Helena F. Florindo

Immune system targeting by biodegradable nanoparticles for cancer vaccines

The concept of therapeutic cancer vaccines is based on the activation of the immune system against tumor cells after the presentation of tumor antigens. Nanoparticles (NPs) have shown great potential as delivery systems for cancer vaccines as they potentiate the co-delivery of tumor-associated antigens and adjuvants to dendritic cells (DCs), insuring effective activation of the immune system against tumor cells. In this review, the immunological mechanisms behind cancer vaccines, including the role of DCs in the stimulation of T lymphocytes and the use of Toll-like receptor (TLR) ligands as adjuvants will be discussed. An overview of each of the three essential components of a therapeutic cancer vaccine - antigen, adjuvant and delivery system - will be provided with special emphasis on the potential of particulate delivery systems for cancer vaccines, in particular those made of biodegradable aliphatic polyesters, such as poly(lactic-co-glycolic acid) (PLGA) and poly-ε-caprolactone (PCL). Some of the factors that can influence NP uptake by DCs, including size, surface charge, surface functionalization and route of administration, will also be considered.

The enhancement of the immune response against S. equi antigens through the intranasal administration of poly-epsilon-caprolactone-based nanoparticles

Strangles is a bacterial infection of the Equidae family that affects the nasopharynx and draining lymph nodes, caused by Streptococcus equi subspecies equi. This agent is responsible for 30% of all worldwide equine infections and is quite sensitive to penicillin and other antibiotics. However, prevention is still the best option because the current antibiotic therapy and vaccination is often ineffective. As S. equi induces very strong systemic and mucosal responses in convalescent horses, an effective and economic strangles vaccine is still a priority. In this study the humoral, cellular and mucosal immune responses to S. equi antigens encapsulated or adsorbed onto poly-epsilon-caprolactone nanospheres were evaluated in mice. Particles were produced by a double (w/o/w) emulsion solvent evaporation technique and contained mucoadhesive polymers (alginate or chitosan) and absorption enhancers (spermine, oleic acid). Their intranasal administration, particularly those constituted by the mucoadhesive polymers, increased the immunogenicity and mucosal immune responses (SIgA) to the antigen. The inclusion of cholera toxin B subunit in the formulations successfully further activated the paths leading to Th1 and Th2 cells. Therefore, those PCL nanospheres are potential carriers for the delivery of S.equi antigens to protect animals against strangles.

Cancer immunotherapy: nanodelivery approaches for immune cell targeting and tracking

Cancer is one of the most common diseases afflicting people globally. New therapeutic approaches are needed due to the complexity of cancer as a disease. Many current treatments are very toxic and have modest efficacy at best. Increased understanding of tumor biology and immunology has allowed the development of specific immunotherapies with minimal toxicity. It is important to highlight the performance of monoclonal antibodies, immune adjuvants, vaccines and cell-based treatments. Although these approaches have shown varying degrees of clinical efficacy, they illustrate the potential to develop new strategies. Targeted immunotherapy is being explored to overcome the heterogeneity of malignant cells and the immune suppression induced by both the tumor and its microenvironment. Nanodelivery strategies seek to minimize systemic exposure to target therapy to malignant tissue and cells. Intracellular penetration has been examined through the use of functionalized particulates. These nano-particulate associated medicines are being developed for use in imaging, diagnostics and cancer targeting. Although nano-particulates are inherently complex medicines, the ability to confer, at least in principle, different types of functionality allows for the plausible consideration these nanodelivery strategies can be exploited for use as combination medicines. The development of targeted nanodelivery systems in which therapeutic and imaging agents are merged into a single platform is an attractive strategy. Currently, several nanoplatform-based formulations, such as polymeric nanoparticles, micelles, liposomes and dendrimers are in preclinical and clinical stages of development. Herein, nanodelivery strategies presently investigated for cancer immunotherapy, cancer targeting mechanisms and nanocarrier functionalization methods will be described. We also intend to discuss the emerging nano-based approaches suitable to be used as imaging techniques and as cancer treatment options.

