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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.

Despite the remarkable efficiency of immune checkpoint modulators against primary and metastatic brain neoplasms, there is a low percentage of responders and clinical trials report severe immune-mediated side effects and disease relapse. An accumulated body of evidence show that non-tumor cells within the tumor microenvironment (TME), specifically the brain microenvironment (BME), including tumor vasculature and immune stromal cells, such as astrocytes, microglia and pericytes, dictate the overall therapeutic efficacy. We synthesized a biodegradable, off-the-shelf and cost-effective nano-sized polymeric platform that combines a cancer vaccine with the targeted inhibition of molecular and/or cellular immune suppressive players. These precision nano-sized medicines aim to re-educate and harness patient T-cell response against tumors, leading to an immunological memory able to control tumor relapse without any follow-up treatment. The design of these advanced peptide and RNA immunotherapeutics is guided by the identification of lead immune suppressor factors and tumor-specific antigens and proinflammatory cytokines using novel 3D bio-printed brain tumor-immune models developed in our lab. Using this unique model platform we identified P-selectin as a novel immune checkpoint in the brain regulating cancer cells-microglia and tumor-associated macrophages interactions. Combining a P-selectin inhibitor with our first nano-immunotherapy candidates sensitized brain malignancies mouse models to immune-checkpoint modulators, dramatically increasing disease-free survival rates. Citation Format: Eilam Yeini, Daniella Vaskovich-Koubi, Ron Kleiner, Helena F. Florindo, Ronit Satchi-Fainaro. Modulating the brain immune system to fight cancer in 3 dimensions [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 3243.

This commentary article conveys the views of the board of the Nanomedicine and Nanoscale Delivery Focus Group of the Controlled Release Society regarding the decision of the United States National Cancer Institute (NCI) in halting funding for the Centers of Cancer Nanotechnology Excellence (CCNEs), and the subsequent editorial articles that broadened this discussion.

The small intestine hosts specialized lymphoid structures, the Peyer's patches, that face the gut lumen and are overlaid with unique epithelial cells, called microfold (M) cells. M cells are considered to constitute an important route for antigen uptake in the mucosal immune system. Here, we used intravital microscopy to define immune cell populations, which are in close contact with M cells and potentially sample antigen. We present live evidence that DCs enter M cell pockets and highlight the abundance of mononuclear phagocytes in these structures. Taking advantage of the respective reporter animals, we focused on classical DCs that express Zbtb46 and analyzed how these cells interact with M cells in steady state and sample antigen for T cell activation in the Peyer's patches following challenge.

Development of resistance to chemo- and immunotherapies often occurs following treatment of melanoma brain metastasis (MBM). The brain microenvironment (BME), particularly astrocytes, cooperate toward MBM progression by upregulating secreted factors, among which we found that monocyte chemoattractant protein-1 (MCP-1) and its receptors, CCR2 and CCR4, were overexpressed in MBM compared with primary lesions. Among other sources of MCP-1 in the brain, we show that melanoma cells altered astrocyte secretome and evoked MCP-1 expression and secretion, which in turn induced CCR2 expression in melanoma cells, enhancing in vitro tumorigenic properties, such as proliferation, migration, and invasion of melanoma cells. In vivo pharmacological blockade of MCP-1 or molecular knockout of CCR2/CCR4 increased the infiltration of cytotoxic CD8+ T cells and attenuated the immunosuppressive phenotype of the BME as shown by decreased infiltration of Tregs and tumor-associated macrophages/microglia in several models of intracranially injected MBM. These in vivo strategies led to decreased MBM outgrowth and prolonged the overall survival of the mice. Our findings highlight the therapeutic potential of inhibiting interactions between BME and melanoma cells for the treatment of this disease.

