A Step Forward for the Treatment of Localized Prostate Cancer Using Gold Nanoparticles Combined with Laser Irradiation.

Prostate cancer (PCA) is the second most common cancer diagnosis in men and the fifth leading cause of death worldwide. The conventional treatments available are beneficial to only a few patients and, in those, some present adverse side effects that eventually affect the quality of life of most patients. Thus, there is an urgent need for effective, less invasive and targeted specific treatments for PCA. Photothermal therapy (PTT) is a minimally invasive therapy that provides a localized effect for tumour cell ablation by activating photothermal agents (PTA) that mediate the conversion of the light beam's energy into heat at the site. As tumours are unable to easily dissipate heat, they become more susceptible to temperature increases. In the PTT field, gold nanoparticles (AuNPs) have been attracting interest as PTA. The aim of this study was to formulate AuNPs capable of remaining retained in the tumour and subsequently generating heat at the tumour site. AuNPs were synthesized and characterized in terms of size, polydispersity index (PdI), zeta potential (ZP), morphology and the surface plasmon resonance (SPR). The safety of AuNPs and their efficacy were assessed using in vitro models. A preliminary in vivo safety assessment of AuNPs with a mean size lower than 200 nm was confirmed. The morphology was spherical-like and the SPR band showed good absorbance at the laser wavelength. Without laser, AuNPs proved to be safe both in vitro (>70% viability) and in vivo. In addition, with laser irradiation, they proved to be relatively effective in PCA cells. Overall, the formulation appears to be promising for use in PTT.

Polyoxazoline-Based Nanovaccine Synergizes with Tumor-Associated Macrophage Targeting and Anti-PD-1 Immunotherapy against Solid Tumors.

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)-ß 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.

Safety of Gold Nanoparticles: From In Vitro to In Vivo Testing Array Checklist.

In recent years, gold nanoparticles (AuNPs) have aroused the interest of many researchers due to their unique physicochemical and optical properties. AuNPs are being explored in a variety of biomedical fields, either in diagnostics or therapy, particularly for localized thermal ablation of cancer cells after light irradiation. Besides the promising therapeutic potential of AuNPs, their safety constitutes a highly important issue for any medicine or medical device. For this reason, in the present work, the production and characterization of physicochemical properties and morphology of AuNPs coated with two different materials (hyaluronic and oleic acids (HAOA) and bovine serum albumin (BSA)) were firstly performed. Based on the above importantly referred issue, the in vitro safety of developed AuNPs was evaluated in healthy keratinocytes, human melanoma, breast, pancreatic and glioblastoma cancer cells, as well as in a three-dimensional human skin model. Ex vivo and in vivo biosafety assays using, respectively, human red blood cells and Artemia salina were also carried out. HAOA-AuNPs were selected for in vivo acute toxicity and biodistribution studies in healthy Balb/c mice. Histopathological analysis showed no significant signs of toxicity for the tested formulations. Overall, several techniques were developed in order to characterize the AuNPs and evaluate their safety. All these results support their use for biomedical applications.

Pluronic® F127 Hydrogel Containing Silver Nanoparticles in Skin Burn Regeneration: An Experimental Approach from Fundamental to Translational Research.

Presently, skin burns are considered one of the main public health problems and lack therapeutic options. In recent years, silver nanoparticles (AgNPs) have been widely studied, playing an increasingly important role in wound healing due to their antibacterial activity. This work is focused on the production and characterization of AgNPs loaded in a Pluronic® F127 hydrogel, as well as assessing its antimicrobial and wound-healing potential. Pluronic® F127 has been extensively explored for therapeutic applications mainly due to its appealing properties. The developed AgNPs had an average size of 48.04 ± 14.87 nm (when prepared by method C) and a negative surface charge. Macroscopically, the AgNPs solution presented a translucent yellow coloration with a characteristic absorption peak at 407 nm. Microscopically, the AgNPs presented a multiform morphology with small sizes (~50 nm). Skin permeation studies revealed that no AgNPs permeated the skin after 24 h. AgNPs further demonstrated antimicrobial activity against different bacterial species predominant in burns. A chemical burn model was developed to perform preliminary in vivo assays and the results showed that the performance of the developed AgNPs loaded in hydrogel, with smaller silver dose, was comparable with a commercial silver cream using higher doses. In conclusion, hydrogel-loaded AgNPs is potentially an important resource in the treatment of skin burns due to their proven efficacy by topical administration.

