The human microbiome has emerged as a key player in health and disease, including cancer, which remains one of the leading causes of mortality worldwide. Although advances in understanding the tumor immune microenvironment and the development of immunotherapies have transformed cancer treatment, clinical efficacy remains limited by suboptimal response rates and severe side effects. Recent integrative research in cancer biology, immune-oncology, and cancer microbiome research, enabled by omics technologies and advanced bioinformatics, has begun to reveal intricate links between the microbiome, cancer progression, and immune modulation. These findings underscore the microbiome's pivotal role in shaping both therapeutic efficacy and resistance mechanisms.Currently, nanotechnology, propelled into mainstream success through the development of COVID-19 mRNA vaccines, is offering new tools for precision oncology. Nanomaterials are now being explored not only for targeted drug delivery but also for monitoring and modulating the microbiome, with significant potential for biomarker discovery and personalized medicine. In this article, we explore the role of the microbiota in tumorigenesis and cancer therapy, with a particular focus on its crosstalk with the immune system. We highlight emerging microbiota-targeted therapeutic strategies and discuss how nanotechnology-based systems are being designed to modulate the microbiome-immune-cancer axis. Finally, we discuss future directions in leveraging the convergence of microbiome science, nanotechnology, and immunotherapy to advance cancer treatment.

Tumor-associated antigen-based cancer vaccines suffer from limited clinical success compared to alternative immunotherapies in melanoma, an aggressive skin cancer with an immunosuppressive tumor microenvironment. The anti-tumor potential of a multivalent nanoconjugate cancer vaccine platform - a cross-linked star-shaped polyglutamate carrier (StCl) with marked lymphotropic character conjugated with melanoma-associated peptide antigens is evaluated through redox-responsive linkers. The co-delivery of melanoma-associated peptide antigens by the nanoconjugate platform induced significant effector immune responses in a mouse melanoma model. The nanoconjugate platform synergized with a PD-1 inhibitor to revert the immunosuppressive melanoma tumor microenvironment by improving cytotoxic T-cell infiltration, which prompted a superior anti-tumor effect with prolonged overall survival without acute organ toxicity. The antigen-specific anti-tumor immune response induced by the nanoconjugate platform is also validated in a melanoma patient-derived xenograft mouse model. A promising, versatile StCl-based platform is reported for generating off-the-shelf multivalent nanoconjugate cancer vaccines for the safe and efficient immunotherapeutic treatment of melanoma.

Programmed cell death protein 1 (PD-1) and its ligand PD-L1 are targets of immune checkpoint blockade (ICB) therapies that have shown promise in cancer treatment. However, their effectiveness is often hindered by resistance mechanisms such as poor tumor immunogenicity, T-cell exhaustion, insufficient T-cell infiltration, and an immunosuppressive tumor microenvironment (TME). Overcoming these barriers requires strategies to enhance tumor immunogenicity and modulate the TME. In this study, we present a nanoparticle-based strategy to enhance melanoma immunotherapy and overcome ICB resistance. We developed a mannose-grafted poly(lactic-co-glycolic) acid (PLGA) nanovaccine designed to target dendritic cells and deliver melanoma neoantigens, thereby promoting T-cell activation. When combined with PD-1/PD-L1 pathway modulators, including a monoclonal antibody (αPD-L1) and a novel small-molecule inhibitor (SM56), this nanovaccine significantly suppressed tumor growth in an aggressive, ICB-resistant B16F10 melanoma mouse model and enhanced T-cell infiltration into the TME. Notably, only the combination with SM56 reduced the infiltration of immunosuppressive cell populations within the TME. These findings highlight the potential of polymeric nanovaccines to overcome key resistance mechanisms limiting ICB efficacy and underscore the promise of novel small-molecule inhibitors as effective alternatives to monoclonal antibodies in melanoma immunotherapy.