In current times, DNA vaccines are seen as a promising approach to treat and prevent diseases, such as virus infections and cancer. Aiming at the production of a functional and effective plasmid DNA (pDNA) delivery system, four chitosan polymers, differing in the molecular weight, were studied using the design of experiments (DoE) tool. These gene delivery systems were formulated by ionotropic gelation and exploring the chitosan and TPP concentrations as DoE inputs to maximize the nanoparticle positive charge and minimize their size and polydispersity index (PDI) as DoE outputs. The obtained linear and quadratic models were statistically significant (p-value &lt, 0.05) and non-significant lack of fit, with suitable coefficient of determination and the respective optimal points successfully validated. Furthermore, morphology, stability and cytotoxicity assays were performed to evaluate the endurance of these systems over time and their further potential for future in vitro studies. The subsequent optimization process was successful achieved for the delivery systems based on the four chitosan polymers, in which the smallest particle size was obtained for the carrier containing the 5 kDa chitosan (~82 nm), while the nanosystem prepared with the high molecular weight (HMW) chitosan displayed the highest zeta potential (~+26.8 mV). Delivery systems were stable in the formulation buffer after a month and did not exhibit toxicity for the cells. In this sense, DoE revealed to be a powerful tool to explore and tailor the characteristics of chitosan/pDNA nanosystems significantly contributing to unraveling an optimum carrier for advancing the DNA vaccines delivery field.

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.

Selenium (Se) is an essential element to human health that can be obtained in nature through several sources. In the human body, it is incorporated into selenocysteine, an amino acid used to synthesize several selenoproteins, which have an active center usually dependent on the presence of Se. Although Se shows several beneficial properties in human health, it has also a narrow therapeutic window, and therefore the excessive intake of inorganic and organic Se-based compounds often leads to toxicity. Nanoparticles based on Se (SeNPs) are less toxic than inorganic and organic Se. They are both biocompatible and capable of effectively delivering combinations of payloads to specific cells following their functionalization with active targeting ligands. Herein, the main origin of Se intake, its role on the human body, and its primary biomedical applications are revised. Particular focus will be given to the main therapeutic targets that are explored for SeNPs in cancer therapies, discussing the different functionalization methodologies used to improve SeNPs stability, while enabling the extensive delivery of drug-loaded SeNP to tumor sites, thus avoiding off-target effects.

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.

Human pluripotent stem cells , which comprise embryonic stem cells and induced pluripotent stem cells (iPSCs), may become a potential renewable source of tissues and cells in the future, given their ability to expand and differentiate into multiple somatic cell types. In particular, iPSCs offer the possibility of growing patient-specific cells with the aim to achieve potential therapies or even cures for a variety of diseases. In the past years, progress has been made in the field of iPSCs regarding their differentiation into pancreatic cell phenotypes with a great focus on insulin-producing cells. In this chapter, we will give a brief background of the pancreatic niche and discuss iPSCs potential in pancreatic regeneration. Further, iPSCs differentiation into β- and α-cells will be highlighted, as well as their prospective role in diabetes treatment. Lastly, current challenges regarding pancreatic cell differentiation and future perspectives will also be addressed.

Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-related death. Low response rate, acquired resistance, severe side effects, highly desmoplastic, and immunosuppressive microenvironment have limited the clinical outcomes of immunotherapy in PDAC. To overcome these hurdles, we set to design novel immunotherapy that would be safe, efficacious, and selective to PDAC. The failure of immunotherapies to yield clinical response in PDAC is partially attributed to the inefficient antigen presentation by dendritic cells (DC) which results in insufficient T cells stimulation despite their expression of tumor-associated antigens (TAA). DC have an important role in the initiation and regulation of the innate and adaptive immune system. By efficient presentation of antigens, DC elicit an antigen-specific immune response. Trp53R172H is among the most common TP53 mutations in human PDAC, and is known as one of PDAC TAA. Despite the poor clinical outcomes, longer survival time was observed in PDAC patients with tumors that express high number of TAA combined with an abundant presentation of CD8+ T cell. To that end, we designed a DC-targeted nanoparticle (NP) containing PDAC Trp53R172H TAA and immune potentiators, with the DC targeting moiety, mannose, that was covalently conjugated to the NP, to enhance TAA presentation on DC. Thus, promote specific activation of T cells against PDAC’s TAA. The therapeutic activity of the NP was evaluated in vitro by our newly established 3D PDAC spheroids model, which composed of freshly isolated murine stromal cells, therefore, better imitates the complex microenvironment found in PDAC in vitro. Along with an in vivo PDAC model, we evaluated the safety profile and therapeutic effect of our NP. The NP proposed herein successfully activated splenocytes and induced a specific cytotoxic reaction against PDAC cells ex vivo, which may result in an improved survival rate. Citation Format: Daniella Vaskovich-Koubi, Ron kleiner, Anat Eldar-Boock, Sabina Pozzi, Dikla Dikla Ben-Shushan, Helena F. Florindo, Ronit Satchi-Fainaro. Designing a dendritic cell-targeted immunotherapy for the treatment of pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3520.

