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.

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.

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.

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.

Formation and Properties of Membrane-Ordered Domains by Phytoceramide: Role of Sphingoid Base Hydroxylation.

Phytoceramide is the backbone of major sphingolipids in fungi and plants and is essential in several tissues of animal organisms, such as human skin. Its sphingoid base, phytosphingosine, differs from that usually found in mammals by the addition of a hydroxyl group to the 4-ene, which may be a crucial factor for the different properties of membrane microdomains among those organisms and tissues. Recently, sphingolipid hydroxylation in animal cells emerged as a key feature in several physiopathological processes. Hence, the study of the biophysical properties of phytosphingolipids is also relevant in that context since it helps us to understand the effects of sphingolipid hydroxylation. In this work, binary mixtures of N-stearoyl-phytoceramide (PhyCer) with palmitoyloleoylphosphatidylcholine (POPC) were studied. Steady-state and time-resolved fluorescence of membrane probes, X-ray diffraction, atomic force microscopy, and confocal microscopy were employed. As for other saturated ceramides, highly rigid gel domains start to form with just ∼5 mol % PhyCer at 24 °C. However, PhyCer gel-enriched domains in coexistence with POPC-enriched fluid present additional complexity since their properties (maximal order, shape, and thickness) change at specific POPC/PhyCer molar ratios, suggesting the formation of highly stable stoichiometric complexes with their own properties, distinct from both POPC and PhyCer. A POPC/PhyCer binary phase diagram, supported by the different experimental approaches employed, is proposed with complexes of 3:1 and 1:2 stoichiometries which are stable at least from ∼15 to ∼55 °C. Thus, it provides mechanisms for the in vivo formation of sphingolipid-enriched gel domains that may account for stable membrane compartments and diffusion barriers in eukaryotic cell membranes.

A biomimetic platform to study the interactions of bioelectroactive molecules with lipid nanodomains.

In this work, we developed a biomimetic platform where the study of membrane associated redox processes and high-resolution imaging of lipid nanodomains can be both performed, based on a new functional gold modification, l-cysteine self-assembled monolayer. This monolayer proved to be ideal for the preparation of defect-free planar supported lipid bilayers (SLBs) where nanodomains with height difference of ∼1.5 nm are clearly resolved by atomic force microscopy. Single and multicomponent lipid compositions were used, leading to the formation of different phases and domains mimicking the lateral organization of cellular membranes, and in all cases stable and continuous bilayers were obtained. These platforms were tested toward the interaction with bioelectroactive molecules, the antioxidant quercetin, and the hormone epinephrine. Despite the weak interaction detected between epinephrine and lipid bilayers, our biomimetic interface was able to sense the redox process of membrane-bound epinephrine, obtain its surface concentration (9.36 × 10(-11) mol/cm(2) for a fluid bilayer), and estimate a mole fraction membrane/water partition coefficient (Kp) from cyclic voltammetric measurements (1.13 × 10(4) for a fluid phase membrane). This Kp could be used to quantitatively describe the minute changes observed in the photophysical properties of epinephrine intrinsic fluorescence upon its interaction with liposome suspensions. Moreover, we showed that the lipid membrane stabilizes epinephrine structure, preventing its oxidation, which occurs in neutral aqueous solution, and that epinephrine partition and mobility in membranes depends on lipid phase, expanding our knowledge on hormone membrane interactions.

Polynorepinephrine and polydopamine-bacterial laccase coatings for phenolic amperometric biosensors.

The successful fabrication of biosensors is greatly limited by the immobilization of their bioreceptor, thus we propose a facile and reproducible two-step method to modify graphite electrodes with a bacterial laccase, relying on a fast and controllable potentiostatic process to coat graphite surfaces with biomolecule-compatible thin films of polynorepinephrine (ePNE) and polydopamine (ePDA). Both polymers, synthesized with a similar thickness, were functionalized with bacterial laccase, displaying distinct electrochemical transducing behaviours at pH 5.0 and 7.0. ePNE layer enables adequate electron transfer of anionic and cationic species in acidic and neutral media, whereas transduction across ePDA strongly depends on pH and redox probe charge. ePNE stands out by improving the amperometric responses of the biosensing interface towards a phenolic acid (gallic acid) and a flavonoid (catechin), in respect to ePDA. The optimal graphite/ePNE/laccase interface outperforms biosensing interfaces based on fungal laccases at neutral pH, displaying detection sensitivities of 104 and 14.4 µA cm-2 mM-1for gallic acid and catechin, respectively. The fine synthetic control of the ePNE bio-inspired transduction layer and the use of an alkaliphilic bacterial laccase enabled the construction of an amperometric biosensing interface with extended pH range of polyphenols detection present in food products and agro-industrial waste.

