the activation effect of antigen-specific cytotoxic T lymphocytes and antigen presentation by the S/O/W emulsion, indicating that the S/O/W emulsion consisting of already approved materials is a promising vaccine carrier to produce both humoral and cellular immunity.

Biocompatible Ionic Liquid Enhances Transdermal Antigen Peptide Delivery and Preventive Vaccination Effect.

Ionic liquids (ILs) attract significant attention as novel solvents for drug delivery systems because of their ability to solubilize poorly soluble drugs and tune the physiological properties of active pharmaceutical ingredients. For the next generation of IL-based drug delivery systems, biocompatibility is a high priority. In the current study, choline-fatty acids ([Cho][FA]) were used as a biocompatible IL to mediate the dissolution of a water-soluble antigen peptide in an oil-based skin penetration enhancer. Among the candidate fatty acids (C8, C10, C12, C14, C16, C18:0, and C18:1), C18:1 was selected because of its low cytotoxicity and mediation of skin permeability for an antigen peptide. Using IL[Cho][C18:1] and an oil-based penetration enhancer, the flux of transdermal delivery of the peptide increased 28-fold compared with delivery using an aqueous vehicle. Furthermore, the IL-mediated transcutaneous vaccination succeeded in suppressing tumor growth in vivo compared to injection. The skin irritation produced by this formulation was tested using an in vitro 3D constructed skin tissue model and an in vivo histological study, which concluded that the formulation did not cause skin irritation. The results suggest that biocompatible IL-mediated dissolution in an oil-based skin penetration enhancer is a promising strategy for transdermal drug delivery.

In Vivo Regeneration of Large Bone Defects by Cross-Linked Porous Hydrogel: A Pilot Study in Mice Combining Micro Tomography, Histological Analyses, Raman Spectroscopy and Synchrotron Infrared Imaging.

The transplantation of engineered three-dimensional (3D) bone graft substitutes is a viable approach to the regeneration of severe bone defects. For large bone defects, an appropriate 3D scaffold may be necessary to support and stimulate bone regeneration, even when a sufficient number of cells and cell cytokines are available. In this study, we evaluated the in vivo performance of a nanogel tectonic 3D scaffold specifically developed for bone tissue engineering, referred to as nanogel cross-linked porous-freeze-dry (NanoCliP-FD) gel. Samples were characterized by a combination of micro-computed tomography scanning, Raman spectroscopy, histological analyses, and synchrotron radiation-based Fourier transform infrared spectroscopy. NanoCliP-FD gel is a modified version of a previously developed nanogel cross-linked porous (NanoCliP) gel and was designed to achieve highly improved functionality in bone mineralization. Spectroscopic imaging of the bone tissue grown in vivo upon application of NanoCliP-FD gel enables an evaluation of bone quality and can be employed to judge the feasibility of NanoCliP-FD gel scaffolding as a therapeutic modality for bone diseases associated with large bone defects.

Lactic Acid Permeation through Deep Eutectic Solvents-Based Polymer Inclusion Membranes.

Lactic acid that is prepared by fermentation is a compound in food, cosmetic pharmaceutical, and chemical industries. Since a simple technique is desired that separates lactic acid from the cultures, we propose lactic acid permeation through a poly(vinyl chloride)(PVC)-based membrane that contains deep eutectic solvents (DESs) as a carrier. Lactic acid was successfully permeated through polymer inclusion membranes (PIMs) containing hydrophilic DESs, urea-choline chloride and glucose-choline chloride. The permeation behavior was explained by the facilitated transport mechanism based on the solution-diffusion model. Simple preparation of thinner membranes in the PIM process and higher permeation rates are advantages over the supported liquid membrane process. The PVC-based membrane process containing environmentally benign hydrophilic DESs is promising for lactic acid separation on an industrial scale.

Solid-in-oil nanodispersions as a novel delivery system to improve the oral bioavailability of bisphosphate, risedronate sodium.