Nanoparticulate vaccines are promising tools to overcome cancer immune evasion. However, a deeper understanding on nanoparticle-immune cell interactions and treatments regime is required for optimal efficacy. We provide a comprehensive study of treatment schedules and mode of antigen-association to nanovaccines on the modulation of T cell immunity in vivo, under steady-state and tumor-bearing mice. The coordinated delivery of antigen and two adjuvants (Monophosphoryl lipid A, oligodeoxynucleotide cytosine-phosphate-guanine motifs (CpG)) by nanoparticles was crucial for dendritic cell activation. A single vaccination dictated a 3-fold increase on cytotoxic memory-T cells and raised antigen-specific immune responses against B16.M05 melanoma. It generated at least a 5-fold increase on IFN-γ cytokine production, and presented over 50% higher lymphocyte count in the tumor microenvironment, compared to the control. The number of lymphocytes at the tumor site doubled with triple immunization. This lymphocyte infiltration pattern was confirmed in mammary huHER2 carcinoma, with significant tumor reduction.

Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-related death. Low response rate, acquired resistance, severe side effects, highly desmoplastic and immunosuppressive microenvironment have limited the clinical outcomes of immunotherapy in PDAC. To overcome these hurdles, we set to design a novel immunotherapy that would be safe, efficacious, and selective to PDAC. The failure of immunotherapies to yield clinical response in PDAC is partially attributed to the inefficient antigen presentation by dendritic cells (DC) which results in insufficient T cells stimulation despite their expression of tumor-associated antigens (TAA). DC have an important role in the initiation and regulation of the innate and adaptive arms of the immune system. By efficient presentation of antigens, DC elicit an antigen-specific immune response. Trp53R172H is among the most common TP53 mutations in human PDAC, and is known as one of PDAC TAA. Despite the poor clinical outcomes, longer survival time was observed in PDAC patients with tumors that express high number of TAA combined with an abundant presentation of CD8+ T cell. To that end, we designed a DC-targeted nanoparticle (NP) containing PDAC Trp53R172H TAA and immune potentiators, with the DC targeting moiety, mannose, that was covalently conjugated to the NP, to enhance TAA presentation on DC. This approach led to specific activation of T cells against PDAC's TAA. The therapeutic activity of the NP was evaluated in vitro by our newly established 3D PDAC spheroids model, which is composed of freshly isolated murine stromal cells, therefore, better imitates the complex microenvironment found in PDAC. Along with an in vivo PDAC model, we evaluated the safety profile and therapeutic efficacy of our NP. The NP proposed herein successfully activated splenocytes and induced a specific cytotoxic reaction against PDAC cells ex vivo, which may result in improved survival rate. Citation Format: Ron Kleiner, Daniella Vaskovich, Sabina Pozzi, Anat Eldar-Boock, Dikla Ben-Shushan, Helena Florindo, Ronit Satchi-Fainaro. Designing a dendritic cell-targeted immunotherapy for the treatment of pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1746.