Immune checkpoint blockade reaches remarkable clinical responses. However, even in the most favorable cases, half of these patients do not benefit from these therapies in the long term. This work hypothesized that the activation of host immunity by co-delivering peptide antigens, adjuvants, and regulators of the transforming growth factor (TGF)-β expression using a polyoxazoline (POx)-poly(lactic-co-glycolic) acid (PLGA) nanovaccine, while modulating the tumor-associated macrophages (TAM) function within the tumor microenvironment (TME) and blocking the anti-programmed cell death protein 1 (PD-1) can constitute an alternative approach for cancer immunotherapy. Nanoparticles (NP) prepared by the double emulsion solvent evaporation method were modified with mannose to target the mannose receptor (CD206) expressed at the dendritic cells (DC) surface, thus promoting receptor-ligand interaction and subsequently improving payload delivery. NP physicochemical properties were characterized, and the loading of antigens, adjuvants, immune modulators was quantified by fluorescence. The synergistic immunotherapeutic potential of our multifunctional nanovaccine, isolated and in combination with the TGF-β downregulation and Pexidartinib (colony-stimulating factor 1 receptor (CSF-1R) inhibitor that modulates TAM), was assessed in the immune-competent MC38 colorectal (CRC) mouse model. The added value of combining the immune checkpoint inhibitor anti-PD-1 monoclonal antibody to control the CRC (MC38 and CT26) and the highly aggressive melanoma (B16F10) growth and survival was also evaluated.NP presented a mean diameter close to 200 nm, low polydispersity index, neutral surface charge, homogenous spherical shape, and high loadings for antigens, immune adjuvants, and modulators. POx-Mannose (Man) nanovaccines generated stronger antigen-specific T-cell responses, which led to significant tumor growth inhibition when compared with poly(ethylene glycol) (PEG)-Man nanovaccines. This anti-tumor effect induced by the POx-Man nanovaccines is mediated by a CD8+-T cell-dependent mechanism, in contrast to the PEG-Man nanovaccines. POx-Man nanovaccine combined with Pexidartinib restricted the MC38 tumor growth and synergized with PD-1 blockade, controlling MC38 and CT26 tumor growth and survival. This data was further validated in B16F10-bearing mice, a highly aggressive and poorly immunogenic melanoma mouse model for immunotherapy studies.This innovative approach discloses the synergy among the targeted cancer nanovaccine and immune modulatory and checkpoint therapies (αCSF-1R and αPD-1) within the immunosuppressive TME, which overall outcome may constitute a promising nanotechnology-enhanced immunotherapy for solid cancer patients. Ana I. Matos, Bárbara Carreira, Liane I. F. Moura, Rita C. Acúrcio, Ronit Satchi-Fainaro, Helena F. Florindo. Turning up the heat: nanoimmunotherapy transforming outcomes in breast cancer treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4474.

Abstract Despite the remarkable efficiency of cancer immunotherapies, only a low percentage of patients achieve long-lasting clinical responses. Non-tumor cells within the tumor microenvironment (TME), including tumor vasculature and immune stromal cells, dictate therapeutic efficacy. The presence of germinal centers (GC) within the TME of cancer patients, including infiltrating T follicular helper (Tfh) cells and B cells, has been considered predictive of response to immunotherapies. We are developing nano-immunotherapies to induce immunological memory to control tumor relapse without any follow-up treatment.Nanomaterials co-incorporated tumor-associated antigens, clinically relevant toll-like receptor ligands, and regulators of tumor progression, namely the PD-L1/PD-1 expression and the secretion of TGF-β. Nanoparticle (NP) physicochemical properties were fully addressed. The immunotherapeutic potential of this nano-immunotherapy was addressed in melanoma, colorectal cancer (CRC), pancreatic cancer, and triple-negative breast cancer (TNBC) 3D models. These were created using patient-derived tumor cells, cancer-associated fibroblasts, and endothelial cells, to which paired patients' peripheral blood mononuclear cells were added to assess T-cell infiltration and tumor sprouting to identify the best nano-immunotherapy dosage/schedule. Our in vivo studies evaluated tumor volume, and animals’ survival, and characterized the tumor-infiltrating immune cells within TME, including the differentiation of Tfh cells and primary GC by flow cytometry.Our nano-immunotherapy remodeled the TME of B16F10 melanoma, MC38 CRC, 4T1 and E0771 TNBC, and KPC PDAC. Different TME subpopulations were identified as major blockers of anti-tumor immunity, and this knowledge guided the selection of combination approaches using modulators of those tumor immune evasion-related pathways (e.g., PD-L1, FAK). Our combination nano-immunotherapies tailored to address the tumor immune suppression profile found in vaccinated animals, significantly delayed tumor development, and increased disease-free survival rates. We found that the adjuvants delivered by our NP led to a ~4-fold increase in antibody production, being far more effective in generating GC responses. Thus, the effectiveness of combinational immunotherapy is at least partially related to the modulation of Tfh cells driving the formation of GC responses.Our nano-immunotherapy is a promising clinically translatable approach to defeating solid tumors. Acknowledgments: This work was supported by PTDC/BTM-SAL/4350/2021 and LCF/PR/HR22/52420016. Citation Format: Barbara Carreira, Rita C. Acúrcio, Ana I. Matos, Liane M. Moura, Ana C. Santos, Raquel Gouveia, Jéssica Cordeiro, Carina Peres, Daniela Vaskovich-Koubi, Ron Kleiner, Sabina Pozzi, Ronit Satchi-Fainaro, Helena F. Florindo. Unlocking solid tumors to nano-immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 493.