Enhanced Cytotoxicity against a Pancreatic Cancer Cell Line Combining Radiation and Gold Nanoparticles.

The present work consisted of an exploratory study aiming to evaluate in vitro the potential of AuNPs during Radiation Therapy (RT) in human pancreatic adenocarcinoma cells. AuNPs coated with hyaluronic and oleic acids (HAOA-AuNPs) or with bombesin peptides (BBN-AuNPs) were used. AuNPs were characterized by Atomic Force Microscopy (AFM) and Dynamic Light Scattering. BxPC-3 tumor cells were irradiated with a 6 MV X-rays beam, in the absence or presence of AuNPs. AFM showed that HAOA-AuNPs and BBN-AuNPs are spherical with a mean size of 83 ± 20 nm and 49 ± 12 nm, respectively. For RT alone, a reduction in cell viability of up to 33 ± 12% was obtained compared to the control (p ≤ 0.0001). HAOA-AuNPs alone at 200 and 400 µM showed a reduction in cell viability of 20 ± 4% and 35 ± 4%, respectively, while for BBN-AuNPs, at 50 and 200 µM, a reduction in cell viability of 25 ± 3% and 37 ± 3% was obtained, respectively, compared to the control (p < 0.0001). At 72 h post-irradiation, a decrease in cell viability of 26 ± 3% and 22 ± 2% between RT + HAOA-AuNPs at 400 µM and RT + BBN-AuNPs at 50 µM, compared to RT alone, was obtained (p < 0.004). The combination of RT with AuNPs led to a significant decrease in cell viability compared to the control, or RT alone, thus representing an improved effect.

Intranasal Multiepitope PD-L1-siRNA-Based Nanovaccine: The Next-Gen COVID-19 Immunotherapy.

The first approved vaccines for human use against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are nanotechnology-based. Although they are modular, rapidly produced, and can reduce disease severity, the currently available vaccines are restricted in preventing infection, stressing the global demand for novel preventive vaccine technologies. Bearing this in mind, we set out to develop a flexible nanovaccine platform for nasal administration to induce mucosal immunity, which is fundamental for optimal protection against respiratory virus infection. The next-generation multiepitope nanovaccines co-deliver immunogenic peptides, selected by an immunoinformatic workflow, along with adjuvants and regulators of the PD-L1 expression. As a case study, we focused on SARS-CoV-2 peptides as relevant antigens to validate the approach. This platform can evoke both local and systemic cellular- and humoral-specific responses against SARS-CoV-2. This led to the secretion of immunoglobulin A (IgA), capable of neutralizing SARS-CoV-2, including variants of concern, following a heterologous immunization strategy. Considering the limitations of the required cold chain distribution for current nanotechnology-based vaccines, it is shown that the lyophilized nanovaccine is stable for long-term at room temperature and retains its in vivo efficacy upon reconstitution. This makes it particularly relevant for developing countries and offers a modular system adaptable to future viral threats.

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.

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.

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.

Ethanol effects on binary and ternary supported lipid bilayers with gel/fluid domains and lipid rafts.

