Application of Doxorubicin-loaded PLGA nanoparticles targeting both tumor cells and cancer-associated fibroblasts on 3D human skin equivalents mimicking melanoma and cutaneous squamous cell carcinoma.

Nanoparticle (NP) use in cancer therapy is extensively studied in skin cancers. Cancer-associated fibroblasts (CAFs), a major tumor microenvironment (TME) component, promote cancer progression, making dual targeting of cancer cells and CAFs an effective therapy. However, dual NP-based targeting therapy on both tumor cells and CAFs is poorly investigated in skin cancers. Herein, we prepared and characterized doxorubicin-loaded PLGA NPs (DOX@PLGA NPs) and studied their anti-tumor effects on cutaneous melanoma (SKCM)(AN, M14) and cutaneous squamous cell carcinoma (cSCC) (MET1, MET2) cell lines in monolayer, as well as their impact on CAF deactivation. Then, we established 3D full thickness models (FTM) models of SKCM and cSCC using AN or MET2 cells on dermis matrix populated with CAFs respectively, and assessed the NPs' tumor penetration, tumor-killing ability, and CAF phenotype regulation through both topical administration and intradermal injection. The results show that, in monolayer, DOX@PLGA NPs inhibited cancer cell growth and induced apoptosis in a dose- and time-dependent manner, with a weaker effect on CAFs. DOX@PLGA NPs reduced CAF-marker expression and had successful anti-tumor effects in 3D skin cancer FTMs, with decreased tumor-load and invasion. DOX@PLGA NPs also showed great delivery potential in the FTMs and could be used as a platform for future functional study of NPs in skin cancers using human-derived skin equivalents. This study provides promising evidence for the potential of DOX@PLGA NPs in dual targeting therapy for SKCM and cSCC.

Correction: Yu et al. Achieving Effective Multimodal Imaging with Rare-Earth Ion-Doped CaF2 Nanoparticles. Pharmaceutics 2022, 14, 840.

There was an error in the original publication [...].

Correction: Yu et al. Rare-Earth-Metal (Nd3+, Ce3+ and Gd3+)-Doped CaF2: Nanoparticles for Multimodal Imaging in Biomedical Applications. Pharmaceutics 2022, 14, 2796.

In the original publication [...].

Dual-Targeting Nanoliposome Improves Proinflammatory Immunomodulation of the Tumor Microenvironment.

Immunotherapies targeting immune checkpoints have revolutionized cancer treatment by normalizing the immunosuppressive microenvironment of tumors and reducing adverse effects on the immune system. Indoleamine 2,3-dioxygenase (IDO) inhibitors have garnered attention as a promising therapeutic agent for cancer. However, their application alone has shown limited clinical benefits. Cabozantinib, a multitarget tyrosine kinase inhibitor, holds immunomodulatory potential by promoting infiltration and activation of effector cells and inhibiting suppressive immune cells. Despite its potential, cabozantinib as a monotherapy has shown limited efficacy in terms of objective response rate. In this study, IDO-IN-7 and cabozantinib are coencapsulated into liposomes to enhance tumor accumulation and minimize adverse effects. The liposomal combination exhibits potent cytotoxicity and inhibits the function of IDO enzyme. Furthermore, the dual-targeted treatment effectively inhibits tumor development and reverses the suppressive tumor microenvironment by regulating both adaptive and innate branch of immune system. This is evidenced by pronounced infiltration of T cells and B cells, a decrease of regulatory T lymphocytes, a shift to a proinflammatory phenotype of tumor-associated macrophages, and increases levels of neutrophils. This is the first developed of a liposome-delivered combination of IDO inhibitors and cabozantinib, and holds great potential for future clinical application as a promising anticancer strategy.

Erythrocyte-cancer hybrid membrane-coated reduction-sensitive nanoparticles for enhancing chemotherapy efficacy in breast cancer.

