Photoacoustic Imaging Using Polysaccharide-Porphyrin Complexes by Photoirradiation at Long Wavelengths.

Photoacoustic (PA) imaging is a novel biological imaging technique with superior depth resolution compared to fluorescence imaging. The efficacy of PA imaging depends on contrast agents that possess considerable absorbance at longer wavelengths, coupled with high permeability in biological tissue and minimal fluorescence, achieved through mitigating aggregation-caused quenching (ACQ) that attenuates PA intensity. Despite the successful transfer of porphyrin 2 featuring amino moieties from polysaccharides to liposomes, most of 2 incorporated within λ-carrageenan (CGN-2 complex) remained in CGN under acidic lysosomal conditions (pH 5.0). Consequently, the CGN-2 complex exhibited a strong PA signal under 680 nm photoirradiation in Colon26 cells owing to the ACQ of 2. Moreover, the PA intensity of the CGN-2 complex was further enhanced under 780 nm photoirradiation owing to the increased absorbance at 780 nm facilitated by the redshift of the Q-band at pH 5.0.

Theranostic Agent Combining Fullerene Nanocrystals and Gold Nanoparticles for Photoacoustic Imaging and Photothermal Therapy.

Developing photoactivatable theranostic platforms with integrated functionalities of biocompatibility, targeting, imaging contrast, and therapy is a promising approach for cancer diagnosis and therapy. Here, we report a theranostic agent based on a hybrid nanoparticle comprising fullerene nanocrystals and gold nanoparticles (FGNPs) for photoacoustic imaging and photothermal therapy. Compared to gold nanoparticles and fullerene crystals, FGNPs exhibited stronger photoacoustic signals and photothermal heating characteristics by irradiating light with an optimal wavelength. Our studies demonstrated that FGNPs could kill cancer cells due to their photothermal heating characteristics in vitro. Moreover, FGNPs that are accumulated in tumor tissue via the enhanced permeation and retention effect can visualize tumor tissue due to their photoacoustic signal in tumor xenograft model mice. The theranostic agent with FGNPs shows promise for cancer therapy.

Assessment of Surface Glycan Diversity on Extracellular Vesicles by Lectin Microarray and Glycoengineering Strategies for Drug Delivery Applications.

Extracellular vesicles (EVs) are released by all types of mammalian cells for cell-cell communication. In this study, surface glycans on EVs are compared in terms of their cell type, size, and isolation method to examine whether EV glycan profiles by lectin microarray can be used to define EV subpopulations. Moreover, EVs are glycoengineered with four distinctive surface glycan patterns and evaluated their cellular uptake efficiencies for potential drug delivery applications. Both similarities and differences in glycan patterns are identified on EVs obtained under each experimental condition. EV size- and isolation method-dependent lectin-binding patterns are observed. Moreover, cellular uptake behaviors of EVs are affected by EV glycan profiles and acceptor cells. The in vivo biodistribution of EVs is also dependent on their glycan profile. These results suggest that EV surface glycans are a potential novel indicator of EV heterogeneity, and glycoengineering is a useful approach to regulate cell-EV interactions for biomedical applications.

Synergistic anti-tumor efficacy by combination therapy of a self-assembled nanogel vaccine with an immune checkpoint anti-PD-1 antibody.

Therapeutic strategies for cancer involving immune checkpoint inhibitors (ICIs) have been gaining widespread attention, but their efficacy remains limited. Thus, combination of ICI therapies with other therapeutic modalities may be required to improve their outcomes. In this study, we examined the improved efficacy of a CHP nanogel-based vaccine delivery system after combination with ICI therapy. For this, we evaluated the therapeutic efficacy of combining an anti-PD-1 antibody as an ICI with an OVA antigen-complexed CHP nanogel vaccine delivery system in a mouse E.G7-OVA tumor model. Mice were subcutaneously inoculated with E.G7-OVA tumor cells on one side of the back, and subcutaneously injected with OVA or the OVA/CHP nanogel vaccine on the other side of the back. Anti-PD-1 antibody was administered at defined intervals. Tumor volume, immune responses, and tumor-infiltrating cells were evaluated. Mice treated with OVA vaccine alone showed weak tumor suppression compared with untreated control mice. Mice receiving combined OVA/CHP nanogel vaccine and anti-PD-1 antibody therapy exhibited strong tumor growth suppression and markedly improved survival, suggesting that PD-1 signaling blockade by the anti-PD-1 antibody enhanced the anti-tumor efficacy of the OVA vaccine. Furthermore, tumor-infiltrating cells and immune responses were increased in the combined therapy group. No serious side effects were observed for any of the treatments. Taken together, the immune system activation induced by the CHP nanogel vaccine was synergistically enhanced by the anti-PD-1 antibody. The present findings suggest the potential for enhanced therapeutic efficacy by combining the CHP nanogel vaccine delivery system with ICI therapy for various cancer types.

