Carina Peres

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.

Poly(lactic acid)-based particulate systems are promising tools for immune modulation.

Poly(lactic acid) (PLA) is one of the most successful and versatile polymers explored for controlled delivery of bioactive molecules. Its attractive properties of biodegradability and biocompatibility in vivo have contributed in a meaningful way to the approval of different products by the FDA and EMA for a wide range of biomedical and pharmaceutical applications, in the past two decades. This polymer has been widely used for the preparation of particles as delivery systems of several therapeutic molecules, including vaccines. These PLA vaccine carriers have shown to induce a sustained and targeted release of different bacterial, viral and tumor-associated antigens and adjuvants in vivo, triggering distinct immune responses. The present review intends to highlight and discuss the major advantages of PLA as a promising polymer for the development of potent vaccine delivery systems against pathogens and cancer. It aims to provide a critical discussion based on preclinical data to better understand the major effect of PLA-based carrier properties on their interaction with immune cells and thus their role in the modulation of host immunity. STATEMENT OF SIGNIFICANCE During the last decades, vaccination has had a great impact on global health with the control of many severe diseases. Polymeric nanosystems have emerged as promising strategies to stabilize vaccine antigens, promoting their controlled release to phagocytic cells, thus avoiding the need for multiple administrations. One of the most promising polymers are the aliphatic polyesters, which include the poly(lactic acid). This is a highly versatile biodegradable and biocompatible polymer. Products containing this polymer have already been approved for all food and some biomedical applications. Despite all favorable characteristics presented above, PLA has been less intensively discussed than other polymers, such as its copolymer PLGA, including regarding its application in vaccination and particularly in tumor immunotherapy. The present review discusses the major advantages of poly(lactic acid) for the development of potent vaccine delivery systems, providing a critical view on the main properties that determine their effect on the modulation of immune cells.

Nanoparticle impact on innate immune cell pattern-recognition receptors and inflammasomes activation

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.

α-Galactosylceramide and peptide-based nano-vaccine synergistically induced a strong tumor suppressive effect in melanoma

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.

Optimization of protein loaded PLGA nanoparticle manufacturing parameters following a quality-by-design approach

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.

Functional Moieties for Intracellular Traffic of Nanomaterials

Abstract 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.

Polymer-Based Nanoparticles as Modern Vaccine Delivery Systems

Nanoparticulate carriers are particularly promising and versatile in stimulating immune response. Indeed, nanotechnology allows the creation of structures that exploit the inherent ability of the immune system to recognize nanoscale systems. This chapter will review the most recent developments of polymeric nanoparticles with a special emphasis on their ability to interact with the immune system network. Polymer-based vaccines allow effective encapsulation and controlled release of one or several antigens. They promote antigen processing and further association with major histocompatibility complex class molecules, and they modulate the induced immune response. Moreover, they can also contain and regulate the intracellular trafficking of other agents such as adjuvants with a strong potential for modulating both the nature and strength of an immune response. The recent developments of polymeric nanovaccines will be discussed as will the various parameters that play a role in their efficiency.

Translational Peptide-associated Nanosystems: Promising Role as Cancer Vaccines.

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.