Dendritic cells (DC) are antigen-presenting cells with an important role in the initiation and regulation of the innate and adaptive arms of the immune system. By efficient presentation of antigens, DC elicit an antigen-specific immune response. Therefore, we previously established in our laboratory a melanoma-DC-targeted NP that was evaluated and validated against aggressive melanoma. Although DC-therapy is mainly exploited in the treatment of cancer, DC are becoming a key therapeutic target in other pathologies such as infectious diseases. Viral pathogens, similarly, to cancer, modulate the host defense mechanisms to their benefit. COVID-19, an ongoing pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread globally and is already infecting tens of millions of people worldwide. Historically, vaccines are among therapies with the greatest impact in health and constitute an unmet medical need against coronavirus disease-19 (COVID-19) pandemic. To that end, we repurposed our recently published melanoma-targeted NP to co-deliver modulators of host immune response and SARS-CoV-2 specific antigen epitopes discovered by a computational approach integrating 3D information with large-scale statistical analysis. The immune response kinetics in patients infected with SARS-CoV-2 is poorly understood, but recent reports show that germinal B cells and active follicular T-helper cells, IgM/IgG SARS-CoV-2 antibodies, in addition to an enhanced cellular immunity, are found in patients with mild-to-moderate SARS-CoV-2 infection. Our preliminary data demonstrated that our nano-vaccine was able to activate specific immune cell response of effector T cells and B cells and decrease the regulatory activity of T cells, compared to controls. Moreover, we found that our DC-targeted SARS-CoV-2 NP successfully induced antigen-specific secretion of IgM and IgG antibodies with high binding affinity to the antigens. Importantly, our nano-vaccine elicited specific neutralization of Spike virus like particles (VLPs) by sera of immunized mice. We hypothesize that implementing our novel nano-vaccine platform for COVID-19 will lead to a safe and efficacious COVID-19 vaccine ready for first-in-human clinical trials. Citation Format: Daniella Vaskovich-Koubi, Ron Kleiner, Yulia Liubomirski, Eilam Yeini, Galia Tiram, Sabina Pozzi, Anat Eldar-Boock, Helena F. Florindo, Ronit Satchi-Fainaro. From cancer to COVID-19- development of a dendritic cell-targeted nano-vaccine for prevention and therapy of COVID-19 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 714.

Nanotechnology-based strategies can dramatically impact the treatment, prevention and diagnosis of a wide range of diseases. Despite the unprecedented success achieved with the use of nanomaterials to address unmet biomedical needs and their particular suitability for the effective application of a personalized medicine, the clinical translation of those nanoparticulate systems has still been impaired by the limited understanding on their interaction with complex biological systems. As a result, unexpected effects due to unpredicted interactions at biomaterial and biological interfaces have been underlying the biosafety concerns raised by the use of nanomaterials. This review explores the current knowledge on how nanoparticle (NP) physicochemical and surface properties determine their interactions with innate immune cells, with particular attention on the activation of pattern-recognition receptors and inflammasome. A critical perspective will additionally address the impact of biological systems on the effect of NP on immune cell activity at the molecular level. We will discuss how the understanding of the NP-innate immune cell interactions can significantly add into the clinical translation by guiding the design of nanomedicines with particular effect on targeted cells, thus improving their clinical efficacy while minimizing undesired but predictable toxicological effects.

Nuno Martinho,1–3 Liana C Silva,1,4 Helena F Florindo,1 Steve Brocchini,2 Mire Zloh,3 Teresa S Barata2 1Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, Lisbon, Portugal; 2Department of Pharmaceutics, UCL School of Pharmacy, London, 3School of Life and Medical Sciences, University of Hertfordshire, Hatfield, UK; 4Centro de Química-Física Molecular and IN – Institute of Nanoscience and Nanotechnology, Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal Abstract: Dendrimers are hyperbranched polymers with a multifunctional architecture that can be tailored for the use in various biomedical applications. Peptide dendrimers are particularly relevant for drug delivery applications due to their versatility and safety profile. The overall lack of knowledge of their three-dimensional structure, conformational behavior and structure–activity relationship has slowed down their development. Fluorophores are often conjugated to dendrimers to study their interaction with biomolecules and provide information about their mechanism of action at the molecular level. However, these probes can change dendrimer surface properties and have a direct impact on their interactions with biomolecules and with lipid membranes. In this study, we have used computer-aided molecular design and molecular dynamics simulations to identify optimal topology of a poly(L-glutamic acid) (PG) backbone dendrimer that allows incorporation of fluorophores in the core with minimal availability for undesired interactions. Extensive all-atom molecular dynamic simulations with the CHARMM force field were carried out for different generations of PG dendrimers with the core modified with a fluorophore (nitrobenzoxadiazole and Oregon Green 488) and various surface groups (glutamic acid, lysine and tryptophan). Analysis of structural and topological features of all designed dendrimers provided information about their size, shape, internal distribution and dynamic behavior. We have found that four generations of a PG dendrimer are needed to ensure minimal exposure of a core-conjugated fluorophore to external environment and absence of undesired interactions regardless of the surface terminal groups. Our findings suggest that NBD-PG-G4 can provide a suitable scaffold to be used for biophysical studies of surface-modified dendrimers to provide a deeper understanding of their intermolecular interactions, mechanisms of action and trafficking in a biological system. Keywords: dendrimers, peptide dendrimers, molecular dynamics, fluorescence, CHARMM, structure-activity, surface properties