The aim of the current study was to modify the oral absorption of risedronate sodium (RS) using solid-in-oil nanodispersions (SONDs) technology. The oral therapeutic effect of RS is limited in vivo because of its low membrane permeability and the formation of insoluble precipitates with bivalent cations (such as Ca2+) in the gastrointestinal (GI) tract.We used SONDs to prepare medium-chain triglyceride (MCT)-based nanodispersions of the hydrophilic drug, which used the oral absorption mechanism of MCT digestion to improve bioavailability of RS in vivo. SONDs exhibited high encapsulation efficiency of RS and excellent enzymatic degradation-dependent release behavior. The result of an everted gut sac test showed that the Papp value of the SONDs was 6.29-fold (p<0.05) higher than that of RS aqueous solutions in simulated intestinal fluid containing 5 mM Ca2+, this was because MCT can be digested to form the fatty acids C8 and C10, which have an adsorption-promoting effect on RS. Further, solid-in-oil-in-water (S/O/W) emulsion droplets formedafter emulsification by bile salts and MCT digestionwere effective in disrupting epithelial tight junctions (TJs), facilitating the paracellular permeation of RS throughout the intestine. Moreover, in vivo absorption study in rats revealed that the AUC0-12h of RS in SONDs was approximately 4.56-fold (p<0.05) higher than with RS aqueous solutions at the same dose (15 mg/kg). This approach demonstrates a potential drug delivery system to improve the bioavailability of risedronate sodium.

Choline and amino acid based biocompatible ionic liquid mediated transdermal delivery of the sparingly soluble drug acyclovir.

Transdermal delivery of drugs is more challenging for drugs that are insoluble or sparingly soluble in water and most organic solvents. To overcome this problem, ionic liquid (IL)-mediated ternary systems have been suggested as potential drug carriers. Here, we report potent ternary (IL-EtOH-IPM) systems consisting of biocompatible ILs, ethanol (EtOH), and isopropyl myristate (IPM) that can dissolve a significant amount of the sparingly soluble drug acyclovir (ACV). The ternary systems were optically transparent and thermodynamically stable with a wide range of IL pertinence. An in vitro drug permeation study showed that the ILs in the ternary systems dramatically enhanced ACV permeation into and across the skin. Fourier Transform Infrared spectroscopy of the stratum corneum (sc) after treatment with ternary systems showed that the skin barrier function was reduced by disturbance of the regularly ordered arrangement of corneocytes and modification of the surface properties of the sc during permeation. Histological analysis, and skin irritation studies using a reconstructed human epidermis model showed the safety profile of the ternary system, and there were no significant changes in the structures of the sc, epidermis, and dermis. Therefore, ternary systems containing biocompatible ILs are promising for transdermal delivery of insoluble or sparingly soluble drugs.

Solid-in-Oil Nanodispersions for Transcutaneous Immunotherapy of Japanese Cedar Pollinosis.

Japanese cedar pollinosis (JCP) is a common affliction caused by an allergic reaction to cedar pollen and is considered a disease of national importance in Japan. Antigen-specific immunotherapy (AIT) is the only available curative treatment for JCP. However, low compliance and persistence have been reported among patients subcutaneously or sublingually administered AIT comprising a conventional antigen derived from a pollen extract. To address these issues, many research studies have focused on developing a safer, simpler, and more effective AIT for JCP. Here, we review the novel antigens that have been developed for JCP AIT, discuss their different administration routes, and present the effects of anti-allergy treatment. Then, we describe a new form of AIT called transcutaneous immunotherapy (TCIT) and its solid-in-oil (S/O) nanodispersion formulation, which is a promising antigen delivery system. Finally, we discuss the applications of S/O nanodispersions for JCP TCIT. In this context, we predict that TCIT delivery by using a S/O nanodispersion loaded with novel antigens may offer an easier, safer, and more effective treatment option for JCP patients.

A Solid-in-Oil Nanodispersion System for Transcutaneous Immunotherapy of Cow's Milk Allergies.

