Construction of brain-like spheroids containing endothelial tubular networks by an adhesive culture system.

Spheroids which are composed of several types of cells have been widely studied in the pharmaceutical field as their structure and functions are similar to human organs. Three-dimensional brain-like tissues are one of the most important tissues for the development of medicines to treat brain diseases and for in vitro brain models. In this study, spheroids mainly containing neurons, astrocytes, and endothelial cells were fabricated using a novel 3D culture plate, "MicoCell™" to construct a brain mimicking tissue. Due to the multicavity structures of MicoCell, ∼102 of attached spheroids were fabricated in a single plate. Spheroids in MicoCell were attached onto a mild cell adhesive surface, allowing for easy immunostaining and microscopic observation. Spheroid formation was improved by adding a Rho-Kinase inhibitor during cultivation. Endothelial cells formed vascular network structures in spheroids and some parts of the vascular structures attached onto the bottom of a culture plate. Co-culture of multiple cell types required optimization of the culture medium during spheroid formation. The mixture of neural stem cell medium and endothelial growth medium showed good spheroid formation and a vascular network. These results indicated that our culture plates and brain mimicking spheroids would be a suitable candidate for pharmaceutical applications such as drug screening and for in vitro brain models.

3D-cultured small size adipose-derived stem cell spheroids promote bone regeneration in the critical-sized bone defect rat model.

Adipose-derived stem cells (ADSCs), due to their regenerative ability, have beneficial effects on bone and cartilage defects. In addition, spheroid formation of ADSCs obtained using three-dimensional (3D) culture accelerates the regenerative ability of ADSCs. The study investigated the regenerative effect of 3D-cultured small size ADSC spheroids without a scaffold in rats with defects in the critical-sized calvarial bone. ADSC-single cells, ADSC-spheroids, or PBS (as control) were implanted in rats, and radiological and histological assessment of bone regeneration was performed. Bone defects were significantly regenerated in the ADSC-spheroid group compared to that in the control group. ADSC-spheroids also showed the most significant bone regeneration in histological assessment. Immunohistochemistry assessment showed that ADSC-spheroids could survive 12 weeks after cell implantation. In vitro, cell apoptosis in ADSC-spheroids was significantly suppressed compared to that in ADSC-single cells. In addition, gene expression related to bone morphogenesis, angiogenesis, and stemness in ADSC-spheroids was elevated. The scaffold-free 3D-cultured small ADSC-spheroids survived in in vitro and in vivo conditions and promoted bone regeneration. Therefore, injectable small size ADSC-spheroids are a novel and less-invasive therapeutic option for treating bone defects.

Fabrication of Spheroids with Dome-Shaped Endothelial Tube Networks by an Adhesive Culture System.

3D functional tissues, such as spheroids fabricated by mesenchymal stem cells (MSCs), which can mimic parts of tissues and organs, have recently been extensively studied in the fields of regenerative medicine and drug discovery. In this study, spheroids containing endothelial tubular structures are fabricated by use of a novel 3D culture plate, "MicoCell." As MicoCell has a mild cell adhesive surface and multicavity structures, it can provide multiple attached spheroids at the same time (about ≈102 to ≈104 spheroids). Spheroids can be fabricated without using serum, and are easily collected by simple pipetting and no use of enzyme. For the fabrication of spheroids containing endothelial tubular structures, MSCs and endothelial cells are co-cultured with MicoCell. Surprisingly, endothelial tubular structures are found to extend upward from the bottom where the spheroids attach onto, forming a dome-shaped morphology. Notably, some tubular structures in the spheroids have a basement membrane and markedly improved oxygen level of the inner part of spheroids. Moreover, as spheroids attach onto the bottom, they do not require any pre-treatment such as embedding into gel before microscopic observation using an optical clearing reagent. These results indicate that the culture plates will be suitable for clinical and pharmaceutical applications.

3D-Printing of Structure-Controlled Antigen Nanoparticles for Vaccine Delivery.

Targeted delivery of antigens to immune cells using micro/nanocarriers may serve as a therapeutic application for vaccination. However, synthetic carriers have potential drawbacks including cytotoxicity, low encapsulation efficiency of antigen, and lack of a morphological design, which limit the translation of the delivery system to clinical use. Here, we report a carrier-free and three-dimensional (3D)-shape-designed antigen nanoparticle by multiphoton lithography-based 3D-printing. This simple, versatile 3D-printing approach provides freedom for the precise design of particle shapes with a nanoscale resolution. Importantly, shape-designed antigen nanoparticles with distinct aspect ratios show shape-dependent immune responses. The 3D-printing approach for the rational design of nanomaterials with increasing safety, complexity, and efficacy offers an emerging platform to develop vaccine delivery systems and mechanistic understanding.

Micro Vacuum Chuck and Tensile Test System for Bio-Mechanical Evaluation of 3D Tissue Constructed of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes (hiPS-CM).

