A dual fluorescent reverse targeting drug delivery system based on curcumin-loaded ovalbumin nanoparticles for allergy treatment.

A reverse targeting drug delivery based on antigen-modified nanoparticles provided an innovative strategy for effectively alleviating or inhibiting immune response. In this study, a dual fluorescent reverse targeting drug delivery system based on curcumin-loaded ovalbumin nanoparticles is developed for allergy treatment. The self-crosslinked ovalbumin nanoparticles achieved the double function of reverse targeting and sustained delivery carriers to maximize the anti-allergy of curcumin. Using a murine model of ovalbumin-induced allergy, this drug delivery system suppressed antigen-specific IgG1 and IgE production, inhibited CD4+ T activity, and decreased the level of ovalbumin-sensitized memory B cells. The curcumin-loaded ovalbumin nanoparticles exert stronger and more effective treatment on the immunomodulatory role. Furthermore, fluorescence imaging in vivo can reveal the precise delivery process for the effective allergy immunotherapy. The dual fluorescent reverse targeting delivery system provided a significant potential for the visible treatment of allergy with the merged functions of targeting, vehicles and fluorescence.

Photothermally triggered disassembly of a visible dual fluorescent poly(ethylene glycol)/α-cyclodextrin hydrogel.

The real-time tracking and adjustment of the disassembly and status of hydrogels in vivo are important challenges to accurate and precise assessment. In this article, a photothermally controllable, visible, dual fluorescent thermosensitive hydrogel was designed and developed based on a porphyrin-poly(ethylene glycol)/IR-820-α-cyclodextrin hydrogel. Due to the photothermal effect and fluorescence emission of IR-820, it can exert the dual functions of photothermal control and fluorescence imaging tracking. The IR-820 conjugated hydrogel can regulate the hydrogel disassembly by the photothermal effect of IR-820. Furthermore, each component of the hydrogel can be tracked by the fluorescence of IR-820 and porphyrin. Fluorescence imaging tracking and remote photothermal control were merged into the visible and controlled hydrogel disassembly after subcutaneous injection using mice as models. The dual fluorescence imaging visualization of cyclodextrin/poly(ethylene glycol) hydrogels revealed the disassembly process by tracking each component, and the hydrogel disassembly can be efficiently accelerated under laser irradiation with the photothermal effect of IR-820. This affords an important basis for understanding the disassembly process of the poly(ethylene glycol)/α-cyclodextrin hydrogel.

An "IgG-hitchhiking" approach for rapid tumor accumulation and clearance of photosensitizers.

Photodynamic therapy (PDT) has been widely used for the local treatment of a variety of cancer. To improve the therapeutic effect, delicate nanoparticles loading photosensitizers (PSs) have been designed to improve the accumulation of PSs in tumor. Different from the anti-cancer drugs for chemotherapy or immunotherapy, the delivery of PSs requires rapid tumor accumulation followed by quick elimination to reduce the potential risk of phototoxicity. However, owing to the nature of prolonged blood circulation of the nanoparticles, the conventional nanoparticulate delivery systems may decelerate the clearance of PSs. Here, we present a tumor-targeted delivery approach termed "IgG-hitchhiking" strategy through a self-assembled PSs nanostructure, according to the intrinsic binding between the photosensitizer pheophorbide A (PhA) and immunoglobulin (IgG). We utilize the intravital fluorescence microscopic imaging to uncover that the nanostructures (IgG:PhA NPs) increase the extravasation of PhA into tumor within the first hour post intravenous injection compared with free PhA, correlating with an improved efficacy of PDT. After ∼1 h post-injection, a quick decrease in the PhA amount in the tumor is observed, while the tumor IgG level is continuously increasing. The disparity of the tumor distribution between PhA and IgG allows the quick elimination of the PSs for minimized skin phototoxicity. Our results provide a direct evidence of the enhanced accumulation and elimination of the PSs in the tumor microenvironment through the "IgG-hitchhiking" approach. This strategy presents a promising tumor-targeted delivery approach for the PSs in lieu of the existing strategy for enhanced PDT with minimal toxicity in clinic.

A rationally designed cancer vaccine based on NIR-II fluorescence image-guided light-triggered remote control of antigen cross-presentation and autophagy.