Dendrimers and dendrons offer an excellent platform for developing novel drug delivery systems and medicines. The rational design and further development of these repetitively branched systems are restricted by difficulties in scalable synthesis and structural determination, which can be overcome by judicious use of molecular modelling and molecular simulations. A major difficulty to utilise in silico studies to design dendrimers lies in the laborious generation of their structures. Current modelling tools utilise automated assembly of simpler dendrimers or the inefficient manual assembly of monomer precursors to generate more complicated dendrimer structures. Herein we describe two novel graphical user interface toolkits written in Python that provide an improved degree of automation for rapid assembly of dendrimers and generation of their 2D and 3D structures. Our first toolkit uses the RDkit library, SMILES nomenclature of monomers and SMARTS reaction nomenclature to generate SMILES and mol files of dendrimers without 3D coordinates. These files are used for simple graphical representations and storing their structures in databases. The second toolkit assembles complex topology dendrimers from monomers to construct 3D dendrimer structures to be used as starting points for simulation using existing and widely available software and force fields. Both tools were validated for ease-of-use to prototype dendrimer structure and the second toolkit was especially relevant for dendrimers of high complexity and size.

Polymer-directed enzyme prodrug therapy (PDEPT) and polymer enzyme liposome therapy (PELT) are two-step therapies developed to provide anticancer drugs site-selective intratumoral accumulation and release. Nanomedicines, such as polymer-drug conjugates and liposomal drugs, accumulate in the tumor site due to extravasation-dependent mechanism (enhanced permeability and retention - EPR - effect), and further need to cross the cellular membrane and release their payload in the intracellular compartment. The subsequent administration of a polymer-enzyme conjugate able to accumulate in the tumor tissue and to trigger the extracellular release of the active drug showed promising preclinical results. The development of polymer-enzyme, polymer-drug conjugates and liposomal drugs had undergone a vast advancement over the past decades. Several examples of enzyme mimics for in vivo therapy can be found in the literature. Moreover, polymer therapeutics often present an enzyme-sensitive mechanism of drug release. These nanomedicines can thus be optimal substrates for PDEPT and this review aims to provide new insights and stimuli toward the future perspectives of this promising combination.

Herein is described a new modular platform for the construction of cancer-cell-targeting drug conjugates. Tripodal boronate complexes featuring reversible covalent bonds were designed to accommodate a cytotoxic drug (bortezomib), poly(ethylene glycol) (Peg) chains, and folate targeting units. The B-complex core was assembled in one step, proved stable under biocompatible conditions, namely, in human plasma (half-life up to 60 h), and underwent disassembly in the presence of glutathione (GSH). Stimulus-responsive intracellular cargo delivery was confirmed by confocal fluorescence microscopy, and a mechanism for GSH-induced B-complex hydrolysis was proposed on the basis of mass spectrometry and DFT calculations. This platform enabled the modular construction of multivalent conjugates with high selectivity for folate-positive MDA-MB-231 cancer cells and IC50 values in the nanomolar range.