Breast cancer is the primary cause of cancer-related death in women worldwide. Breast cancer subtypes are characterized by different gene expression patterns, which drive their prognostic factors and therapeutic options. Among them, triple-negative breast cancer (TNBC) is one of the deadliest due to its aggressiveness, high rate of early recurrence and distant metastases, and limited therapeutic options. Despite the recent approval of monoclonal antibodies targeting programmed cell death protein 1 (PD-1) or its ligand (PD-L1) for the treatment of TNBC patients with a locally recurrent unresectable or metastatic tumor expressing PD-L1, their response rate is very modest. It is reported that polymeric nanoparticle (NP)-based cancer vaccines, co-entrapping tumor-associated antigens, Toll-like receptor ligands and small interfering RNA (siRNA) targeting the expression of the immunosuppressive cytokine transforming growth factor (TGF)-β1 by dendritic cells, sensitized TNBC to the agonist immune checkpoint OX40, inhibiting tumor growth and increasing overall survival. This anti-tumor immune-mediated effect is also observed in a luminal type of mammary cancer similar to human disease. Therefore, these synergistic anticancer effects of αOX40 and the antigen-specific adaptive immunity induced by nanovaccine-mediated TGF-β silencing may guide the development of novel combination regimens able to improve the response rate to this aggressive tumor.

Immune checkpoint blockade reaches remarkable clinical responses. However, even in the most favorable cases, half of these patients do not benefit from these therapies in the long term. It is hypothesized that the activation of host immunity by co-delivering peptide antigens, adjuvants, and regulators of the transforming growth factor (TGF)-β expression using a polyoxazoline (POx)-poly(lactic-co-glycolic) acid (PLGA) nanovaccine, while modulating the tumor-associated macrophages (TAM) function within the tumor microenvironment (TME) and blocking the anti-programmed cell death protein 1 (PD-1) can constitute an alternative approach for cancer immunotherapy. POx-Mannose (Man) nanovaccines generate antigen-specific T-cell responses that control tumor growth to a higher extent than poly(ethylene glycol) (PEG)-Man nanovaccines. This anti-tumor effect induced by the POx-Man nanovaccines is mediated by a CD8+ -T cell-dependent mechanism, in contrast to the PEG-Man nanovaccines. POx-Man nanovaccine combines with pexidartinib, a modulator of the TAM function, restricts the MC38 tumor growth, and synergizes with PD-1 blockade, controlling MC38 and CT26 tumor growth and survival. This data is further validated in the highly aggressive and poorly immunogenic B16F10 melanoma mouse model. Therefore, the synergistic anti-tumor effect induced by the combination of nanovaccines with the inhibition of both TAM- and PD-1-inducing immunosuppression, holds great potential for improving immunotherapy outcomes in solid cancer patients.