Antigen Delivery to Antigen-Presenting Cells for Adaptive Immune Response by Self-Assembled Anionic Polysaccharide Nanogel Vaccines.

Although current vaccine technology induces sufficient antibody responses to prophylactically ward off viral infections, an anticancer vaccine that directs the patient's immune system to directly fight extant malignant cells will require inducing Th1 and cytotoxic T lymphocyte responses in addition to antibody-mediated activities. Thus, new mechanisms are necessary to deliver antigen to cells in the lymphatic system that will induce these responses. To this end, we have developed a cholesterol-bearing pullulan (CHP) self-assembly nanogel of less than 100 nm, which we have now further modified to be anionic by carboxyl group substitution. Overall, the nanogel-protected antigens during transport to the lymphatic system and converting the vehicle to an anionic charge improved interactions with antigen-presenting cells. We further show that these modified nanogels are a more efficient system for delivering antigen to antigen-presenting cells, particularly langerin-expressing cells, and that this induced significant adaptive immunity. Therefore, we think that this technology could be used to improve anticancer immunotherapies.

Engineered collagen-binding serum albumin as a drug conjugate carrier for cancer therapy.

Serum albumin (SA) is used as a carrier to deliver cytotoxic agents to tumors via passive targeting. To further improve SA's tumor targeting capacity, we sought to develop an approach to retain SA-drug conjugates within tumors through a combination of passive and active targeting. SA was recombinantly fused with a collagen-binding domain (CBD) of von Willebrand factor to bind within the tumor stroma after extravasation due to tumor vascular permeability. Doxorubicin (Dox) was conjugated to the CBD-SA via a pH-sensitive linker. Dox-CBD-SA treatment significantly suppressed tumor growth compared to both Dox-SA and aldoxorubicin treatment in a mouse model of breast cancer. Dox-CBD-SA efficiently stimulated host antitumor immunity, resulting in the complete eradication of MC38 colon carcinoma when used in combination with anti-PD-1 checkpoint inhibitor. Dox-CBD-SA decreased adverse events compared to aldoxorubicin. Thus, engineered CBD-SA could be a versatile and clinically relevant drug conjugate carrier protein for treatment of solid tumors.

Synthesis of oxytocin derivatives lipidated via a carbonate or carbamate linkage as a long-acting therapeutic agent for social impairment-like behaviors.

In the course of our studies of hydrophobic oxytocin (OT) analogues, we newly synthesized lipidated OT (LOT-4a-c and LOT-5a-c), in which a long alkyl chain (C14-C16) is conjugated via a carbonate or carbamate linkage at the Tyr-2 phenolic hydroxy group and a palmitoyl group at the terminal amino group of Cys-1. These LOTs did not activate OT and vasopressin receptors. Among the LOTs, however, LOT-4c, having a C16-chain via a carbonate linkage at the phenolic hydroxyl group of the Tyr-2, showed very long-lasting action for the recovery of impaired social behavior in CD38 knockout mice, a rodent model of autistic phenotypes, whereas the effect of OT itself rapidly diminished. These results indicate that LOT-4c may serve as a potential prodrug in mice.

Structural effects and lymphocyte activation properties of self-assembled polysaccharide nanogels for effective antigen delivery.

The success of immunotherapeutic vaccines is often limited by their inability to activate the cytotoxic T lymphocyte (CTL)-inducing Th1 pathway. We investigated the ability of self-assembled nanogels (CHP or CH-CDex) to activate this pathway, and characterised them chemically and biologically. Once loaded with antigen (ovalbumin, OVA) their OVA encapsulation and dissociation rates suggested the possibility of effective antigen delivery. The DC2.4 dendritic cell line took up either vaccine time-dependently, but both vaccines required CpG DNA for class I MHC presentation. The nanogel vaccines interacted with RAW264.7, a Balb/c mouse-derived macrophage cell line, and co-localised with lysosomes, suggesting their endocytotic internalization in RAW264.7. Both vaccines activated CTLs better than OVA alone. Unlike OVA alone, the nanogel vaccines induced IgG2a antibody production in mice, whereas the former induced IgG1 antibodies. OVA-nanogel delivery to the draining lymph nodes (DLNs) was higher than that for OVA alone, reaching a deeper medullary area. Furthermore, Langerin+ CD103+ DCs interacted with the nanogel vaccines effectively, which is a subset of cross-presentation DC, in the DLNs. The nanogel vaccines each had good anti-tumour efficacy in OVA tumour-bearing mice compared with the OVA alone. Thus, CHP and CH-CDex nanogels should be investigated further because of the great potential they offer for immunotherapy.