Vaccination is a promising strategy to trigger and boost immune responses against cancer or infectious disease. We have designed, synthesized and characterized aliphatic-polyester (poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NP) to investigate how the nature of protein association (adsorbed versus entrapped) and polymer/surfactant concentrations impact on the generation and modulation of antigen-specific immune responses. The ability of the NP formulations to target dendritic cells (DC), be internalized and activate the T cells was characterized and optimized in vitro and in vivo using markers of DC activation and co-stimulatory molecules. Ovalbumin (OVA) was used as a model antigen in combination with the engraftment of CD4+ and CD8+ T cells, carrying a transgenic OVA-responding T cell receptor (TCR), to trace and characterize the activation of antigen-specific CD4+ and CD8+ lymph node T cells upon NP vaccination. Accordingly, the phenotype and frequency of immune cell stimulation induced by the NP loaded with OVA, isolated or in combination with synthetic unmethylated cytosine-phosphate-guanine (CpG) oligodeoxynucleotide (ODN) motifs, were characterized. DC-NP interactions increased with incubation time, presenting internalization values between 50 and 60% and 30-40%, in vitro and in vivo, respectively. Interestingly, animal immunization with antigen-adsorbed NP up-regulated major histocompatibility complex (MHC) class II (MHCII), while NP entrapping the antigen up-regulated MHCI, suggesting a more efficient cross-presentation. On the other hand, rather surprisingly, the surfactant used in the NP formulation had a major impact on the activation of antigen presenting cells (APC). In fact, DC collected from lymph nodes of animals immunized with NP prepared using poly(vinil alcohol) (PVA), as a surfactant, expressed significantly higher levels of CD86, MHCI and MHCII. In addition, those NP prepared with PVA and co-entrapping OVA and the toll-like receptor (TLR) ligand CpG, induced the most profound antigen-specific T cell response, by both CD4+ and CD8+ T cells, in vivo. Overall, our data reveal the impact of NP composition and surface properties on the type and extension of induced immune responses. Deeper understanding on the NP-immune cell crosstalk can guide the rational development of nano-immunotherapeutic systems with improved and specific therapeutic efficacy and avoiding off-target effects.

Dendritic cells (DC) are unrivaled in their potential to prime naive T cells by presenting antigen and providing costimulation. DC are furthermore believed to decode antigen context by virtue of pattern recognition receptors and to polarize T cells through cytokine secretion toward distinct effector functions. Diverse polarized T helper (TH) cells have been explored in great detail. In contrast, studies of instructing DC have to date largely been restricted to in vitro settings or adoptively transferred DC. Here we report efforts to unravel the DC response to cognate T cell encounter in antigen-challenged lymph nodes (LN). Mice engrafted with antigen-specific T cells were immunized with nanoparticles (NP) entrapping adjuvants and absorbed with antigen to study the immediate DC response to T cell encounter using bulk and single cell RNA-seq profiling. NP induced robust antigen-specific TH1 cell responses with minimal bystander activation. Fluorescent-labeled NP allowed identification of antigen-carrying DC and focus on transcriptional changes in DC that encounter T cells. Our results support the existence of a bi-directional crosstalk between DC and T cells that promotes TH1 responses, including involvement of the ubiquitin-like molecule Isg15 that merits further study.

Cisplatin and other platinum(II) analogs are widely used in clinical practice as anti-cancer drugs for a wide range of tumors. The primary mechanism by which they exert their action is through the formation of adducts with genomic DNA. However, multiple cellular targets by platinum(II) complexes have been described. In particular, the early events occurring at the plasma membrane (PM), i.e., platinum-membrane interactions seem to be involved in the uptake, cytotoxicity and cell-resistance to cisplatin. In fact, PM influences signaling events, and cisplatin-induced changes on membrane organization and fluidity were shown to activate apoptotic pathways. This review critically discusses the sequence of events caused by lipid membrane-platinum interactions, with emphasis on the mechanisms that lead to changes in the biophysical properties of the membranes (e.g., fluidity and permeability), and how these correlate with sensitivity and resistance phenotypes of cells to platinum(II) complexes.

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.