Sixty percent of melanoma patients present brain metastases, having low response rates to current systemic therapies, which do not prolong survival. New therapeutic options to address this clinical need are urgent. Here, we report the development of a polymeric nanoparticle as a cancer nanovaccine capable of selectively co-targeting toll-like receptor function and PD-L1 expression to improve antigen presentation and subsequent effector immune cell function within brain microenvironment. Mannose-poly(lactic-co-glycolic acid)/poly(lactic acid) (man-PLGA/PLA) nanoparticles (NP) were prepared by a modified double emulsion solvent evaporation method, to deliver combinations of melanoma neoantigens, toll-like receptor ligands and regulators of the PD-1/PD-L1 axis. NP physicochemical properties were fully characterized, including size, surface charge and morphology. The amount of melanoma neoantigens and immune regulators entrapped within NP was determined by HPLC. Immature dendritic cells (DC) were used to evaluate the impact of NP on cell viability, and to assess NP uptake kinetics by flow cytometry. NP ability to target and trigger the activation of DC and T cells, was assessed in the lymph nodes and spleen of immunized mice. The NP anti-tumor immune-mediated effect was evaluated in vivo in two primary melanoma-bearing immunocompetent mouse models (B16F10 and B16MO5), and in a melanoma brain metastasis (MBM) mouse model (B16F10), which also included the immune profiling within tumor site by flow cytometry, before and after treatment.NP presented an average diameter of 180 nm, narrow polydispersity index, surface charge close to neutrality, spherical morphology, and high loadings of the neoantigens and immunoregulators. NP did not affect DC viability and were extensively internalized by immature DC. NP were preferentially taken-up in vivo by DC, increasing the expression of activation/maturation markers at DC surface, such as CD80 and CD86. NP elicited antigen-specific T-cell responses, by the significant increase in the expression of TNF-alpha and IFN-gamma (TH1-guided response). In vivo combination of NP with anti-PD-L1 induced a potent immune-mediated anti-tumor response in both primary and preclinical models of MBM, overcoming tumor development with an increased overall survival. This combination re-shaped immune and stroma cell populations in primary tumor and MBM microenvironment, which presented a marked infiltration of cytotoxic T cells that correlated with a decreased expression of PD-1 and PD-L1 within the TME. The synergy between our nanovaccine and anti-PD-L1 provide essential insights to devise alternative combinations regiments to improve the efficacy of immune checkpoint inhibitors in metastatic melanoma, thus opening new line for this unmet medical need. Citation Format: Barbara Carreira, Rita A. Acúrcio, Sabina Pozzi, Ron Kleiner, Liane I. Moura, Ana I. Matos, Daniela vaskovich, Carina Peres, Adelaide Fernandes, Sara Xapelli, Ronit Satchi-Fainaro, Helena F. Florindo. Translational nanotechnology-based cancer vaccine to re-educate host immune response against metastatic cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 835.

Background: The cannabinoid receptor type-1 (CB1R) is a major regulator of metabolism, growth and inflammation. Yet, its potential role in the skin is not well understood. Our aim was to evaluate the role of CB1R in aging-like diabetic skin changes by using a CB1R knockout mouse model. Methods: We evaluated several signals of skin aging in wild-type control (WT), WT streptozotocin-induced type 1 diabetic mice (WT DM), CB1R knockout (CB1RKO) and CB1RKO DM mice. We quantified markers of inflammation, angiogenesis, antioxidant enzymes and collagen content. Moreover, we evaluate reactive oxygen species (ROS) levels and macrophage phenotype, M1 and M2. Results: CB1R expression is decreased in the skin of WT DM mice and collagen levels are decreased in the skin of WT DM, CB1RKO and CB1RKO DM mice. Additionally, the absence of CB1R correlated with higher expression of pro-inflammatory markers, also evident in WT DM or CB1RKO DM mice. Moreover, the M1/M2 macrophage ratio and ROS levels were significantly elevated but in the diabetic WT and the CB1RKO mice, consistent with a significant decrease in the antioxidant capacity of the skin. Conclusions: Our results indicate that CB1R deficiency in the skin may lead to accelerated skin aging due to the increased production of ROS, a decrease in the antioxidant defenses and a higher pro-inflammatory environment. A significant decrease in the CB1R expression may be a significant contributing factor to the early aging-like changes in diabetes.

The remarkable success of targeted immunotherapies is revolutionizing cancer treatment. However, tumor heterogeneity and low immunogenicity, in addition to several tumor-associated immunosuppression mechanisms are among the major factors that have precluded the success of cancer vaccines as targeted cancer immunotherapies. The exciting outcomes obtained in patients upon the injection of tumor-specific antigens and adjuvants intratumorally, reinvigorated interest in the use of nanotechnology to foster the delivery of vaccines to address cancer unmet needs. Thus, bridging nano-based vaccine platform development and predicted clinical outcomes the selection of the proper preclinical model will be fundamental. Preclinical models have revealed promising outcomes for cancer vaccines. However, only few cases were associated with clinical responses. This review addresses the major challenges related to the translation of cancer nano-based vaccines to the clinic, discussing the requirements for ex vivo and in vivo models of cancer to ensure the translation of preclinical success to patients.