An allergy to cow's milk proteins is the most common food allergy in infants and toddlers. Conventional oral immunotherapy for cow's milk allergies requires hospital admission due to the risk of severe allergic reactions, including anaphylaxis. Therefore, a simpler and safer immunotherapeutic method is desirable. We examined transcutaneous immunotherapy with a solid-in-oil (S/O) system. In the S/O system, nano-sized particles of proteins are dispersed in an oil-vehicle with the assistance of nonionic surfactants. In the present study, the S/O system enhanced the skin permeation of the allergen molecule ß-lactoglobulin (BLG), as compared with a control PBS solution. The patches containing BLG in the S/O nanodispersion skewed the immune response in the allergy model mice toward T helper type 1 immunity, indicating the amelioration of allergic symptoms. This effect was more pronounced when the immunomodulator resiquimod (R-848) was included in the S/O system.

Transcutaneous Cancer Vaccine Using a Reverse Micellar Antigen Carrier.

Skin dendritic cells (DCs) such as Langerhans cells and dermal dendritic cells have a pivotal role in inducing antigen-specific immunity; therefore, transcutaneous cancer vaccines are a promising strategy to prophylactically prevent the onset of a variety of diseases, including cancers. The largest obstacle to delivering antigen to these skin DC subsets is the barrier function of the stratum corneum. Although reverse micellar carriers are commonly used to enhance skin permeability to hydrophilic drugs, the transcutaneous delivery of antigen, proteins, or peptides has not been achieved to date because of the large molecular weight of drugs. To achieve effective antigen delivery to skin DCs, we developed a novel strategy using a surfactant as a skin permeation enhancer in a reverse micellar carrier. In this study, glyceryl monooleate (MO) was chosen as a skin permeation enhancer, and the MO-based reverse micellar carrier enabled the successful delivery of antigen to Langerhans cells and dermal dendritic cells. Moreover, transcutaneous vaccination with the MO-based reverse micellar carrier significantly inhibited tumor growth, indicating that it is a promising vaccine platform against tumors.

Transcutaneous Delivery of Immunomodulating Pollen Extract-Galactomannan Conjugate by Solid-in-Oil Nanodispersions for Pollinosis Immunotherapy.

Japanese cedar pollinosis is a type I allergic disease and has already become a major public health problem in Japan. Conventional subcutaneous immunotherapy (SCIT) and sublingual immunotherapy (SLIT) cannot meet patients' needs owing to the side effects caused by both the use of conventional whole antigen molecules in the pollen extract and the administration routes. To address these issues, a surface-modified antigen and transcutaneous administration route are introduced in this research. First, the pollen extract (PE) was conjugated to galactomannan (PE-GM) to mask immunoglobulin E (IgE)-binding epitopes in the PE to avoid side effects. Second, as a safer alternative to SCIT and SLIT, transcutaneous immunotherapy (TCIT) with a solid-in-oil (S/O) nanodispersion system carrying PE-GM was proposed. Hydrophilic PE-GM was efficiently delivered through mouse skin using S/O nanodispersions, reducing the antibody secretion and modifying the type 1 T helper (Th1)/ type 2 T helper (Th2) balance in the mouse model, thereby demonstrating the potential to alleviate Japanese cedar pollinosis.

Solid-in-oil nanodispersions for intranasal vaccination: Enhancement of mucosal and systemic immune responses.

En masse vaccination is a promising strategy for combatting infectious diseases. Intranasal vaccination is a viable route of mass vaccination, and it could be performed easily via needle-free administration. However, it is not widely used because it tends not to evoke sufficient immunity. The aim of the present study was to improve the performance of intranasal vaccination by extending the amount of time that administered antigens remain in the nasal cavity, and enhancing immune responses via a nanocarrier-based adjuvant. A simple and safe solid-in-oil (S/O) system was investigated as a nanocarrier in intranasal vaccination. S/O nanodispersions are oil-based dispersions of antigens coated with surfactants. Because of the mucoadhesive capacities of surfactant and oil they have high potential to extend the amount of time that administered antigens remain in the nasal cavity, and can induce strong immune responses due to a nanocarrier-based adjuvant effect. In nasal absorption experiments antigens administered intranasally via S/O nanodispersions remained in the nasal cavity longer and induced strong mucosal and systemic immune responses. Histopathology analysis indicated that S/O nanodispersions did not modify the nasal epithelium or cilia, suggesting non-toxicity of the carrier. These results indicate the potential of intranasal vaccination using S/O nanodispersions for future vaccination.