The Role of Rosmarinic Acid on the Bioproduction of Gold Nanoparticles as Part of a Photothermal Approach for Breast Cancer Treatment.

Breast cancer is a high-burden malignancy for society, whose impact boosts a continuous search for novel diagnostic and therapeutic tools. Among the recent therapeutic approaches, photothermal therapy (PTT), which causes tumor cell death by hyperthermia after being irradiated with a light source, represents a high-potential strategy. Furthermore, the effectiveness of PTT can be improved by combining near infrared (NIR) irradiation with gold nanoparticles (AuNPs) as photothermal enhancers. Herein, an alternative synthetic method using rosmarinic acid (RA) for synthesizing AuNPs is reported. The RA concentration was varied and its impact on the AuNPs physicochemical and optical features was assessed. Results showed that RA concentration plays an active role on AuNPs features, allowing the optimization of mean size and maximum absorbance peak. Moreover, the synthetic method explored here allowed us to obtain negatively charged AuNPs with sizes favoring the local particle accumulation at tumor site and maximum absorbance peaks within the NIR region. In addition, AuNPs were safe both in vitro and in vivo. In conclusion, the synthesized AuNPs present favorable properties to be applied as part of a PTT system combining AuNPs with a NIR laser for the treatment of breast cancer.

Interfacing the enzyme multiheme cytochrome c nitrite reductase with pencil lead electrodes: Towards a disposable biosensor for cyanide surveillance in the environment.

The present study reports a novel voltammetric biosensor for cyanide based on its inhibitory effect on cytochrome c nitrite reductase (ccNiR). Interestingly, the earlier development of a point-of-care test for nitrite based on the direct electrochemistry of ccNiR has shown that the cyanide inhibition depends on the type of carbon material employed as transducer (Monteiro et al., 2019). In this work, commercial graphite pencil leads were employed in the construction of both working and pseudo-reference electrodes, with ccNiR being simply drop casted onto the former. In this way, we produced a functional and fully integrated voltammetric biosensor for nitrite quantification that also allows to observe a decrease in the catalytic current due to cyanide addition. Under turnover conditions, the biosensor showed a linear response with the logarithm of cyanide concentration in the 5-76 µM (cyclic voltammetry) and 1-40 µM (square-wave voltammetry) ranges, with a sensitivity of 20-25% ln [cyanide µM]-1 and a detection limit of 0.86-4.4 µM. The application of the pencil lead as a putative pseudo-reference was very promising, since the potentials profile matched those observed with a true reference electrode (Ag/AgCl). Overall, the direct electron transfer between ccNiR and a pencil lead electrode was demonstrated for the first time, with cyanide-induced inhibition being easily monitored, paving the way for the employment of these low-cost bioelectrodes as cyanide probes for on-site surveillance of aquatic environments.

Proof-of-Concept Study of Multifunctional Hybrid Nanoparticle System Combined with NIR Laser Irradiation for the Treatment of Melanoma.

The global impact of cancer emphasizes the importance of developing innovative, effective and minimally invasive therapies. In the context of superficial cancers, the development of a multifunctional nanoparticle-based system and its in vitro and in vivo safety and efficacy characterization are, herein, proposed as a proof-of-concept. This multifunctional system consists of gold nanoparticles coated with hyaluronic and oleic acids, and functionalized with epidermal growth factor for greater specificity towards cutaneous melanoma cells. This nanoparticle system is activated by a near-infrared laser. The characterization of this nanoparticle system included several phases, with in vitro assays being firstly performed to assess the safety of gold nanoparticles without laser irradiation. Then, hairless immunocompromised mice were selected for a xenograft model upon inoculation of A375 human melanoma cells. Treatment with near-infrared laser irradiation for five minutes combined with in situ administration of the nanoparticles showed a tumor volume reduction of approximately 80% and, in some cases, led to the formation of several necrotic foci, observed histologically. No significant skin erythema at the irradiation zone was verified, nor other harmful effects on the excised organs. In conclusion, these assays suggest that this system is safe and shows promising results for the treatment of superficial melanoma.