Here we aimed to unify some previous controversial reports on changes in both cannabinoid CB1 receptor (CB1R) expression and glucose metabolism in the forebrain of rodent models of diabetes. We determined how glucose metabolism and its modulation by CB1R ligands evolve in the frontal cortex of young adult male Wistar rats, in the first 8 weeks of streptozotocin-induced type-1 diabetes (T1D). We report that frontocortical CB1R protein density was biphasically altered in the first month of T1D, which was accompanied with a reduction of resting glucose uptake ex vivo in acute frontocortical slices that was normalized after eight weeks in T1D. This early reduction of glucose uptake in slices was also restored by ex vivo treatment with both the non-selective CB1R agonists, WIN55212 - 2 (500 nM) and the CB1R-selective agonist, ACEA (3 mu M) while it was exacerbated by the CB1R-selective antagonist, O-2050 (500 nM). These results suggest a gain-of-function for the cerebrocortical CB(1)Rs in the control of glucose uptake in diabetes. Although insulin and IGF-1 receptor protein densities remained unaffected, phosphorylated GSK alpha and GSK beta levels showed different profiles 2 and 8 weeks after T1D induction in the frontal cortex. Altogether, the biphasic response in frontocortical CB1R density within a month after T1D induction resolves previous controversial reports on forebrain CB1R levels in T1D rodent models. Furthermore, this study also hints that cannabinoids may be useful to alleviate impaired glucoregulation in the diabetic cortex.

Colorectal cancer (CRC) is among the five most commonly diagnosed cancers worldwide, constituting 6% of all cancers and the third leading cause of cancer death. CRC is the third and second most frequent cancer in men and women worldwide, accounting for 14% and 13% of all cancer incidence rates, respectively. CRC incidence is decreasing in older populations, but it has been significantly rising worldwide in adolescents and adults younger than 50 years old. Significant advances in the screening methods and surgical procedures have been underlying the reduction of the CRC incidence rate in older populations. However, there is an urgent demand for the development of alternative effective therapeutic options to overcome advanced metastatic CRC, while preventing disease recurrence. This review addresses the immune and CRC biology, summarizing the recent advances on the immune and/or therapeutic regimens currently in clinical use. We will focus on the emerging role of nanotechnology in the development of combinational therapies targeting and thereby regulating the function of the major players in CRC progression and immune evasion.

A low response rate, acquired resistance and severe side effects have limited the clinical outcomes of immune checkpoint therapy. Here, we show that combining cancer nanovaccines with an anti-PD-1 antibody (αPD-1) for immunosuppression blockade and an anti-OX40 antibody (αOX40) for effector T-cell stimulation, expansion and survival can potentiate the efficacy of melanoma therapy. Prophylactic and therapeutic combination regimens of dendritic cell-targeted mannosylated nanovaccines with αPD-1/αOX40 demonstrate a synergism that stimulates T-cell infiltration into tumours at early treatment stages. However, this treatment at the therapeutic regimen does not result in an enhanced inhibition of tumour growth compared to αPD-1/αOX40 alone and is accompanied by an increased infiltration of myeloid-derived suppressor cells in tumours. Combining the double therapy with ibrutinib, a myeloid-derived suppressor cell inhibitor, leads to a remarkable tumour remission and prolonged survival in melanoma-bearing mice. The synergy between the mannosylated nanovaccines, ibrutinib and αPD-1/αOX40 provides essential insights to devise alternative regimens to improve the efficacy of immune checkpoint modulators in solid tumours by regulating the endogenous immune response.

The review focus on branched polymers as promising delivery systems with immunomodulatory properties against cancer and infectious diseases. It covers their physicochemical properties and on how those can be tailored to modulate the immune system.

Both endocannabinoids and insulin regulate peripheral and cerebral glucose homeostasis via convergent signaling pathways that are impacted by diabetes. Here we asked how glucose metabolism and important facets of insulin signaling are affected in the forebrain of cannabinoid CB1 receptor knockout mice (CB1R-KO) and their wild-type (WT) littermates, seven weeks after the induction of insulinopenia/hyperglycemia (diabetes) with intraperitoneal streptozotocin injection. Sham-injected animals served as control. Diabetes caused milder weight loss in the WT mice compared to the phenotypically (similar to)11% leaner CB1R-KO, while hyperglycemia was similar. Resting [H-3]deoxyglucose uptake was significantly reduced by (similar to)20% in acute ex vivo frontocortical and hippocampal slices obtained from both the sham-injected CB1R-KO and the diabetic WT mice. Surprisingly, the third cohort, the diabetic CB1R-KO showed no further impairment in glucose uptake, as compared to the sham-injected CB1R-KO. Depolarization-induced [H-3]deoxyglucose uptake was proportional to the respective resting values only in the cortex in all four cohorts. The dissipative metabolism of [C-14]-U-glucose remained largely unaffected in all cohorts of animals. However, diabetes reduced cortical CB1R density by (similar to)20%, as assessed by Western blotting. Albeit the changes in insulin signaling did not reflect the glucose uptake profile in each cohort, there were significant interactions between diabetes and genotype. In conclusion, a chronic decrease or lack of CB1R expression reduces glucose uptake in the mouse brain. Additionally, diabetes failed to cause further impairment in cerebral glucose uptake in the CB1R-KO. These suggest that diabetic encephalopathy may be in part associated with lower CB1R expression.