Development of a novel ionic liquid-curcumin complex to enhance its solubility, stability, and activity.

We report a one-step emulsification and rapid freeze-drying process to develop a curcumin-ionic liquid (CCM-IL) complex that could be readily dispersed in water with a significantly enhanced solubility of ∼8 mg mL-1 and half-life (t1/2) of ∼260 min compared with free CCM (solubility ∼30 nM and t1/2 ∼ 20 min). This process using an IL consisting of a long chain carbon backbone as a surfactant, may provide an alternative way of enhancing the solubility of poorly water-soluble drugs.

Synthesis and characterization of choline-fatty-acid-based ionic liquids: A new biocompatible surfactant.

Ionic liquid (IL) surfactants have attracted great interest as promising substitutes for conventional surfactants owing to their exceptional and favorable physico-chemical properties. However, most IL surfactants are not eco-friendly and form unstable micelles, even when using a high concentration of the surfactant. In this study, we prepared a series of halogen-free and biocompatible choline-fatty-acid-based ILs with different chain lengths and degrees of saturation, and we then investigated their micellar properties in aqueous solutions. Characterization of the synthesized surface-active ILs (SAILs) was performed by 1H and 13C nuclear magnetic resonance spectroscopy, Fourier transform infrared spectroscopy, differential scanning calorimetry, and elemental analysis. The surface-active properties of the SAILs were investigated by tensiometry, conductometry, and dynamic light scattering measurements. The critical micelle concentration of the SAILs was found to be 2-4 times lower than those of conventional surfactants. The thermodynamic properties of micellization (ΔG0m, ΔH0m, and ΔS0m) indicate that the micellization process of the SAILs is spontaneous, stable, and entropy-driven at room temperature. The cytotoxicity of the SAILs was evaluated using mammalian cell line NIH 3T3. Importantly, [Cho][Ole] shows lower toxicity than the analogous ILs with conventional surfactants. These results clearly suggest that these environmentally friendly SAILs can be used as a potential alternative to conventional ILs for various purposes, including biological applications.

In vivo biocompatibility, pharmacokinetics, antitumor efficacy, and hypersensitivity evaluation of ionic liquid-mediated paclitaxel formulations.

In order to prevent common hypersensitivity reactions to paclitaxel injections (Taxol), we previously reported an ionic liquid-mediated paclitaxel (IL-PTX) formulation with small particle size and narrow size distribution. The preliminary work showed high PTX solubility in the IL, and the formulation demonstrated similar antitumor activity to Taxol, while inducing a smaller hypersensitivity effect in in vitro cell experiments. In this study, the stability of the IL-PTX formulation was monitored by quantitative HPLC analysis, which showed that IL-PTX was more stable at 4 °C than at room temperature. The in vivo study showed that the IL-PTX formulation could be used in a therapeutic application as a biocompatible component of a drug delivery system. To assess the in-vivo biocompatibility, IL or IL-mediated formulations were administered intravenously by maintaining physiological buffered conditions (neutral pH and isotonic salt concentration). From in vivo pharmacokinetics data, the IL-PTX formulation was found to have a similar systemic circulation time and slower elimination rate compared to cremophor EL mediated paclitaxel (CrEL-PTX). Furthermore, in vivo antitumor and hypersensitivity experiments in C57BL/6 mice revealed that IL-PTX had similar antitumor activity to CrEL-PTX, but a significantly smaller hypersensitivity effect compared with CrEL-PTX. Therefore, the IL-mediated formulation has potential to be an effective and safe drug delivery system for PTX.

Osteogenic response of mesenchymal progenitor cells to natural polysaccharide nanogel and atelocollagen scaffolds: A spectroscopic study.