Cancer vaccines represent a promising immunotherapeutic treatment modality. The promotion of cross-presentation of extracellular tumor-associated antigens on the major histocompatibility complex (MHC) class I molecules and dendritic cell maturation at the appropriate time and place is crucial for cancer vaccines to prime cytolytic T cell response with reduced side effects. Current vaccination strategies, however, are not able to achieve the spatiotemporal control of antigen cross-presentation. Here, we report a liposomal vaccine loading the second near-infrared window (NIR-II, 1000-1700 nm) fluorophore BPBBT with an efficient photothermal conversion effect that offers an NIR-light-triggered endolysosomal escape under the imaging guidance. The NIR-II image-guided vaccination strategy specifically controls the cytosolic delivery of antigens for cross-presentation in the draining lymph nodes (DLNs). Moreover, the photothermally induced endolysosomal rupture initiates autophagy. We also find that the adjuvant simvastatin acts as an autophagy activator through inhibiting the PI3K/AKT/mTOR pathway. The light-induced autophagy in the DLNs together with simvastatin treatment cooperatively increase MHC class II expression by activating autophagy machinery for dendritic cell maturation. This study presents a paradigm of NIR-II image-guided light-triggered vaccination. The approach for remote control of antigen cross-presentation and autophagy represents a new strategy for vaccine development.

Pheophorbide A-Mediated Photodynamic Therapy Potentiates Checkpoint Blockade Therapy of Tumor with Low PD-L1 Expression.

Although the immune checkpoint blockade (ICB) has made a great success in cancer immunotherapy, the overall response rate to the ICB, such as anti-programmed death ligand 1 (PD-L1) therapy, remains only at 20-30%. One major reason is the low expression level of the immune checkpoint in a certain type of tumor cells and its insufficient activation of the host immune system. Herein, we reported a cyclic RGD (cRGD)-modified liposomal delivery system loading the anti-PD-L1 antibody and the photosensitizer pheophorbide A (Pa), allowing a targeting of the low PD-L1 expressing 4T1 mouse breast cancer cells through the recognition of an overexpression of αvß3 integrin on the tumor cells. The Pa-mediated photodynamic therapy (PDT) elevated the expression of PD-L1 on the tumor cells. PDT, in combination with the anti-PD-L1 therapy, promoted the activation and maturation of dendritic cells as well as the infiltration of cytotoxic T lymphocytes, resulting in the augmented antitumor immune response for the enhanced therapeutic effect. These results demonstrated the combined therapeutic effects of PDT and ICB on the tumor with low PD-L1 levels. Our study suggested that an increase in the PD-L1 expression in tumor cells by PDT would be a promising adjuvant treatment to overcome the ICB irresponsiveness.

Hydrogel/nanoparticles-mediated cooperative combination of antiangiogenesis and immunotherapy.

Vascular abnormalities are directly related to the tumor immunosuppressive microenvironment, which is an important obstacle to effective immunotherapy. The combination of antiangiogenesis therapy and immunotherapy may promote a mutually reinforcing cycle of immune reprogramming and vascular normalization to increase the effectiveness of immunotherapy. Herein, a hydrogel/nanosystem-mediated antiangiogenesis combined immunotherapy strategy was used to regulate the tumor microenvironment by the controlled release of apatinib, CD47 antibody (aCD47), and CpG. The combination of hydrogel with nanoparticles protected drug activity and maintained a long-term slow release of the drug for maximum synergistic efficacy. Apatinib promotes vascular normalization in tumors and enhances the efficacy of aCD47-based immunotherapy. The addition of immunoadjuvant CpG further enhanced antigen presentation and stimulated the anti-tumor activity of macrophages to strengthen the efficacy of antiangiogenesis combined immunotherapy. The main effector immune cells, including CD4+ T, CD8+ T, NK, and activity DCs, were significantly increased after combination treatment, while the proportion of various immunosuppressive cells decreased significantly, especially MDSCs and M2-polarized macrophages. Based on an effective systemic immune response, the hydrogel/nanoparticle-mediated cooperative combination of antiangiogenesis and immunotherapy enhanced the synergistic effect for primary tumors and prevented metastasis for tumor treatment. The biomaterial-mediated antiangiogenesis combined immunotherapy strategy is a promising strategy for effective immunotherapy. STATEMENT OF SIGNIFICANCE: Relieving immunosuppression of the tumor microenvironment is the key to restoring and rebuilding the normal anti-tumor immune defense of the body. Vascular abnormalities are directly related to the tumor immunosuppressive microenvironment, which is an important obstacle to effective immunotherapy. The combination of antiangiogenesis and immunotherapy may promote a mutually reinforcing cycle of immune reprogramming and vascular normalization to increase the effectiveness of immunotherapy. For the combination of antiangiogenesis and immunotherapy, effective drug delivery to overcome local immune tolerance and regulate the tumor microenvironment to increase therapeutic effects is an important issue. The hydrogel/nanomaterial composite system constructs a dual sustained-release system to achieve step-by-step controlled release of antiangiogenic drugs and immune immunotherapy drugs to promote cooperative combination therapy.