Cell-membrane-coated biomimetic nanoparticles (NPs) have attracted great attention due to their prolonged circulation time, immune escape mechanisms and homotypic targeting properties. Biomimetic nanosystems from different types of cell -membranes (CMs) can perform increasingly complex tasks in dynamic biological environments thanks to specific proteins and other properties inherited from the source cells. Herein, we coated doxorubicin (DOX)-loaded reduction-sensitive chitosan (CS) NPs with 4T1 cancer cell -membranes (CCMs), red blood cell -membranes (RBCMs) and hybrid erythrocyte-cancer membranes (RBC-4T1CMs) to enhance the delivery of DOX to breast cancer cells. The physicochemical properties (size, zeta potential and morphology) of the resulting RBC@DOX/CS-NPs, 4T1@DOX/CS-NPs and RBC-4T1@DOX/CS-NPs, as well as their cytotoxic effect and cellular NP uptake in vitro were thoroughly characterized. The anti-cancer therapeutic efficacy of the NPs was evaluated using the orthotopic 4T1 breast cancer model in vivo. The experimental results showed that DOX/CS-NPs had a DOX-loading capacity of 71.76 ± 0.87 %, and that coating of DOX/CS-NPs with 4T1CM significantly increased the NP uptake and cytotoxic effect in breast cancer cells. Interestingly, by optimizing the ratio of RBCMs:4T1CMs, it was possible to increase the homotypic targeting properties towards breast cancer cells. Moreover, in vivo tumor studies showed that compared to control DOX/CS-NPs and free DOX, both 4T1@DOX/CS-NPs and RBC@DOX/CS-NPs significantly inhibited tumor growth and metastasis. However, the effect of 4T1@DOX/CS-NPs was more prominent. Moreover, CM-coating reduced the uptake of NPs by macrophages and led to rapid clearance from the liver and lungs in vivo, compared to control NPs. Our results suggest that specific self-recognition to source cells resulting in homotypic targeting increased the uptake and the cytotoxic capacity of 4T1@DOX/CS-NPs by breast cancer cells in vitro and in vivo. In conclusion, tumor-disguised CM-coated DOX/CS-NPs exhibited tumor homotypic targeting and anti-cancer properties, and were superior over targeting with RBC-CM or RBC-4T1 hybrid membranes, suggesting that the presence of 4T1-CM is critical for treatment outcome.

Enhancing anti-tumor immunity through liposomal oxaliplatin and localized immunotherapy via STING activation.

The cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway is a promising approach for anti-cancer immunotherapy by bridging innate and adaptive immunity. Recent evidence suggests that chemotherapy-induced DNA damage can directly induce dendritic cell (DC) maturation and recruitment, which synergizes with STING activation to enhance anti-tumor effects. As an immunogenic cell death (ICD) inducer, oxaliplatin generates massive double-stranded DNA (dsDNA) crosslinks, release of tumor-associated antigens and promoting the "eat me" signal. STING activation improves antigen immunogenicity, which can promote T cell activation and infiltration. In this study, we developed liposomes encapsulating oxaliplatin and combine this formulation with a STING agonist (ADU-S100) for treating colorectal cancer. The liposomes efficiently inhibited the proliferation of tumor cells while induced ICD in CT26 colorectal cancer cells, which enhanced dendritic cell maturation and phagocytosis in vitro. The liposome-based immunochemotherapy exhibited the strongest efficacy, resulting in complete remission upon tumor inoculation. Mechanistic studies showed this potent anti-cancer effect was related to the significant recruitment of infiltrating CD8 and CD4 T cells, reduction of suppressive Treg cells, and a shift in the phenotype of tumor-associated suppressive macrophages that promote cancer to immune stimulating macrophages. Thus, our study demonstrated the potential of combining oxaliplatin-loaded liposomes with a STING agonist to reduce tumor growth by regulating the immunosuppressive state in the tumor.

Retinoic acid-loaded PLGA nanocarriers targeting cell cholesterol potentialize the antitumour effect of PD-L1 antibody by preventing epithelial-mesenchymal transition mediated by M2-TAM in colorectal cancer.