Well-defined synthetic branched nanostructures form an emerging subclass of macromolecular structures, whose 3D structure and multivalency offer unique opportunities for fine-tuning their internalization and cellular targeting. In particular, dendrimers possess a well-defined 3D-globular backbone with highly versatile functional surface groups and exhibit a range of chemical and biological properties. Branched polymers present unique opportunities for the targeted delivery of diverse bioactive molecules (including targeting ligands, imaging probes, and drugs) via conjugation to multiple sites within the structure. The inherent versatility and multifunctionality of these architectures make them potentially useful for the modulation of multiple immune-related pathways for the treatment of a wide range of disease and disorders, including cancer and human immunodeficiency virus infection. Herein, we describe the key components of the immune system whose targeting can help to overcome immune-related disorders and discuss branched polymers (including dendrimers) as promising delivery systems with unique immunomodulatory properties against cancer and infectious diseases. &nbsp

In preceding decades, different mechanisms have been proposed to "instruct" dendritic cells (DCs) to induce immune responses against tumor antigens (TAs), thus breaking immune tolerance. Immunotherapy has been, for the last two decades, an attractive and promising therapeutic approach to fight cancer. This review will approach the nature of the immune response during cancer development and its correlation with DC function, as well as cancer vaccine principles and limitations. An overview of several delivery strategies used for in vivo modulation of DCs and direct activation of T cells will be provided, highlighting their advantages, limitations, and optimization strategies. This manuscript also presents a critical and systematic review of recent clinical trials that are investigating the therapeutic effect of these approaches, discussing prognostic outcomes of combined-treatment modalities.

This paper discusses the development of a multivariate-based regression model for estimating the critical attributes to establish a design-space for poly(lactic-co-glycolic acid) (PLGA) nanoparticles formulated by a double emulsion–solvent evaporation method. A three-level, full factorial experimental design is used to assess the impact of three different manufacturing conditions (polymer viscosity, surfactant concentration and amount of model antigen ovalbumin) on five critical particle attributes (zeta potential, polydispersity index, hydrodynamic diameter, loading capacity and entrapment efficiency). The optimized formulation was achieved with a viscosity of 0.6 dl g−1, a surfactant concentration of 11% (w/v) in the internal phase and 2.5% (w/w) of ovalbumin. The design-space that is satisfied for nanoparticles with the targeted attributes was obtained with a polymer viscosity between 0.4 and 0.9 dl g−1, a surfactant concentration ranging from 8 to 15% (w/v) and 2.5% (w/w) of ovalbumin. The nanoparticles were spherical and homogenous and were extensively taken up by JAWS II murine immature dendritic cells without affecting the viability of these phagocytic cells. Better understanding was achieved by multivariate regression to control process manufacturing to optimize PLGA nanoparticle formulation. Utilization of multivariate regression with a defined control space is a good tool to meet product specifications, particularly over a narrow variation range.

α-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-γ 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 α-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.

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.

The modulation of immune checkpoint receptors has been one of the most successful, exciting, and explored approaches for cancer immunotherapy. Currently, several immune checkpoint modulators, mainly monoclonal antibodies, are showing remarkable results. However, the failure to show a response in most patients and the induction of severe immune-related adverse effects are the major drawbacks. Novel approaches concerning the development of immune modulatory small molecules have emerged as an alternative. Nevertheless, the lack of structural information about immune checkpoint receptors has hindered the rational design of those small-molecule modulators by preventing the use of methodologies such as computer-aided drug design. Herein, we provide an overview and critical analysis of the structural and dynamic details of immune checkpoint receptors (cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4), programmed cell death protein 1 (PD-1), and glucocorticoid-induced TNFR-related protein (GITR)) and their interaction with known modulators. This knowledge is essential to advance the understanding of their binding mode and guide the design of novel effective targeted anticancer medicines.