Hydrogel/nanoadjuvant-mediated combined cell vaccines for cancer immunotherapy.

Combined cell vaccines of tumor whole cells and dendritic cells (DCs) provide an effective individualized immunotherapy for malignant tumors. We propose an innovative strategy termed "biomaterial-mediated combined cell vaccines for immunotherapy," which combines tumor cell and DC vaccines with a cyclodextrin-polyethylene glycol hydrogel and a cytosine-phosphate-guanine (CpG) nanoadjuvant. The nanoadjuvant promotes antigen presentation and amplifies immune-eliciting potency by co-delivery of antigens and adjuvants. The hydrogel scaffold provides a better growth microenvironment for injected exogenous DCs and recruits endogenous DCs to maintain their viability for synergistic effect. The results indicated that, relative to live tumor cells, the immunogenically dying tumor cells activated DC maturation effectively with the auxiliary effect of immune adjuvant CpG nanoparticles. The increased T cell percentage, proliferation ability, cytokine secretion, and cytotoxic effect revealed the enhanced immunogenicity of the combined cell vaccines. The combined hydrogel/nanoadjuvant system showed the best efficiency in inhibiting tumor growth. Moreover, vaccination with a single dose of hydrogel-based combined vaccines significantly delayed the development of tumors. The biomaterial-mediated combined cell vaccines remarkably increased the infiltration of effector T cells, alleviated the intratumoral immunosuppressive microenvironment, and maximized the immune effect of the vaccines, thus improving cancer immunotherapy. STATEMENT OF SIGNIFICANCE: Cell-based vaccines, including tumor whole-cell vaccine or DC vaccine, have attracted wide attention as an effective method for cancer immunotherapy. However, it is difficult to gain satisfactory outcomes in clinical trials because of the low immunogenicity of tumor whole cell vaccine and the short-term survival of transferred DC vaccine. Therefore, improving the ability of cell-based vaccines to induce a strong and durable immune response is the primary objective for vaccine development. Biomaterial-mediated combined cell vaccines is an innovative strategy for cancer immunotherapy. The combined hydrogel/ nanoadjuvant system comprises immunogenically dying tumor cells, DCs, and nanoadjuvants. Nanoadjuvant-loaded immunogenically dying tumor cells can induce efficient immune response as the tumor cell vaccine. The hydrogel-based combined tumor cell/DC vaccine could be used for individualized immunotherapy.

Dual fluorescence imaging-guided programmed delivery of doxorubicin and CpG nanoparticles to modulate tumor microenvironment for effective chemo-immunotherapy.

Chemo-immunotherapy has gained increasing attention as one of most promising combination therapy strategies to battle against malignant cancer. In order to achieve a more effective synergistic chemo-immunotherapy and explain the process and mechanism of action, it is an attractive idea to merge chemo-immunotherapy with imaging-guidance and biomaterials assistance. Herein, we designed a dual fluorescence imaging-guided programmed delivery system including doxorubicin and CpG nanoparticles to modulate tumor microenvironment for effective chemo-immunotherapy. CpG self-crosslinking nanoparticles from a hydrogel ensured the long-lasting immune stimulating effect compared to the direct delivery of doxorubicin from the hydrogel. Chemotherapy drug and immunoadjuvant were co-delivery with spatio-temporal release. The immune cells from tumor microenvironment were further analyzed to reveal the possible mechanism of chemo-immunotherapy including cytotoxic CD8+ T lymphocytes, myeloid-derived suppressor cells and M2-like tumor-associated macrophages. Based on the co-stimulation of doxorubicin and CpG nanoparticles, the tumor microenvironment was positively regulated toward tumor-suppressive condition to generate stronger immune response for efficient chemo-immunotherapy. Moreover, dual fluorescence imaging-guided programmed delivery was tracked by own fluorescence of doxorubicin and genipin crosslinking CpG nanoparticles, respectively. Fluorescence imaging-guided programmed delivery of doxorubicin and CpG nanoparticles revealed the dynamic process of chemo-immunotherapy, providing a promising strategy for premise cancer therapy.