Electrosynthesis of polydopamine-ethanolamine films for the development of immunosensing interfaces.

We report a straightforward and reproducible electrochemical approach to develop polydopamine-ethanolamine (ePDA-ETA) films to be used as immunosensing interfaces. ETA is strongly attached to polydopamine films during the potentiodynamic electropolymerization of dopamine. The great advantage of the electrochemical methods is to generate the oxidized species (quinones), which can readily react with ETA amine groups present in solution, with the subsequent incorporation of this molecule in the polymer. The presence of ETA and its effect on the electrosynthesis of polydopamine was accessed by cyclic voltammetry, ellipsometry, atomic force microscopy, FTIR and X-ray photoelectron spectroscopy. The adhesive and biocompatible films enable a facile protein linkage, are resilient to flow assays, and display intrinsic anti-fouling properties to block non-specific protein interactions, as monitored by real-time surface plasmon resonance, and confirmed by ellipsometry. Immunoglobulin G (IgG) and Anti-IgG were used in this work as model proteins for the affinity sensor. By using the one-step methodology (ePDA-ETA), the lower amount of immobilized biorecognition element, IgG, compared to that deposited on ePDA or on ETA post-modified film (ePDA/ETA), allied to the presence of ETA, improved the antibody-antigen affinity interaction. The great potential of the developed platform is its versatility to be used with any target biorecognition molecules, allowing both optical and electrochemical detection.

Development and Mechanistic Insight into the Enhanced Cytotoxic Potential of Parvifloron D Albumin Nanoparticles in EGFR-Overexpressing Pancreatic Cancer Cells.

Pancreatic cancer is one of the most lethal cancers, with an extremely poor prognosis. The development of more effective therapies is thus imperative. Natural origin compounds isolated from Plectranthus genus, such as parvifloron D (PvD), have cytotoxic and antiproliferative activity against human tumour cells. However, PvD is a very low water-soluble compound, being nanotechnology a promising alternative strategy to solve this problem. Therefore, the aim of this study was to optimize a nanosystem for preferential delivery of PvD to pancreatic tumour cells. Albumin nanoparticles (BSA NPs) were produced through a desolvation method. Glucose cross-linking and bioactive functionalization profiles of BSA platform were elucidated and analysed using static lattice atomistic simulations in vacuum. Using the optimized methodology, PvD was encapsulated (yield higher than 80%) while NPs were characterized in terms of size (100-400 nm) and morphology. Importantly, to achieve a preferential targeting to pancreatic cancer cells, erlotinib and cetuximab were attached to the PvD-loaded nanoparticle surface, and their antiproliferative effects were evaluated in BxPC3 and Panc-1 cell lines. Erlotinib conjugated NPs presented the highest antiproliferative effect toward pancreatic tumour cells. Accordingly, cell cycle analysis of the BxPC3 cell line showed marked accumulation of tumour cells in G1-phase and cell cycle arrest promoted by NPs. As a result, erlotinib conjugated PvD-loaded BSA NPs must be considered a suitable and promising carrier to deliver PvD at the tumour site, improving the treatment of pancreatic cancer.

Immunization with mannosylated nanovaccines and inhibition of the immune-suppressing microenvironment sensitizes melanoma to immune checkpoint modulators.

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.

In Situ AFM Imaging of Adsorption Kinetics of DPPG Liposomes: A Quantitative Analysis of Surface Roughness.