Tumour-associated macrophages (TAMs) often promote cancer progression through immunosuppression in the tumour microenvironment (TME). However, the signalling pathways crosstalk responsible for this mechanism remain unclear. The aim of our study was to investigate whether the interaction between TAMs and colorectal cancer cells could be down-regulated by nanoparticles (NPs) loaded with retinoic acid (RA) and coated with cholesterol (CHO), in combination with an anti-PD-L1 immune checkpoint inhibitor. Tumours were evaluated by qRT-PCR and immunohistochemistry from allographic tumour growth model. In addition, human tumours were evaluated by Tissue Microarray (TMA) and immunohistochemistry. Complementary analysis of epithelial-mesenchymal transition, cell migration, and macrophage polarisation were evaluated in vitro. We showed that the IL-10R/IL-10 axis is involved in overstimulation of the STAT3 pathway as well as downregulation of the NF-κB signalling pathway, which supports a loop of immunosuppressive cytokines that induces the M2-TAM phenotype. Furthermore, our combined findings suggest that the upregulation of STAT3/NF-κB pathways crosstalk mediated by immunosuppressive cytokines, such as IL-10/PD-L1/TGF-ß, via M2-TAMs in the TME, leads to immunosuppression and epithelial-mesenchymal-transition of the colorectal cancer for stimulating Vimentin, CXCL12 and CD163 in the primary tumours. Importantly, NPs holding RA and coated with CHO in combination with anti-PD-L1 were more efficient in blocking this signalling pathway. These results contribute to our understanding of the immunological mechanisms, especially the re-educating of TAMs, and provide a novel management strategy for aggressive colorectal cancers using anti-PD-L1-conjugated nanocarriers.

Current Challenges and Opportunities of Photodynamic Therapy against Cancer.

BACKGROUND: Photodynamic therapy (PDT) is an established, minimally invasive treatment for specific types of cancer. During PDT, reactive oxygen species (ROS) are generated that ultimately induce cell death and disruption of the tumor area. Moreover, PDT can result in damage to the tumor vasculature and induce the release and/or exposure of damage-associated molecular patterns (DAMPs) that may initiate an antitumor immune response. However, there are currently several challenges of PDT that limit its widespread application for certain indications in the clinic. METHODS: A literature study was conducted to comprehensively discuss these challenges and to identify opportunities for improvement. RESULTS: The most notable challenges of PDT and opportunities to improve them have been identified and discussed. CONCLUSIONS: The recent efforts to improve the current challenges of PDT are promising, most notably those that focus on enhancing immune responses initiated by the treatment. The application of these improvements has the potential to enhance the antitumor efficacy of PDT, thereby broadening its potential application in the clinic.

Effective breast cancer therapy based on palmitic acid-loaded PLGA nanoparticles.

Although new strategies for breast cancer treatment have yielded promising results, most drugs can lead to serious side effects when applied systemically. Doxorubicin (DOX), currently the most effective chemotherapeutic drug to treat breast cancer, is poorly selective towards tumor cells and treatment often leads to the development of drug resistance. Recent studies have indicated that several fatty acids (FAs) have beneficial effects on inhibiting tumorigenesis. The saturated FA palmitic acid (PA) showed anti-tumor activities in several types of cancer, as well as effective repolarization of M2 macrophages towards the anti-tumorigenic M1 phenotype. However, water insolubility and cellular impermeability limit the use of PA in vivo. To overcome these limitations, here, we encapsulated PA into a poly(d,l-lactic co-glycolic acid) (PLGA) nanoparticle (NP) platform, alone and in combination with DOX, to explore PA's potential as mono or combinational breast cancer therapy. Our results showed that PLGA-PA-DOX NPs and PLGA-PA NPs significantly reduced the viability and migratory capacity of breast cancer cells in vitro. In vivo studies in mice bearing mammary tumors demonstrated that PLGA-PA-NPs were as effective in reducing primary tumor growth and metastasis as NPs loaded with DOX, PA and DOX, or free DOX. At the molecular level, PLGA-PA NPs reduced the expression of genes associated with multi-drug resistance and inhibition of apoptosis, and induced apoptosis via a caspase-3-independent pathway in breast cancer cells. In addition, immunohistochemical analysis of residual tumors showed a reduction in M2 macrophage content and infiltration of leukocytes after treatment of PLGA-PA NPs and PLGA-PA-DOX NPs, suggesting immunomodulatory properties of PA in the tumor microenvironment. In conclusion, the use of PA alone or in combination with DOX may represent a promising novel strategy for the treatment of breast cancer.