In this report, we propose a micro vacuum chuck (MVC) which can connect three-dimensional (3D) tissues to a tensile test system by vacuum pressure. Because the MVC fixes the 3D tissue by vacuum pressure generated on multiple vacuum holes, it is expected that the MVC can fix 3D tissue to the system easily and mitigate the damage which can happen by handling during fixing. In order to decide optimum conditions for the size of the vacuum holes and the vacuum pressure, various sized vacuum holes and vacuum pressures were applied to a normal human cardiac fibroblast 3D tissue. From the results, we confirmed that a square shape with 100 µm sides was better for fixing the 3D tissue. Then we mounted our developed MVCs on a specially developed tensile test system and measured the bio-mechanical property (beating force) of cardiac 3D tissue which was constructed of human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CM); the 3D tissue had been assembled by the layer-by-layer (LbL) method. We measured the beating force of the cardiac 3D tissue and confirmed the measured force followed the Frank-Starling relationship. This indicates that the beating property of cardiac 3D tissue obtained by the LbL method was close to that of native cardiac tissue.

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.

Fabrication of Orientation-Controlled 3D Tissues Using a Layer-by-Layer Technique and 3D Printed a Thermoresponsive Gel Frame.

Herein, we report the fabrication of orientation-controlled tissues similar to heart and nerve tissues using a cell accumulation and three-dimensional (3D) printing technique. We first evaluated the 3D shaping ability of hydroxybutyl chitosan (HBC), a thermoresponsive polymer, by using a robotic dispensing 3D printer. HBC polymer could be laminated to a height of 1124 ± 14 µm. Based on this result, we fabricated 3D gel frames of various shapes, such as square, triangular, rectangular, and circular, for shape control of 3D tissue and then normal human cardiac fibroblasts (NHCFs) coated with extracellular matrix nanofilms were seeded in the frames. Observation of shape-controlled tissues after 1 day of cultivation showed that the orientation of fibroblasts was in one direction when a short-sided, thin, rectangular-shaped frame was used. Next, we tried to fabricate orientation-controlled tissue with a vascular network by coculturing NHCF and normal human cardiac microvascular endothelial cells. As a consequence of cultivation for 4 days, observation of cocultured tissue confirmed aligned cells and blood capillaries in orientation-controlled tissue. Our results clearly demonstrated that it would be possible to control the cell orientation by controlling the shape of the tissues by combining a cell accumulation technique and a 3D printing system. The results of this study suggest promising strategies for the fabrication of oriented 3D tissues in vitro. These tissues, mimicking native organ structures, such as muscle and nerve tissue with a cell alignment structure, would be useful for tissue engineering, regenerative medicine, and pharmaceutical applications.

Construction and histological analysis of a 3D human arterial wall model containing vasa vasorum using a layer-by-layer technique.

There is considerable global demand for three-dimensional (3D) functional tissues which mimic our native organs and tissues for use as in vitro drug screening systems and in regenerative medicine. In particular, there has been an increasing number of patients who suffer from arterial diseases such as arteriosclerosis. As such, in vitro 3D arterial wall models that can evaluate the effects of novel medicines and a novel artificial graft for the treatment are required. In our previous study, we reported the rapid construction of 3D tissues by employing a layer-by-layer (LbL) technique and revealed their potential applications in the pharmaceutical fields and tissue engineering. In this study, we successfully constructed a 3D arterial wall model containing vasa vasorum by employing a LbL technique for the first time. The cells were coated with extracellular matrix nanofilms and seeded into a culture insert using a cell accumulation method. This model had a three-layered hierarchical structure: a fibroblast layer, a smooth muscle layer, and an endothelial layer, which resembled the native arterial wall. Our method could introduce vasa vasorum into a fibroblast layer in vitro and the 3D arterial wall model showed barrier function which was evaluated by immunostaining and transendothelial electrical resistance measurement. Furthermore, electron microscopy observations revealed that the vasa vasorum was composed of single-layered endothelial cells, and the endothelial tubes were surrounded by the basal lamina, which are known to promote maturation and stabilization in native blood capillaries. These models should be useful for tissue engineering, regenerative medicine, and pharmaceutical applications. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 814-823, 2017.

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.

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.

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.

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.

Comparative activity of biodegradable nanoparticles with aluminum adjuvants: antigen uptake by dendritic cells and induction of immune response in mice.

Biodegradable poly(γ-glutamic acid) (γ-PGA) nanoparticles (NPs) are considered to be an excellent antigen carrier. Antigen-carrying γ-PGA NPs were examined for their uptake by murine dendritic cells (DCs) and subsequent induction of antigen-specific immune responses in mice and compared with aluminum (AL) adjuvants. Ovalbumin (OVA)-carrying NPs (FITC-OVA-NPs) were taken up much more efficiently by DCs than OVA alone or its AL-associated form. Both OVA-NPs and OVA+AL were detected in an intracellular lysosome compartment of DCs. Furthermore, the uptake of γ-PGA NPs was inhibited in the presence of pinocytosis and phagocytosis inhibitors. Significantly higher induction of antigen-specific CD8(+) T cells was observed in mice immunized with OVA-carrying γ-PGA NPs than in those immunized with OVA alone, OVA+AL, OVA+3-O-desacyl-4'-monophosphoryl lipid A (MPL), and OVA+AL+MPL. Thus, γ-PGA NPs may have great potential as an effective vaccine carrier and adjuvant for clinical use.

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.