The adsorption of intact liposomes on surfaces is of great importance for the development of sensors and drug delivery systems and, also, strongly dependent on the surface roughness where the liposomes are adsorbed. In this paper, we analyzed, by using atomic force microscopy in liquid, the evolution of the morphology of gold surfaces and of poly(allylamine hydrochloride) (PAH) surfaces with different roughness during the adsorption of liposomes prepared with the synthetic phospholipid 1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)]. Our results reveal the following. On smooth surfaces of Au only and Au with PAH, the liposomes open and deploy on the substrate, creating a supported-lipid bilayer, with the opening process being faster on the Au/PAH surface. On rough substrates of Au coated with polyelectrolyte multilayers, the liposomes were adsorbed intact on the surface. This was corroborated by power spectral density analysis that demonstrates the presence of superstructures with an average lateral size of 43 and 87 nm, in accordance with two and four times the mean liposome hydrodynamic diameter of about 21 nm. In addition, this work presents an adequate and effective methodology for analysis of adsorption phenomena of liposomes on rough surfaces.

Development of Parvifloron D-loaded Smart Nanoparticles to Target Pancreatic Cancer.

Pancreatic cancer is the eighth leading cause of cancer death worldwide. For this reason, the development of more effective therapies is a major concern for the scientific community. Accordingly, plants belonging to Plectranthus genus and their isolated compounds, such as Parvifloron D, were found to have cytotoxic and antiproliferative activities. However, Parvifloron D is a very low water-soluble compound. Thus, nanotechnology can be a promising delivery system to enhance drug solubility and targeted delivery. The extraction of Parvifloron D from P. ecklonii was optimized through an acetone ultrasound-assisted method and isolated by Flash-Dry Column Chromatography. Then, its antiproliferative effect was selectivity evaluated against different tumor cell lines (IC50 of 0.15 ± 0.05 µM, 11.9 ± 0.7 µM, 21.6 ± 0.5, 34.3 ± 4.1 µM, 35.1 ± 2.2 µM and 32.1 ± 4.3 µM for BxPC3, PANC-1, Ins1-E, MCF-7, HaCat and Caco-2, respectively). To obtain an optimized stable Parvifloron D pharmaceutical dosage form, albumin nanoparticles were produced through a desolvation method (yield of encapsulation of 91.2%) and characterized in terms of size (165 nm; PI 0.11), zeta potential (-7.88 mV) and morphology. In conclusion, Parvifloron D can be efficiently obtained from P. ecklonii and it has shown selective cytotoxicity to pancreatic cell lines. Parvifloron D nanoencapsulation can be considered as a possible efficient alternative approach in the treatment of pancreatic cancer.

Sugar-based bactericides targeting phosphatidylethanolamine-enriched membranes.

Anthrax is an infectious disease caused by Bacillus anthracis, a bioterrorism agent that develops resistance to clinically used antibiotics. Therefore, alternative mechanisms of action remain a challenge. Herein, we disclose deoxy glycosides responsible for specific carbohydrate-phospholipid interactions, causing phosphatidylethanolamine lamellar-to-inverted hexagonal phase transition and acting over B. anthracis and Bacillus cereus as potent and selective bactericides. Biological studies of the synthesized compound series differing in the anomeric atom, glycone configuration and deoxygenation pattern show that the latter is indeed a key modulator of efficacy and selectivity. Biomolecular simulations show no tendency to pore formation, whereas differential metabolomics and genomics rule out proteins as targets. Complete bacteria cell death in 10 min and cellular envelope disruption corroborate an effect over lipid polymorphism. Biophysical approaches show monolayer and bilayer reorganization with fast and high permeabilizing activity toward phosphatidylethanolamine membranes. Absence of bacterial resistance further supports this mechanism, triggering innovation on membrane-targeting antimicrobials.

Nanoscale characterization of the temporary adhesive of the sea urchin Paracentrotus lividus.