Regulatory aspects on nanomedicines

Nanomedicines have been in the forefront of pharmaceutical research in the last decades, creating new challenges for research community, industry, and regulators. There is a strong demand for the fast development of scientific and technological tools to address unmet medical needs, thus improving human health care and life quality. Tremendous advances in the biomaterials and nanotechnology fields have prompted their use as promising tools to overcome important drawbacks, mostly associated to the non-specific effects of conventional therapeutic approaches. However, the wide range of application of nanomedicines demands a profound knowledge and characterization of these complex products. Their properties need to be extensively understood to avoid unpredicted effects on patients, such as potential immune reactivity. Research policy and alliances have been bringing together scientists, regulators, industry, and, more frequently in recent years, patient representatives and patient advocacy institutions. In order to successfully enhance the development of new technologies, improved strategies for research-based corporate organizations, more integrated research tools dealing with appropriate translational requirements aiming at clinical development, and proactive regulatory policies are essential in the near future. This review focuses on the most important aspects currently recognized as key factors for the regulation of nanomedicines, discussing the efforts under development by industry and regulatory agencies to promote their translation into the market. Regulatory Science aspects driving a faster and safer development of nanomedicines will be a central issue for the next years.

In vivo delivery of peptides and Toll-like receptor ligands by mannose-functionalized polymeric nanoparticles induces prophylactic and therapeutic anti-tumor immune responses in a melanoma model.

We hypothesized that the co-entrapment of melanoma-associated antigens and the Toll-like receptor (TLR) ligands Poly(I:C) and CpG, known to be Th1-immunopotentiators, in mannose-functionalized aliphatic polyester-based nanoparticles (NPs) could be targeted to mannose receptors on antigen-presenting cells and induce anti-tumor immune responses. High entrapment efficiencies of antigens and immunopotentiators in 150nm NPs were obtained. The co-entrapment of the model antigen ovalbumin and the TLR ligands was crucial to induce high IgG2c/IgG1 ratios and high levels of IFN-γ and IL-2. Mannose-functionalization of NPs potentiated the Th1 immune response. The nanoparticulate vaccines decreased the growth rate of murine B16F10 melanoma tumors in therapeutic and prophylatic settings. The combination of mannose-functionalized NPs containing MHC class I- or class II-restricted melanoma antigens and the TLR ligands induced the highest tumor growth delay. Overall, we demonstrate that the multifunctional properties of NPs in terms of targeting and antigen/adjuvant delivery have high cancer immunotherapeutic potential.

Streptococcus equi antigens adsorbed onto surface modified poly-ɛ-caprolactone microspheres induce humoral and cellular specific immune responses

Streptococcus equi subsp. equi is the causative agent of Strangles, which is one of the most costly and widespread infectious diseases, affecting the respiratory tract of Equidae. In this work, polyvinyl alcohol, alginate and chitosan were used in formulations of surface modified poly-epsilon-caprolactone microspheres which were evaluated after adsorption of S.equi enzymatic extract for physicochemical characteristics and in vivo immune responses in mice. After subcutaneous immunisation, the formulations induced higher lymphokines levels, in accordance with cellular and humoral immune responses, as compared to the free antigen, successfully activating the paths leading to Th1 and Th2 cells. The obtained results highlight the role of these microspheres as an adjuvant and their use to protect animals against strangles.

New approach on the development of a mucosal vaccine against strangles: systemic and mucosal immune responses in a mouse model.

Streptococcus equi subspecies equi affects animals of Equidae family and is the causative agent of strangles, an acute, extremely contagious and deadly disease. Prolonged periods of protection associated to absence of serious adverse reactions were not yet achieved. Thus, this experimental work is focused on the study of mucosal, humoral and cellular immune responses developed in a mouse model, after the intranasal administration of S. equi antigens associated by adsorption or encapsulation to poly(lactic acid) nanospheres, modified by mucoadhesive polymers and absorption enhancers. Particles fitted the nanometer range and proteins integrity and antigenicity were not affected. PLA nanospheres induced a mixed Th1 and Th2 response, being therefore potential carriers for the delivery of S. equi antigens.