Fluorescence imaging guided CpG nanoparticles-loaded IR820-hydrogel for synergistic photothermal immunotherapy.

As synergistic photothermal immunotherapy has developed as one of the most attractive strategies for cancer therapy, it is crucial to design an effective photothermal immunotherapy system to enhance the synergistic anti-tumor effect and reveal the essential role of each treatment. In this study, we designed CpG self-crosslinked nanoparticles-loaded IR820-conjugated hydrogel with dual self-fluorescence to exert the combined photothermal-immunotherapy. IR820-hydrogel can be effective for hyperthermia to eliminate the primary tumor based on its comprehensive coverage and generated photothermal-induced tumor antigens for assisted immunotherapy. CpG self-crosslinked nanoparticles improved the immune response of adjuvant against melanoma without extra nano-carriers. The synergistic photothermal immunotherapy was achieved by the merging of CpG self-crosslinked nanoparticles and IR820-hydrogel. A possible mechanism of combined antitumor effect was further revealed by analyzing immune cells including CD8 +T cells, DCs, B cells, Treg and MDSC in tumor microenvironment. The specific antitumor immunity was provoked to remove the tumor residues and ultimately the combined treatment mode achieved more effective systemic therapeutic effect than either photothermal therapy or immunotherapy alone. Furthermore, self-fluorescent IR820-hydrogel and CpG nanoparticles exerted the imaging-guided combined photothermal-immunotherapy by the dual fluorescence imaging method without additional fluorescent labeling. This visible combined photothermal-immunotherapy offers a potential for precise cancer treatment.

A Visible Codelivery Nanovaccine of Antigen and Adjuvant with Self-Carrier for Cancer Immunotherapy.

Codelivery nanovaccines of antigens and adjuvants have achieved positive therapy for cancer immunotherapy. The insufficient immunogenicity of these vaccines leads to the difficulty of eliciting robust immune effects for immune clearance due to the inadequate loading efficiency, complex preparation processes, low safety concerns, and weak immune responses. Herein, a visible codelivery nanovaccine of an antigen and adjuvant based on self-cross-linked antigen nanoparticles (ovalbumin nanoparticles (ONPs)) combined with the adjuvant (CpG) for cancer immunotherapy was prepared using antigens themselves as carriers. ONPs not only provide sufficient antigens for continuous simulation of the immune response with high antigen loading efficiency but also serve as natural carriers of CpG. In vitro and in vivo experiments proved that ONPs-CpG can elicit a robust immune response including DC maturity, T cell activation, and IFN-γ production. ONPs-CpG induced strong tumor-specific immunity and exhibited remarkable antitumor immunotherapy effects in vivo using mouse models of lymphoma. Furthermore, to perform the precise vaccine delivery, the dual fluorescent codelivery nanovaccine was monitored in real time in vivo by the visible imaging method. With regard to migration tracking, fluorescence imaging allowed for both high resolution and sensitivity of visible detection based on the fluorescence of ONPs and CpG. The multifunctional nanovaccine could function as a robust platform for cancer immunotherapy and a visible system for antigen-adjuvant tracking.

In Vivo Imaging Tracking and Immune Responses to Nanovaccines Involving Combined Antigen Nanoparticles with a Programmed Delivery.

Combined nanovaccine can generate robust and persistent antigen-specific immune responses. A combined nanovaccine was developed based on antigen-loaded genipin-cross-linked-polyethyleneimine-antigen nanoparticles and in vivo multispectral fluorescence imaging tracked the antigen delivery of combined nanovaccine. The inner layer antigen nanoparticles carried abundant antigens by self-cross-linking for persistent immune response, whereas the outer antigen on the surface of antigen nanoparticles provided the initial antigen exposure. The delivery of combined nanovaccine was tracked dynamically and objectively by the separation of inner genipin cross-linked antigen nanoparticle and the outer fluorescent antigen. The immune responses of the combined nanovaccine were evaluated including antigen-specific CD4+ and CD8+ T-cell responses, IgG antibody level, immunological memory, and CD8+ cytotoxic T lymphocyte responses. The results indicated that the inner and outer antigens of combined vaccine can be tracked in real time with a programmed delivery by the dual fluorescence imaging. The programmed delivery of the inner and outer antigens induced strong immune responses with a combination of a quick delivery and a persistent delivery. With adequate antigen exposure, the dendritic cells were effectively activated and matured, and following T cells were further activated for immune response. Compared with a single nanoparticle formulation, the combined nanovaccine exactly elicited a stronger antigen-specific immune response.