Fumiaki Shima

Intracellular degradation and distribution of protein-encapsulated amphiphilic poly(amino acid) nanoparticles.

Physicochemical properties, such as particle size, shape, molecular weight, surface charge and composition, play a key role in the cellular uptake of polymeric nanoparticles. Antigen-encapsulated biodegradable nanoparticles have considerable potential for use in vaccine delivery systems. Although it is accepted that particle size is important for the induction of antigen-specific immune responses in vivo, little is known about how their size affects their intracellular fate. Here, we demonstrate that the size effects on the cellular uptake, intracellular degradation and distribution of protein-encapsulated nanoparticles. We prepared size-regulated ovalbumin (OVA)-encapsulated nanoparticles composed of hydrophobically modified poly(γ-glutamic acid) (γ-PGA). These nanoparticles were efficiently taken up by macrophages, and also delivered encapsulated OVA from the endosomes to the cytoplasm. Comparing 40-200 nm-sized nanoparticles, there was no significant difference in their intracellular distribution. Interestingly, the size of the nanoparticles affected the intracellular degradation of the encapsulated OVA. The uptake of OVA alone by macrophages resulted in early degradation of the OVA. In contrast, the degradation of OVA encapsulated into the nanoparticles was attenuated as compared to free OVA. A difference in OVA degradation kinetics was observed between the particle sizes, the degradation of small nanoparticles was slower than for the larger ones. These results indicate that particle size is an important factor for the intracellular degradation of encapsulated proteins and nanoparticles. These results will provide a rational design of nanoparticle-based vaccines to control immune responses.

Manipulating the antigen-specific immune response by the hydrophobicity of amphiphilic poly(γ-glutamic acid) nanoparticles

The new generation vaccines are safe but poorly immunogenic, and thus they require the use of adjuvants. However, conventional vaccine adjuvants fail to induce potent cellular immunity, and their toxicity and side-effects hinder the clinical use. Therefore, a vaccine adjuvant which is safe and can induce an antigen-specific cellular immunity-biased immune response is urgently required. In the development of nanoparticle-based vaccine adjuvants, the hydrophobicity is one of the most important factors. It could control the interaction between the encapsulated antigens and/or nanoparticles with immune cells. In this study, nanoparticles (NPs) composed of amphiphilic poly(γ-glutamic acid)-graft-L-phenylalanine ethyl ester (γ-PGA-Phe) with various grafting degrees of hydrophobic side chains were prepared to evaluate the effect of hydrophobicity of vaccine carriers on the antigen encapsulation behavior, cellular uptake, activation of dendritic cells (DCs), and induction of antigen-specific cellular immunity-biased immune responses. These NPs could efficiently encapsulate antigens, and the uptake amount of the encapsulated antigen by DCs was dependent on the hydrophobicity of γ-PGA-Phe NPs. Moreover, the activation potential of the DCs and the induction of antigen-specific cellular immunity were correlated with the hydrophobicity of γ-PGA-Phe NPs. By controlling the hydrophobicity of antigen-encapsulated γ-PGA-Phe NPs, the activation potential of DCs was able to manipulate about 5 to 30-hold than the conventional vaccine, and the cellular immunity was about 10 to 40-hold. These results suggest that the hydrophobicity of NPs is a key factor for changing the interaction between NPs and immune cells, and thus the induction of cellular immunity-biased immune response could be achieved by controlling the hydrophobicity of them.

Size effect of amphiphilic poly(γ-glutamic acid) nanoparticles on cellular uptake and maturation of dendritic cells in vivo.

We prepared size-regulated nanoparticles (NPs) composed of amphiphilic poly(γ-glutamic acid) (γ-PGA). In this study, 40, 100 and 200 nm γ-PGA-graft-l-phenylalanine ethylester (γ-PGA-Phe) NPs were employed. The size of NPs significantly influenced the uptake and activation behaviors of antigen-presenting cells (APCs). When 40 nm γ-PGA-Phe NPs were applied to these cells in vitro, they were highly activated compared with 100 and 200 nm NPs, while cellular uptake was size dependent. The size of the γ-PGA-Phe NPs also significantly affected their migration to the lymph nodes and uptake behavior of NPs by dendritic cells (DCs) in vivo. The 40 nm γ-PGA-Phe NPs migrated more rapidly to the lymph nodes and were taken up by a greater number of DCs compared with 100 and 200 nm NPs. On the other hand, when the amount of γ-PGA-Phe NPs taken up per DC was evaluated, it was higher for 100 and 200 nm NPs than for 40 nm NPs, which suggests that the larger γ-PGA-Phe NPs can deliver a large amount of antigen to a single DC compared with smaller NPs. Furthermore, when examined the maturation of DCs in lymph nodes, 40 nm γ-PGA-Phe NPs efficiently stimulated DCs. These results suggest that the activation, uptake behavior by APCs, migration to lymph nodes, and DC maturation can be controlled by the size of γ-PGA-Phe NPs.

Synergistic stimulation of antigen presenting cells via TLR by combining CpG ODN and poly(γ-glutamic acid)-based nanoparticles as vaccine adjuvants.