Antibody and cytokine-associated immune responses to S. equi antigens entrapped in PLA nanospheres

Strangles is an infectious disease caused by Streptococcus equi subspecies equi that affects the upper respiratory tract of the Equidae. The control of this disease seems to be dependent on its earlier detection and prevention, but prolonged animal protection without development of strong and severe side effects has not yet been achieved. Convalescent horses exhibit a protective immune response, mainly against SeM (58 kDa), an antiphagocytic and opsonogenic S. equi M-like protein, known as the major protective antigen against strangles. Purified recombinant SeM and S. equi protein extract-entrapped poly(lactic acid) (PLA) nanospheres were developed and their adjuvant potential was studied via the intramuscular route. The effect including molecules with adjuvant properties such as spermine, oleic acid, alginate and glycol-chitosan was also evaluated. Spherical nanometric particles <500 nm containing the protein antigen were prepared by the solvent evaporation method and protein structure was not affected throughout preparation. The humoral immune response induced by nanospheres was markedly higher than that elicited by soluble antigens, isolated or co-admixed with CpG. The IgG and IgG subtypes, along with cytokine titres, indicated that nanospheres composed by glycolchitosan developed a more balanced Th1/Th2 response for both purified SeM and S. equi enzymatic extract proteins, although those induced by the pure antigen-entrapped particles were higher than the S. equi tested vaccines composed by total antigens entrapped in polymeric nanospheres.

A Three‐Component Assembly Promoted by Boronic Acids Delivers a Modular Fluorophore Platform (BASHY Dyes)

Abstract The modular assembly of boronic acids with Schiff‐base ligands enabled the construction of innovative fluorescent dyes [boronic acid salicylidenehydrazone (BASHY)] with suitable structural and photophysical properties for live cell bioimaging applications. This reaction enabled the straightforward synthesis (yields up to 99 %) of structurally diverse and photostable dyes that exhibit a polarity‐sensitive green‐to‐yellow emission with high quantum yields of up to 0.6 in nonpolar environments. These dyes displayed a high brightness (up to 54 000 m −1 cm−1). The promising structural and fluorescence properties of BASHY dyes fostered the preparation of non‐cytotoxic, stable, and highly fluorescent poly(lactide‐co‐glycolide) nanoparticles that were effectively internalized by dendritic cells. The dyes were also shown to selectively stain lipid droplets in HeLa cells, without inducing any appreciable cytotoxicity or competing plasma membrane labeling; this confirmed their potential as fluorescent stains.

Molecular Modeling to Study Dendrimers for Biomedical Applications

Molecular modeling techniques provide a powerful tool to study the properties of molecules and their interactions at the molecular level. The use of computational techniques to predict interaction patterns and molecular properties can inform the design of drug delivery systems and therapeutic agents. Dendrimers are hyperbranched macromolecular structures that comprise repetitive building blocks and have defined architecture and functionality. Their unique structural features can be exploited to design novel carriers for both therapeutic and diagnostic agents. Many studies have been performed to iteratively optimise the properties of dendrimers in solution as well as their interaction with drugs, nucleic acids, proteins and lipid membranes. Key features including dendrimer size and surface have been revealed that can be modified to increase their performance as drug carriers. Computational studies have supported experimental work by providing valuable insights about dendrimer structure and possible molecular interactions at the molecular level. The progress in computational simulation techniques and models provides a basis to improve our ability to better predict and understand the biological activities and interactions of dendrimers. This review will focus on the use of molecular modeling tools for the study and design of dendrimers, with particular emphasis on the efforts that have been made to improve the efficacy of this class of molecules in biomedical applications.