Effective combination of liposome-targeted chemotherapy and PD-L1 blockade of murine colon cancer.

Therapeutic cancer drug efficacy can be limited by insufficient tumor penetration, rapid clearance, systemic toxicity and (acquired) drug resistance. The poor therapeutic index due to inefficient drug penetration and rapid drug clearance and toxicity can be improved by using a liposomal platform. Drug resistance for instance against pemetrexed, can be reduced by combination with docetaxel. Here, we developed a specific liposomal formulation to simultaneously deliver docetaxel and pemetrexed to enhance efficacy and safety. Hydrophobic docetaxel and hydrophilic pemetrexed were co-encapsulated into pH-sensitive liposomes using a thin-film hydration method with high efficiency. The physicochemical properties, toxicity, and immunological effects of liposomes were examined in vitro. Biodistribution, anti-tumor efficacy, and systemic immune response were evaluated in vivo in combination with PD-L1 immune checkpoint therapy using two murine colon cancer models. In cellular experiments, the liposomes exhibited strong cytotoxicity and induced immunogenic cell death. In vivo, the treatment with the liposome-based drug combination inhibited tumor development and stimulated immune responses. Liposomal encapsulation significantly reduced systemic toxicity compared to the delivery of the free drug. Tumor control was strongly enhanced when combined with anti-PDL1 immunotherapy in immunocompetent mice carrying syngeneic MC38 or CT26 colon tumors. We showed that treatment with liposome-mediated chemotherapy of docetaxel and pemetrexed combined with anti-PD-L1 immunotherapy is a promising strategy for the treatment of colon cancers.

Rare-Earth-Metal (Nd3+, Ce3+ and Gd3+)-Doped CaF2: Nanoparticles for Multimodal Imaging in Biomedical Applications.

Here, we describe the synthesis of a novel type of rare-earth-doped nanoparticles (NPs) for multimodal imaging, by combining the rare-earth elements Ce, Gd and Nd in a crystalline host lattice consisting of CaF2 (CaF2: Ce, Gd, Nd). CaF2: Ce, Gd, Nd NPs are small (15-20 nm), of uniform shape and size distribution, and show good biocompatibility and low immunogenicity in vitro. In addition, CaF2: Ce, Gd, Nd NPs possess excellent optical properties. CaF2: Ce, Gd, Nd NPs produce downconversion emissions in the second near-infrared window (NIR-II, 1000-1700 nm) under 808 nm excitation, with a strong emission peak at 1056 nm. Excitation in the first near- infrared window (NIR-I, 700-900 nm) has the advantage of deeper tissue penetration power and reduced autofluorescence, compared to visible light. Thus, CaF2: Ce, Gd, Nd NPs are ideally suited for in vivo fluorescence imaging. In addition, the presence of Gd3+ makes the NPs intrinsically monitorable by magnetic resonance imaging (MRI). Moreover, next to fluorescence and MR imaging, our results show that CaF2: Ce, Gd, Nd NPs can be used as imaging probes for photoacoustic imaging (PAI) in vitro. Therefore, due to their biocompatibility and suitability as multimodal imaging probes, CaF2: Ce, Gd, Nd NPs exhibit great potential as a traceable imaging agent in biomedical applications.

Nanoparticles targeting hematopoietic stem and progenitor cells: Multimodal carriers for the treatment of hematological diseases.