CpG oligodeoxynucleotide (ODN) encapsulated poly(γ-glutamic acid)-graft-l-phenylalanine ethyl ester (γ-PGA-Phe) nanoparticles (NPs) employing polycations were prepared to develop vaccine delivery and adjuvant systems. The CpG ODN was stably encapsulated into the NPs when protamine was used as the polycation. The CpG ODN-encapsulated γ-PGA-Phe NPs were taken up by macrophages and CpG ODN which was encapsulated into the NPs internalized into endo/lysosomes, where the toll-like receptor (TLR) 9, which recognizes CpG ODN, is expressed. The examination of release behavior in vitro revealed that the encapsulated CpG ODN into NPs was released when these NPs were immersed into the early endosomal environment. Interestingly, CpG ODN-encapsulated γ-PGA-Phe NPs synergistically activated macrophages. This may be due to the multiple stimulation of TLRs by γ-PGA-Phe NPs (TLR4 ligand) and CpG ODN (TLR9 ligand). We previously reported that γ-PGA-Phe NPs are excellent vaccine adjuvants for inducing potent innate and adaptive immune responses via TLR4. Moreover, coencapsulated CpG ODN and antigen in γ-PGA-Phe NPs induced potent antigen-specific cellular immunity at a higher level than the mixture of CpG ODN and antigen which is the conventional vaccine system. These findings suggest that the conjugation strategies of biologically derived adjuvant and polymeric NPs will aid the development of a novel approach for safe and effective vaccine delivery and adjuvant systems.

Uptake of biodegradable poly(γ-glutamic acid) nanoparticles and antigen presentation by dendritic cells in vivo.

Poly(γ-glutamic acid) (γ-PGA) nanoparticles (NPs) carrying antigens have been shown to induce potent antigen-specific immune responses. However, in vivo delivery of γ-PGA NPs to dendritic cells (DCs), a key regulator of immune responses, still remains unclear. In this study, γ-PGA NPs were examined for their uptake by DCs and subsequent migration from the skin to the regional lymph nodes (LNs) in mice. After subcutaneous injection of fluorescein 5-isothiocyanate (FITC)-labeled NPs or FITC-ovalbumin (OVA)-carrying NPs (FITC-OVA-NPs), DCs migrated from the skin to the LNs and maturated, resulting in the upregulation of the costimulatory molecules CD80 and CD86 and the chemokine receptor CCR7. However, the migrated DCs were not detected in the spleen. FITC-OVA-NPs were found to be taken up by skin-derived CD103(+) DCs, and the processed antigen peptides were cross-presented by the major histocompatibility complex (MHC) class I molecule of DCs. Furthermore, significant activation of antigen-specific CD8(+) T cells was observed in mice immunized with OVA-carrying NPs (OVA-NPs) but not with OVA alone or OVA with an aluminum adjuvant. The antigen-specific CD8(+) T cells were induced within 7 days after immunization with OVA-NPs. Thus, γ-PGA NPs carrying various antigens may have great potential as an antigen-delivery system and vaccine adjuvant in vivo.

Effect of Hydrophobic Side Chains in the Induction of Immune Responses by Nanoparticle Adjuvants Consisting of Amphiphilic Poly(γ-glutamic acid)

The new generation vaccines are safe but poorly immunogenic, and thus they require the use of adjuvants. Adjuvants that can control the balance and induction level of cellular and humoral immunities are urgently required for the treatment of and/or protection from infectious diseases and cancers. However, there are no adjuvants which can achieve these requirements. In this study, amphiphilic poly(γ-glutamic acid) (γ-PGA) with various kinds of hydrophobic amino acid ethyl esters (AAE) was synthesized (γ-PGA-AAE) and used to prepare antigen-encapsulated nanoparticles (NPs). γ-PGA-graft-Leu (γ-PGA-Leu, where Leu = leucine ethyl ester), γ-PGA-graft-Phe (γ-PGA-Phe, where Phe = phenylalanine ethyl ester), and γ-PGA-graft-Trp (γ-PGA-Trp, where Trp = tryptophan ethyl ester) formed monodispersed NPs that encapsulated ovalbumin (OVA). The type and the induction level of the antigen-specific cellular and humoral immunities could be controlled by the kinds of hydrophobic segments and vaccine formulation (encapsulation or mixture) used. When OVA was encapsulated into NPs, the cellular immunity was dominantly induced, while humoral immunity was dominant when OVA was mixed with NPs. These results are a first report to demonstrate that the balance and induction level of cellular and humoral immunities could be controlled by modifying compositions of NPs and vaccine formulation. Our results suggest that γ-PGA-AAE NPs can provide safe and efficient nanoparticle-based vaccine adjuvants, and the results also provide guidelines in the rational design of amphiphilic polymers as vaccine adjuvants which can control the balance of immune responses.