A natural polysaccharide scaffold, referred to as "freeze-dry nanogel-crosslinked-porous" (FD-NanoCliP) gel, was tested in comparison with an atelocollagen scaffold with respect to osteogenesis versus the mouse mesenchymal progenitor cell line KUSA-A1. The amphiphilic polysaccharide network, engineered in its structure to fit chemically crosslinked nanogels as building blocks into a physically crosslinked porous gel, revealed a superior osteointegrative performance as compared to the soluble atelocollagen network and a peculiar c-plane orientation growth of apatite crystallites, which resembled the structure of natural enamel. Besides evaluating osteogenesis in the FD-NanoCliP gel scaffold, an additional purpose of this study was to assess its chemical composition at the nanoscale and, through its knowledge, to interpret the osteogenic response of mesenchymal cells. In addition to conventional (optical and electron) microscopy and biological evaluation kits, the peculiar chemistry of the FD-NanoCliP gel scaffold and the formation of apatite on it were characterized by means of several independent analytical probes at the molecular scale, which included Raman, cathodoluminescence, energy dispersive X-ray, and X-ray fluorescence spectroscopies. This body of information consistently provided evidence for a peculiar chemistry developed in osteogenesis at the polysaccharide scaffold surface. Such chemistry is not available in soluble atelocollagen and it is key in the superior bioactivity found in the polysaccharide network.

Antigen delivery targeted to tumor-associated macrophages overcomes tumor immune resistance.

Immune checkpoint inhibitors and adoptive transfer of gene-engineered T cells have emerged as novel therapeutic modalities for hard-to-treat solid tumors; however, many patients are refractory to these immunotherapies, and the mechanisms underlying tumor immune resistance have not been fully elucidated. By comparing the tumor microenvironment of checkpoint inhibition-sensitive and -resistant murine solid tumors, we observed that the resistant tumors had low immunogenicity. We identified antigen presentation by CD11b+F4/80+ tumor-associated macrophages (TAMs) as a key factor correlated with immune resistance. In the resistant tumors, TAMs remained inactive and did not exert antigen-presenting activity. Targeted delivery of a long peptide antigen to TAMs by using a nano-sized hydrogel (nanogel) in the presence of a TLR agonist activated TAMs, induced their antigen-presenting activity, and thereby transformed the resistant tumors into tumors sensitive to adaptive immune responses such as adoptive transfer of tumor-specific T cell receptor-engineered T cells. These results indicate that the status and function of TAMs have a significant impact on tumor immune sensitivity and that manipulation of TAM functions would be an effective approach for improving the efficacy of immunotherapies.

Transcutaneous Codelivery of Tumor Antigen and Resiquimod in Solid-in-Oil Nanodispersions Promotes Antitumor Immunity.

Cancer vaccines aim to prevent or inhibit tumor growth by inducing an immune response to tumor-associated antigens (TAAs) encoded by or present in the vaccine. Previous work has demonstrated that effective antitumor immunity can be induced using a codelivery system in which nonspecific immunostimulatory molecules are administered together with TAAs. In this study, we investigated the antitumor effects of a solid-in-oil (S/O) nanodispersion system containing a model TAA, ovalbumin (OVA), and resiquimod (R-848), a small molecular Toll-like receptor 7/8 ligand, which induces an antigen-nonspecific cellular immune response that is crucial for the efficacy of cancer vaccines. R-848 was contained in the outer oil phase of S/O nanodispersion. Analysis of OVA and R-848 permeation in mouse skin after application of an R-848 S/O nanodispersion indicated that R-848 rapidly permeated the skin and preactivated Langerhans cells, resulting in efficient uptake of OVA and migration of antigen-loaded Langerhans cells to the draining lymph nodes. Transcutaneous immunization of mice with an R-848 S/O nanodispersion inhibited the growth of E.G7-OVA tumors and prolonged mouse survival to a greater extent than did immunization with an S/O nanodispersion containing OVA alone. Consistent with this observation, antigen-specific secretion of the Th1 cytokine interferon-γ and cytolytic activity were both high in splenocytes isolated from mice immunized with R-848 S/O. Our results thus demonstrate that codelivery of R-848 significantly amplified the antitumor immune response induced by antigen-containing S/O nanodispersions and further suggest that S/O nanodispersions may be effective formulations for codelivery of TAAs and R-848 in transcutaneous cancer vaccines.