Modern-day hematopoietic stem cell (HSC) therapies, such as gene therapy, modify autologous HSCs prior to re-infusion into myelo-conditioned patients and hold great promise for treatment of hematological disorders. While this approach has been successful in numerous clinical trials, it relies on transplantation of ex vivo modified patient HSCs, which presents several limitations. It is a costly and time-consuming procedure, which includes only few patients so far, and ex vivo culturing negatively impacts on the viability and stem cell-properties of HSCs. If viral vectors are used, this carries the additional risk of insertional mutagenesis. A therapy delivered to HSCs in vivo, with minimal disturbance of the HSC niche, could offer great opportunities for novel treatments that aim to reverse disease symptoms for hematopoietic disorders and could bring safe, effective and affordable genetic therapies to all parts of the world. However, substantial unmet needs exist with respect to the in vivo delivery of therapeutics to HSCs. In the last decade, in particular with the development of gene editing technologies such as CRISPR/Cas9, nanoparticles (NPs) have become an emerging platform to facilitate the manipulation of cells and organs. By employing surface modification strategies, different types of NPs can be designed to target specific tissues and cell types in vivo. HSCs are particularly difficult to target due to the lack of unique cell surface markers that can be utilized for cell-specific delivery of therapeutics, and their shielded localization in the bone marrow (BM). Recent advances in NP technology and genetic engineering have resulted in the development of advanced nanocarriers that can deliver therapeutics and imaging agents to hematopoietic stem- and progenitor cells (HSPCs) in the BM niche. In this review we provide a comprehensive overview of NP-based approaches targeting HSPCs to control and monitor HSPC activity in vitro and in vivo, and we discuss the potential of NPs for the treatment of malignant and non-malignant hematological disorders, with a specific focus on the delivery of gene editing tools.

Upconversion nanoparticle platform for efficient dendritic cell antigen delivery and simultaneous tracking.

Upconversion nanoparticles (UCNPs) represent a group of NPs that can convert near-infrared (NIR) light into ultraviolet and visible light, thus possess deep tissue penetration power with less background fluorescence noise interference, and do not induce damage to biological tissues. Due to their unique optical properties and possibility for surface modification, UCNPs can be exploited for concomitant antigen delivery into dendritic cells (DCs) and monitoring by molecular imaging. In this study, we focus on the development of a nano-delivery platform targeting DCs for immunotherapy and simultaneous imaging. OVA 254-267 (OVA24) peptide antigen, harboring a CD8 T cell epitope, and Pam3CysSerLys4 (Pam3CSK4) adjuvant were chemically linked to the surface of UCNPs by amide condensation to stimulate DC maturation and antigen presentation. The OVA24-Pam3CSK4-UCNPs were thoroughly characterized and showed a homogeneous morphology and surface electronegativity, which promoted a good dispersion of the NPs. In vitro experiments demonstrated that OVA24-Pam3CSK4-UCNPs induced a strong immune response, including DC maturation, T cell activation, and proliferation, as well as interferon gamma (IFN-γ) production. In vivo, highly sensitive upconversion luminescence (UCL) imaging of OVA24-Pam3CSK4-UCNPs allowed tracking of UCNPs from the periphery to lymph nodes. In summary, OVA24-Pam3CSK4-UCNPs represent an effective tool for DC-based immunotherapy.

Thermosensitive Injectable Hydrogels for Intra-Articular Delivery of Etanercept for the Treatment of Osteoarthritis.

The intra-articular administration of drugs has attracted great interest in recent decades for the treatment of osteoarthritis. The use of modified drugs has also attracted interest in recent years because their intra-articular administration has demonstrated encouraging results. The objective of this work was to prepare injectable-thermosensitive hydrogels for the intra-articular administration of Etanercept (ETA), an inhibitor of tumor necrosis factor-α. Hydrogels were prepared from the physical mixture of chitosan and Pluronic F127 with ß-glycerolphosphate (BGP). Adding ß-glycerolphosphate to the system reduced the gelation time and also modified the morphology of the resulting material. In vitro studies were carried out to determine the cytocompatibility of the prepared hydrogels for the human chondrocyte line C28/I2. The in vitro release study showed that the incorporation of BGP into the system markedly modified the release of ETA. In the in vivo studies, it was verified that the hydrogels remained inside the implantation site in the joint until the end of the study. Furthermore, ETA was highly concentrated in the blood of the study mice 48 h after the loaded material was injected. Histological investigation of osteoarthritic knees showed that the material promotes cartilage recovery in osteoarthritic mice. The results demonstrate the potential of ETA-loaded injectable hydrogels for the localized treatment of joints.