The role of hydrophobicity in the disruption of erythrocyte membrane by nanoparticles composed of hydrophobically modified poly(γ-glutamic acid)

Polymeric nanoparticles (NPs) prepared from biocompatible polymers have been studied extensively as carriers for the targeted and controlled delivery of antigens to develop safe and effective vaccines. Especially, the endosomal escape of antigens is essential for the induction of antigen-specific potent immune responses, and the NPs which can control the endosomal escape are urgently required. It has been reported that the hydrophobicity of polymers affected the interactions between the polymer and the membranes, but there have no reports about investigating the effect of the hydrophobicity of the NPs on the membrane disruptive property. In this study, we evaluated the effect of hydrophobicity of NPs on the membrane disruptive property for the first time. We prepared NPs composed of amphiphilic poly(γ-glutamic acid) (γ-PGA) with various grafting degrees of hydrophobic backbone (43–71%), and evaluated the membrane disruptive property. These NPs showed membrane disruptive activity only at the endosomal pH range, and this activity was dependent on the hydrophobicity of γ-PGA. The dependency of the membrane disruptive property on the hydrophobicity of NPs was due to the surface hydrophobicity of them. Our results could provide a guideline for the rational design of amphiphilic polymers as nanoparticle-based vaccine carriers.

Biodegradable nanoparticles composed of enantiomeric poly(γ-glutamic acid)- graft -poly(lactide) copolymers as vaccine carriers for dominant induction of cellular immunity

The design of particulate materials with controlled degradation at desired sites is important in applications for drug/vaccine/gene delivery systems. Amphiphilic biodegradable polymeric nanoparticles are promising vaccine delivery carriers due to their ability to stably maintain antigens, provide tailored release kinetics, effectively target, and function as adjuvants. In this study, we report that stereocomplex nanoparticles (SC NPs) composed of enantiomeric poly(γ-glutamic acid)-graft-poly(lactide) (γ-PGA-PLA) copolymers are excellent protein delivery carriers for vaccines that can deliver antigenic proteins to dendritic cells (DCs) and elicit potent immune responses. We prepared ovalbumin (OVA)-encapsulated γ-PGA-PLA SC NPs (OVA-SC NPs) and isomer NPs. These NPs were efficiently taken up by DCs and also affected the intracellular degradation of the encapsulated OVA. The degradation of OVA encapsulated into the SC NPs was attenuated as compared to free OVA and the corresponding isomer NPs. Interestingly, immunization with OVA-SC NPs predominantly induced antigen-specific cellular immunity. The crystalline structure of inner NPs consisting of PLA had a significant impact on the degradation profiles of NPs and the release/degradation behavior of encapsulated antigens and thus the efficiency of immune induction. Our findings suggest that the γ-PGA-PLA SC NPs are suitable for protein-based vaccines that are used to induce cellular immunity, such as for infectious diseases, cancer, allergies and autoimmune diseases.

The hydrophobic effect of nanoparticles composed of amphiphilic poly(γ-glutamic acid) on the degradability of the encapsulated proteins

For the development of safe and effective next-generation vaccine carriers, their physicochemical properties (size, shape, surface charge, and hydrophobic/hydrophilic balance) are crucial to control their interactions (cellular uptake, intracellular degradability of the loaded antigen, and intracellular localization) with immune cells. Recently, the hydrophobicity of carriers affected the cellular uptake and immune response, which demonstrated that hydrophobicity is one of the most important factors to control the behaviors of the loaded antigens and carriers. In this study, we investigated the effect of the hydrophobicity of nanoparticles (NPs) composed of amphiphilic poly(γ-glutamic acid)-graft-phenylalanine ethyl ester (γ-PGA-Phe) with various grafting degrees of hydrophobic side chains on cellular uptake of the encapsulated antigens, their degradability, and their release behavior in the endosomal environment. These NPs could encapsulate proteins, and the degradability of the encapsulated proteins was changed by the hydrophobicity of NPs. On the other hand, the release behavior of the encapsulated proteins was not changed by the hydrophobicity of NPs. These results suggest that the intracellular behaviors of the encapsulated protein could be controlled by the hydrophobicity of NPs, and could result in the manipulation of the antigen-specific immune responses.

Engraftment and morphological development of vascularized human iPS cell-derived 3D-cardiomyocyte tissue after xenotransplantation

One of the major challenges in cell-based cardiac regenerative medicine is the in vitro construction of three-dimensional (3D) tissues consisting of induced pluripotent stem cell-derived cardiomyocyte (iPSC-CM) and a blood vascular network supplying nutrients and oxygen throughout the tissue after implantation. We have successfully built a vascularized iPSC-CM 3D-tissue using our validated cell manipulation technique. In order to evaluate an availability of the 3D-tissue as a biomaterial, functional morphology of the tissues was examined by light and transmission electron microscopy through their implantation into the rat infarcted heart. Before implantation, the tissues showed distinctive myofibrils within iPSC-CMs and capillary-like endothelial tubes, but their profiles were still like immature. In contrast, engraftment of the tissues to the rat heart led the iPSC-CMs and endothelial tubes into organization of cell organelles and junctional apparatuses and prompt development of capillary network harboring host blood supply, respectively. A number of capillaries in the implanted tissues were derived from host vascular bed, whereas the others were likely to be composed by fusion of host and implanted endothelial cells. Thus, our vascularized iPSC-CM 3D-tissues may be a useful regenerative paradigm which will require additional expanded and long-term studies.