Energy transfer cassettes that build on the platform of boronic acid derived salicylidenehydrazone (BASHY) complexes were prepared. The functional flexibility of the BASHY chromophore was underpinned by its tunable role as energy donor or acceptor, integrated in compact and non‐conjugated bichromophoric dyads. The energy transfer is highly efficient (ΦET>0.95) and is assumed to proceed mainly via a through‐bond mechanism. Both constituent chromophores benefit mutually from their integration in the cassettes: a) The pseudo Stokes shift is increased to 110–200 nm; b) the antenna (donor) chromophore improves the light absorption of the acceptor chromophore; and c) the emission window of the BASHY chromophore is expanded in the BASHY‐Bodipy dye without using strategies that compromise the observation of high quantum yields. The application of the cassettes for the formulation of fluorescent polymeric nanoparticles, that can be internalized in cells and observed by fluorescence imaging, was demonstrated using the BASHY‐Bodipy dye as an example., The financial support of the Portuguese Fundac ̧a ̃o para a Cieˆnciae a Tecnologia (FCT) (grants SFRH/BD/94779/2013, PTDC/QEQ-QOR/1434/2014, PTDC/QEQ-MED/5512/2014, FEDER-029967,SAICTPAC/0019/2015, iMed.ULisboa grant UID/DTP/04138/2013,UTAP-ICDT/DTP-FTO/0016/2014, PhD fellowship PD/BD/113959/20for A.I.M), the Spanish Ministerio de Ciencia, Innovacio ́nyUniversidades (grants CTQ2014-54729-C2-1-P, CTQ2017-89832-Pand PhD fellowship BES-2015-074458 for Z.D.), the Junta deAndalucı ́a (P12-FQM-2140), the ERDF, and the Academy of Finland(decision 287954) is gratefully acknowledged. CSC–IT Center forScience Ltd, Finland is thanked for the allocation of computa-tional resources

Development of RNA interference-based therapies with appropriate therapeutic window remains a challenge for advanced cancers. Because cancer stem cells (CSC) are responsible of sustaining the metastatic spread of the disease to distal organs and the progressive gain of resistance of advanced cancers, new anticancer therapies should be validated specifically for this subpopulation of cells. A new amphihilic-based gene delivery system that combines Pluronic® F127 micelles with polyplexes spontaneously formed by electrostatic interaction between anionic siRNA and cationic polyethylenimine (PEI) 10K, was designed (PM). Resultant PM gather the requirements for an efficient and safe transport of siRNA in terms of its physicochemical characteristics, internalization capacity, toxicity profile and silencing efficacy. PM were loaded with a siRNA against AKT2, an important oncogene involved in breast cancer tumorigenesis, with a special role in CSC malignancy. Efficacy of siAKT2-PM was validated in CSC isolated from two breast cancer cell lines: MCF-7 and Triple Negative MDA-MB-231 corresponding to an aggressive subtype of breast cancer. In both cases, we observed significant reduction on cell invasion capacity and strong inhibition of mammosphere formation after treatment. These results prompt AKT2 inhibition as a powerful therapeutic target against CSC and pave the way to the appearance of more effective nanomedicine-based gene therapies aimed to prevent CSC-related tumor recurrence.

Cancer is a heterogeneous disease that results from a multi-step process, being characterized by uncontrolled proliferation, invasion and metastasis. The understanding that tumor cells can be recognized by host immune cells has highlighted the potential advantages of using vaccination purposes to eliminate cancer cells, while avoiding severe side effects associated to conventional cancer treatments. Interesting outcomes have been obtained with the new identified tumor associated antigens (TAAs), including recombinant proteins and peptides. However, these molecules are weakly immunogenic, demanding the concomitant use of adjuvants to boost and achieve a strong tumor-specific immune response. Different classes of nanosystems have been used to protect and deliver several vaccine components. In vitro and preclinical studies have emphasized their promising role to attain a prolonged eradication of cancer cells, including metastasis. However, some studies support the co-entrapment of multiple adjuvants and TAAs within a single particulate carrier, while others indicate that stronger immune responses were obtained using a mixture of nanocarriers entrapping different combinations of TAAs and adjuvants. These apparently contradictory results may be related to nanocarrier physicochemical properties, which have a profound impact on their interaction with targeted cells and consequent biological effects. This review discusses the application of nanoscale systems as cancer vaccines, highlighting the particular characteristics of tumor biology and immunology that have been used to guide the design of these nanodelivery tools. We also aim to explore the major weaknesses that have prevented their wide application in the clinic to overcome the delivery, efficacy and safety issues associated to biological entities.