Combinatorial therapeutic approaches of photodynamic therapy and immune checkpoint blockade for colon cancer treatment.

Photodynamic therapy (PDT) has shown impressive therapeutic effects on various types of cancers by reactive oxygen species (ROS) generation and induction of immune responses. However, under certain conditions, the immune responses induced by PDT are not always sufficient to eradicate the remaining tumor cells. On the other hand, the photosensitizer indocyanine green (ICG) can mediate PDT under near-infrared (NIR) illumination, thereby enhancing the penetration depth of the excitation light into the tumor. We found that ICG is rapidly taken up in vitro by colorectal MC38 and CT26 tumor cells and it promotes PDT-mediated cell-killing effects. Our results furthermore revealed that ICG induces immunogenic cell death (ICD), as dendritic cells (DCs) were found to engulf ICG-PDT-treated tumor cells and undergo phenotypic maturation. ICG accumulated in tumors 2 h after administration, as measured by fluorescence and photoacoustic imaging. Considering the advantages of ICG as a photosensitizer, we sought to design a therapy that combines PDT and immune checkpoint blockade to maximize tumor control. To this end, a 25% thermosensitive polymer 407 hydrogel was included as a co-delivery platform for this treatment scheme. NIR-PDT under 808 nm irradiation in combination with cytotoxic T-lymphocyte-associated protein 4 (CTLA4)/programmed death-ligand 1 (PD-L1) checkpoint blockade prolonged survival rate of colorectal tumor-bearing mice by inducing a series of immune responses, like the phagocytosis of tumor debris by macrophages and DCs, and induction of acute inflammation, leukocyte infiltration, maturation and activation of DCs. Altogether, our work presents a NIR-triggered PDT strategy in combination with immune checkpoint blockade. Compared to a single treatment, the combination treatment increased efficiency to inhibit solid tumor growth and improved the survival rate of tumor-bearing mice.

PLGA Nanoparticles Grafted with Hyaluronic Acid to Improve Site-Specificity and Drug Dose Delivery in Osteoarthritis Nanotherapy.

Nanoparticles (NPs) have a tremendous potential in medicinal applications, and recent studies have pushed the boundaries in nanotherapy, including in osteoarthritis treatments. The aim of this study was to develop new poly(lactide-co-glycolide) (PLGA) nanoparticles (NPs) surfaces decorated with hyaluronic acid (HA) to enhance targeted drug specificity to the osteoarthritic knee joint. HA was selected since it binds to specific receptors expressed in many cells, such as the cluster determinant 44 (CD44), a major receptor of chondrocytes, and because of its function in the synovial fluid (SF), such as maintenance of high fluid viscosity. The PLGA polymer was grafted to sodium hyaluronate using dimethoxy-PEG (PLGA-HA) and compared with control PLGA NPs (not grafted). NPs were characterized by 1H-NMR and IR spectroscopy. Then, near-infrared (NIR) dye and gold (20 nm) were encapsulated in the formulated NPs and used to access NPs' performance in in vitro, in vivo, and ex vivo experiments. To test the NPs' CD44 receptor specificity, an antibody assay was performed. All NPs presented a size in the range viable for cell-uptake, no cytotoxicity to chondrocytes was registered. Although all the NPs had a high capacity to be absorbed by the cells, PLGA-HA NPs showed significantly higher affinity towards the chondrocytic C28/I2 cell line. In conclusion, PLGA NPs grafted to sodium hyaluronate showed increased binding to cartilage cells and tissue and enhanced accumulation at the target site. Thus, this study presents a safe drug-delivery system with improved receptor specificity, which may represent an advantageous alternative to current nanotherapies.

Micromechanical Model for Predicting the Tensile Properties of Guadua angustifolia Fibers Polypropylene-Based Composites.