alpha-Galactosylceramide (GalCer) is a glycolipid widely known as an activator of Natural killer T (NKT) cells, constituting a promising adjuvant against cancer, including melanoma. However, limited clinical outcomes have been obtained so far. This study evaluated the synergy between GalCer and major histocompatibility complex (MHC) class I and MHC class II melanoma-associated peptide antigens and the Toll-Like Receptor (TLR) ligands CpG and monophosphoryl lipid A (MPLA), which we intended to maximize following their co-delivery by a nanoparticle (NP). This is expected to improve GalCer capture by dendritic cells (DCs) and subsequent presentation to NKT cells, simultaneously inducing an anti-tumor specific T-cell mediated immunity. The combination of GalCer with melanoma peptides and TLR ligands successfully restrained tumor growth. The tumor volume in these animals was 5-fold lower than the ones presented by mice immunized with NPs not containing GalCer. However, tumor growth was controlled at similar levels by GalCer entrapped or in its soluble form, when mixed with antigens and TLR ligands. Those two groups showed an improved infiltration of T lymphocytes into the tumor, but only GalCer-loaded nano vaccine induced a prominent and enhanced infiltration of NKT and NK cells. In addition, splenocytes of these animals secreted levels of IFN-gamma and IL-4 at least 1.5-fold and 2-fold higher, respectively, than those treated with the mixture of antigens and adjuvants in solution. Overall, the combined delivery of the NKT agonist with TLR ligands and melanoma antigens via this multivalent nano-vaccine displayed a synergistic anti-tumor immune-mediated efficacy in B16F10 melanoma mouse model. Statement of Significance Combination of alpha-galactosylceramide (GalCer), a Natural Killer T (NKT) cell agonist, with melanoma associated antigens presented by MHC class I (Melan-A:26) and MHC class II (gp100:44) molecules, and Toll-like Receptor (TLR) ligands (MPLA and CpG), within nanoparticle matrix induced a prominent anti-tumor immune response able to restrict melanoma growth. An enhanced infiltration of NKT and NK cells into tumor site was only achieved when the combination GalCer, antigens and TLR ligands were co-delivered by the nanovaccine. (C) 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Particulate delivery systems can protect entrapped material from chemical and enzymatic degradation, resulting in increased blood circulation time, by avoiding the uptake by the mononuclear phagocyte system and rapid clearance via the kidneys. In addition, those carriers allow the concomitant delivery of multiple components for a sustained release of the entrapped active molecules, prolonging their therapeutic effects. To achieve a specific therapeutic outcome, the scientific community has done considerable efforts on developing different strategies to target tissues and specific cells through the development of site-directed nanocarriers. By modulating nanoparticle (NP) size, surface charge, or hydrophobicity, it is possible to regulate the endocytic pathways and facilitate endosomal escape, leading to the cytosolic delivery of therapeutic molecules. The modification of NPs by organelle-specific targeting macromolecules (drugs, proteins, DNA, short interference RNA, among others) has an extreme potential for the delivery of molecules to intracellular target receptors, constituting a particularly important strategy to develop nanomedicines with extended efficacy and specificity. This chapter addresses the current strategies explored to achieve the delivery and accumulation of bioactive molecules to targeted organelles by nanotechnology-based systems.