Ethanol-lipid bilayer interactions have been a recurrent theme in membrane biophysics, due to their contribution to the understanding of membrane structure and dynamics. The main purpose of this study was to assess the interplay between membrane lateral heterogeneity and ethanol effects. This was achieved by in situ atomic force microscopy, following the changes induced by sequential ethanol additions on supported lipid bilayers formed in the absence of alcohol. Binary phospholipid mixtures with a single gel phase, dipalmitoylphosphatidylcholine (DPPC)/cholesterol, gel/fluid phase coexistence DPPC/dioleoylphosphatidylcholine (DOPC), and ternary lipid mixtures containing cholesterol, mimicking lipid rafts (DOPC/DPPC/cholesterol and DOPC/sphingomyelin/cholesterol), i.e., with liquid ordered/liquid disordered (ld/lo) phase separation, were investigated. For all compositions studied, and in two different solid supports, mica and silicon, domain formation or rearrangement accompanied by lipid bilayer thinning and expansion was observed. In the case of gel/fluid coexistence, low ethanol concentrations lead to a marked thinning of the fluid but not of the gel domains. In the case of ld/lo all the bilayer thins simultaneously by a similar extent. In both cases, only the more disordered phase expanded significantly, indicating that ethanol increases the proportion of disordered domains. Water/bilayer interfacial tension variation and freezing point depression, inducing acyl chain disordering (including opening and looping), tilting, and interdigitation, are probably the main cause for the observed changes. The results presented herein demonstrate that ethanol influences the bilayer properties according to membrane lateral organization.

Polyelectrolyte-assisted noncovalent functionalization of carbon nanotubes with ordered self-assemblies of a water-soluble porphyrin.

The self-assembly and induced supramolecular chirality of meso-tetrakis(4-sulfonatophenyl)porphyrin (TSPP) on both single-wall (SWCNT) and multiwall carbon nanotubes (MWCNT) are investigated. Under mild pH conditions (pH 3), TSPP forms aggregates when CNTs are dispersed in an aqueous solution containing positively charged polyelectrolytes such as poly-L-lysine (PLL) or poly(allylamine hydrochloride) (PAH). Evidence for the geometry of the porphyrin aggregates is obtained from absorption spectra, whereby the fingerprints of J- and H-aggregates are clearly seen only in the presence of smaller-diameter nanotubes. J-aggregates are better stabilized with PLL, whereas in the presence of PAH mainly H-aggregates prevail. Excited-state interactions within these nanohybrids are studied by steady-state and time-resolved fluorescence. The porphyrin emission intensity in the nanohybrid solution is significantly quenched compared to that of TSPP alone, and this implies strong electronic interaction between CNTs and porphyrin molecules. Fluorescence lifetime imaging microscopy (FLIM) further supports that porphyrin arrays are associated with the MWCNT sidewalls wrapped in PLL. In the case of the SWCNT hybrid, spherical structures associated with longer fluorescence lifetime appeared after one week, indicative of H-aggregates of TSPP. The latter are the result of π-π stacking of porphyrin units on neighboring nanotubes facilitated by the strong tendency of these nanotubes to interact with each other. These results highlight the importance of optimum dimensions and surface-area architectures of CNTs in the control/stability of the porphyrin aggregates with promising properties for light harvesting.

Antibody oriented immobilization on gold using the reaction between carbon disulfide and amine groups and its application in immunosensing.

Carbon disulfide (CS(2)) can spontaneously react with amine groups to form dithiocarbamates on gold surface, providing the possibility to immobilize some compounds with primary or secondary amine groups in one step. Using this principle, an immunosensor interface prepared for immunoglobulin G (IgG) sensing surface toward anti-IgG has been fabricated for the first time by simply immersing gold slides into a mixed aqueous solution of CS(2) and protein A, followed by incubation in immunoglobulin G solution. The reaction between CS(2) and protein A has been followed by UV-vis spectroscopy, whereas cyclic voltammetry has been employed in the characterization of the modified gold surface with CS(2) and protein A, both methods indicating that protein A immobilization is implemented by CS(2). Conventional ellipsometry, atomic force microscopy (AFM), as well as surface plasmon resonance (SPR) have been used to evaluate the specific binding of protein A with IgG and IgG with anti-IgG, revealing that IgG is specifically captured to form the biosensing interface, maintaining its bioactivity. Compared to direct adsorption of IgG on the gold surface, the IgG sensing surface constructed of CS(2) and protein A is far more sensitive to capture anti-IgG as its target molecule. In addition, the modified surface is proven to have good capability to inhibit nonspecific adsorption, as supported by control experiments using lysozyme and BSA. To conclude, antibody immobilization using this one-step method has potential as a simple and convenient surface modification approach for immunosensor development.

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.

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.

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.

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.