In this paper, the one-dimensional tensile behavior of Guadua angustifolia Kunth fibre/polypropylene (PP+GAKS) composites is modeled. The classical model of Kelly-Tyson and its Bowyer-Bader's solution is not able to reproduce the entire stress-strain curve of the composite. An integral (In-Built) micromechanical model proposed by Isitman and Aykol, initially for synthetic fiber-reinforced composites, was applied to predict micromechanical parameters in short natural fiber composites. The proposed method integrates both the information of the experimental stress-strain curves and the morphology of the fiber bundles within the composite to estimate the interfacial shear strength (IFSS), fiber orientation efficiency factor ηFOD, fiber length efficiency factor ηFLD and critical fiber length lc. It was possible to reproduce the stress-strain curves of the PP+GAKS composite with low residual standard deviation. A methodology was applied using X-ray microtomography and digital image processing techniques for the precise extraction of the micromechanical parameters involved in the model. The results showed good agreement with the experimental data.

Bacterial Cellulose as Drug Delivery System for Optimizing Release of Immune Checkpoint Blocking Antibodies.

Immune checkpoint blocking therapy is a promising cancer treatment modality, though it has limitations such as systemic toxicity, which can often be traced to uncontrolled antibody spread. Controlling antibody release with delivery systems is, therefore, an attractive approach to reduce systemic antibody spread and potentially mitigate the side effects of checkpoint immunotherapy. Here, bacterial cellulose (BC) was produced and investigated as a delivery system for optimizing checkpoint-blocking antibody delivery. BC was produced in 24-well plates, and afterward, the edges were removed to obtain square-shaped BC samples with a surface of ~49 mm2. This customization was necessary to allow smooth in vivo implantation. Scanning electron microscopy revealed the dense cellulose network within BC. Human IgG antibody was included as the model antibody for loading and release studies. IgG antibody solution was injected into the center of BC samples. In vitro, all IgG was released within 24 to 48 h. Cell culture experiments demonstrated that BC neither exerted cytotoxic effects nor induced dendritic cell activation. Antibody binding assays demonstrated that BC does not hamper antibody function. Finally, antibody-loaded BC was implanted in mice, and serum measurements revealed that BC significantly reduced IgG and anti-CTLA-4 spread in mice. BC implantation did not induce side effects in mice. Altogether, BC is a promising and safe delivery system for optimizing the delivery and release of checkpoint-blocking antibodies.

M1-derived extracellular vesicles enhance photodynamic therapy and promote immunological memory in preclinical models of colon cancer.

Extracellular vesicles (EVs) are promising drug carriers of photosensitizers for photodynamic therapy (PDT) in cancer treatment, due to their ability to circulate in blood and enter cells efficiently. The therapeutic potential of EVs has been suggested to depend on the type and physiological state of their cell of origin. However, the effects of deriving EVs from various cells in different physiological states on their antitumor capacity are rarely evaluated. In the present study, we compared the antitumor efficacy of EV-mediated PDT by incorporating the photosensitizer Zinc Phthalocyanine (ZnPc) into EVs from multiple cells sources. ZnPc was incorporated by a direct incubation strategy into EVs derived from immune cells (M1-like macrophages and M2-like macrophages), cancer cells (B16F10 melanoma cancer cells) and external sources (milk). Our data show that all EVs are suitable carriers for ZnPc and enable efficient PDT in vitro in co-culture models and in vivo. We observed that EV-mediated PDT initiates immunogenic cell death through the release and exposure of damage associated molecular patterns (DAMPs) on cancer cells, which subsequently induced dendritic cell (DC) maturation. Importantly, of all ZnPc-EVs tested, in absence of light only M1-ZnPc displayed toxicity to MC38, but not to DC, in monoculture and in co-culture, indicating specificity for cancer over immune cells. In MC38 tumor-bearing mice, only M1-ZnPc induced a tumor growth delay compared to control in absence of light. Interestingly, M1- but not M2-mediated PDT, induced complete responses against MC38 tumors in murine models (100% versus 38% of cases, respectively), with survival of all animals up to at least 60 days post inoculation. Finally, we show that all cured animals are protected from a rechallenge with MC38 cells, suggesting the induction of immunological memory after EV-mediated PDT. Together, our data show the importance of the cell type from which the EVs are obtained and highlight the impact of the immunological state of these cells on the antitumor efficacy of EV-mediated PDT.