Background: Unlike the thin homogeneous films that are typical for adhesives produced by humans, biological adhesives present complex hierarchical micro- and nanostructures. Most studies on marine adhesives have focused on permanent adhesives, whereas the nanostructures of nonpermanent, temporary or reversible adhesives have only been examined in some organisms such as marine flatworms, barnacle cyprids, freshwater cnidaria and echinoderms such as sea cucumbers and sea stars. In this study, the first nanoscale characterization of sea urchin temporary adhesives was performed using atomic force microscopy (AFM). Results: The adhesive topography was similar under dry and native (seawater) conditions, which was comprised of a honeycomb-like meshwork of aggregated globular nanostructures. In terms of adhesion forces, higher values were obtained in dry conditions, reaching up to 50 nN. Under native conditions, lower adhesive forces were obtained (up to 500 pN) but the adhesive seemed to behave like a functional amyloid, as evidenced by the recorded characteristic sawtooth force-extension curves and positive thioflavin-T labelling. Conclusion: Our results confirm that like other temporary adhesives, the sea urchin adhesive footprint nanostructure consists of a meshwork of entangled globular nanostructures. Under native conditions, the adhesive footprints of the sea urchin behaved like a functional amyloid, suggesting that among its proteinaceous constituents there are most likely proteins with amyloid quaternary structures or rich in ß-sheets. These results extend our knowledge on sea urchin adhesive composition and mechanical properties essential for the engineering of biomimetic adhesives.

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.

Nanoparticulate vaccine inhibits tumor growth via improved T cell recruitment into melanoma and huHER2 breast cancer.

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.

Differential targeting of membrane lipid domains by caffeic acid and its ester derivatives.

Phenolic acids have been associated to a wide range of important health benefits underlain by a common molecular mechanism of action. Considering that significant membrane permeation is prevented by their hydrophilic character, we hypothesize that their main effects result from the interplay with cell membrane surface. This hypothesis was tested using the paradigmatic caffeic acid (CA) and two of its ester derivatives, rosmarinic (RA) and chlorogenic (CGA) acids, for which we predict, based on molecular dynamics simulations, a shallow location in phospholipid bilayers dependent on the protonation-state. Using complementary experimental approaches, an interaction with the membrane was definitely revealed for the three compounds, with RA exhibiting the highest lipid bilayer partition, and the redox signals of membrane-bound RA and CA being clearly detected. Cholesterol decreased the compounds bilayer partition, but not their ability to lower membrane dipole potential. In more complex membrane models containing also sphingomyelin, with liquid disordered (ld)/ liquid ordered (lo) phases coexistence, mimicking domains in the external leaflet of human plasma membrane, all compounds were able to affect nanodomains lateral organization. RA, and to a lesser extent CGA, decreased the size of lo domains. The most significant effect of CA was the possible formation of a rigid gel-like phase, enriched in sphingomyelin. In addition, all phenolic acids decreased the order of lo domains. In sum, phenolic acid effects on the membrane are enhanced in cholesterol-rich lo phases, which predominate in the outer leaflet of human cell membranes and are involved in many key cellular processes.

Tip-Specific Functionalization of Gold Nanorods for Plasmonic Biosensing: Effect of Linker Chain Length.

Gold nanorods are promising platforms for label-free biosensing. We have functionalized gold nanorods with biotin thiol linkers of increasing chain length and evaluated their ability in the molecular detection of streptavidin. We have found an unexpected effect of the increase in linker length, which resulted in a substantial improvement of the plasmon response at surface saturation. The plasmon peak shift increased from 5 to 14 nm, i.e., more than twice the response, between the short and long biotin linkers. This effect is observed only for site-selective tip functionalization, whereas for a full biotin coating there is no improvement observed with the linker length. The improved plasmon response for tip functionalization is attributed to low biotin coverage but is directed to the most sensitive regions, which, combined with a longer chain linker, reduces the steric hindrance for streptavidin binding on the rod's surface. The model sensors were further characterized by measuring their dose-response curves and binding kinetic assays. Simulations of the discrete dipole approximation give theoretical plasmon shifts that compare well with the experimental ones for the long linker but not with those of the short linker, thus suggesting that steric hindrance affects the latter. Our results highlight the importance of specifically functionalizing the plasmonic hot spots in nanoparticle sensors with the adequate density of receptors in order to maximize their response.

Rational design of nanoparticles towards targeting antigen-presenting cells and improved T cell priming.

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.