Introduction Colorectal cancer (CRC) is the third most commonly diagnosed cancer and the fourth cause of cancer death worldwide. It is respossible for approximately 7 00 000 deaths per year. This work focused on the development of a combinatorial multivalent nanoplatform for CRC immunotherapy and immunomodulation based on the design of polymeric nanoparticles (NP) able to deliver a combination of CRC-associated antigen, adjuvants and gene regulators according to targeted cells, dendritic cell (DC) and CRC cells. Material and methods Poly(lactic-co-glycolic) (PLGA)-based NP were prepared by the double emulsion (w/o/w) solvent evaporation method. NP were physicochemically characterised in terms of size, zeta potential and surface morphology. CRC antigen loadings were quantified by fluorescence. Immature DC (ATCC ® CRL-11904 TM ) were used to evaluate the in vitro NP cytotoxicity by the propidium iodide assay, as well as NP cellular uptake profile by flow cytometry. In vivo biodistribution assay of plain NP was also performed using the IVIS Lumina ® Bioimaging system. NP uptake in vivo by myeloid antigen presenting cells and the expression of maturation and co-stimulatory molecules at the surface of these cells sorted within draining lymph nodes, were also evaluated by flow cytometry. Results and discussions PLGA-based NP presented a mean size diameter close to 200 nm, with low polydispersity index (PdI) (≤0.200), a surface charge close to neutrality, as well as, a spherical shape and smooth surface. These multivalent delivery systems presented high loadings for antigen and adjuvants. No cytotoxic effect was observed on immature DC up to 48 hour of incubation. NP were extensively internalised by immature DC in vitro after 48 hour incubation, and by migratory DC in vivo 17 hour after animal immunisation. In vivo real-time monitoring of NP accumulation in mice whole bodies and dissected organs showed a fluorescent signal at 17 hour close to the site of immunisation and in the lymph nodes. No significant differences in the expression of the co-stimulatory CD80, CD86 and MHC class I markers on CD11b + CD11c + MHCII + population at lymph nodes were observed among different polymeric combinations upon mice immunisation with NP carrying CRC antigen and adjuvant. Conclusion According to NP physicochemical characteristics, internalisation and biodistribution patterns, this innovative nanoplatform can lead to a safe multivalent nanomedicine able to modulate dendritic cell activity and T cell expansion against tumour cells expressing entrapped antigens.

Introduction Melanoma is the most dangerous type of skin cancer and novel treatments are needed. Alternative therapeutics should be devised isolated or in combination with targeted immunotherapies, to efficiently stimulate specific anti-tumour responses. Branched polypeptides exhibit advanced engineered complexity and unique structural properties inaccessible to linear polymers that make them ideal drug delivery systems with enhanced biological performance. Branched nanosystems have the ability to activate immune cells, as dendritic cells (DC) and natural killer (NK) cells, constituting potential platforms to modulate the release profile of loaded molecules, including tumour associated antigens (TAA), adjuvants and drugs. This work aims to evaluate the in vivo anti-tumour efficacy of peptide-1 -conjugated polypeptide (pept-1-BP), with special emphasis on their impact on the modulation of the immune cell function. Material and methods BP were synthesised and conjugated with the peptide-1 (pept-1-BP) via reductive-sensitive disulfide linker. To address in vitro and in vivo studies, Cy5.5 was conjugated to platform. To evaluate the effect of the conjugate on melanoma tumour growth, B16.F10 cells were implanted subcutaneously into C57BL/6 mice. At day 7, animals were injected with two doses (1 week apart) of 100 µL of PBS, Toll-like receptor ligands CpG (20 µg/dose) and Poly I:C (40 µg/dose) in solution, BP backbone (575 µg/dose) and pept-1-BP (575 µg/dose) mixed with adjuvants. Every 2 days, weight of the mice and tumour growth was followed. At day 21, mice were sacrificed and tumour and lymph nodes were collected. A cell suspension from tumour cells and lymph nodes of each animal was prepared and analysed for infiltrated lymphocytes (CD45.1, CD3e, CD8α, CD4, CD107, PD-1, CTLA-4) by flow cytometry. Results and discussions The BP presented a size of 81.86±1.63 nm and a zeta potential of −45.10±1.72 mV, while pept-1-BP showed a mean average diameter of 104.1±2.21 nm and a zeta potential of −24.8±0.64 mV, with a pept-1 loading efficiency of 8.7% (w/w). In vivo results showed a significant reduction of tumour size in conjugate treated mice compared with the other groups. In addition, the FACS analysis of infiltrating lymphocytes within tumour site evidenced an increased expression for CD4, CD8α and NK cells. Conclusion Overall, our results support the promising use of this novel conjugate for the delivery of TAA, as an effective anti-tumour immune therapeutic strategy able to decrease and control of tumour growth.