Microfluidic development and biological evaluation of targeted therapy-loaded biomimetic nano system to improve the metastatic melanoma treatment.

Optimizing current therapies is among next steps in metastatic melanoma (MM) treatment landscape. The innovation of this study is the design of production process by microfluidics of cell membrane (CM)-modified nanoparticles (NPs), as an emerging biomimetic platform that allows for reduced immune clearance, long blood circulation time and improved specific tumor targeting. To achieve melanoma selectivity, direct membrane fusion between synthetic liposomes and CMs extracted from MM cell line was performed by microfluidic sonication approach, then the hybrid liposomes were loaded with cobimetinib (Cob) or lenvatinib (Lenva) targeting agents and challenged against MM cell lines and liver cancer cell line to evaluate homotypic targeting and antitumor efficacy. Characterization studies demonstrated the effective fusion of CM with liposome and the high encapsulation efficiency of both drugs, showing the proficiency of microfluidic-based production. By studying the targeting of melanoma cells by hybrid liposomes versus liposomes, we found that both NPs entered cells through endocytosis, whereas the former showed higher selectivity for MM cells from which CM was extracted, with 8-fold higher cellular uptake than liposomes. Hybrid liposome formulation of Cob and Lenva reduced melanoma cells viability to a greater extent than liposomes and free drug and, notably, showed negligible toxicity as demonstrated by bona fide haemolysis test. The CM-modified NPs presented here have the potential to broaden the choice of therapeutic options in MM treatment.

The influence of mannose-based esters on the mesophase behaviour of lyotropic liquid crystalline nanosystems as drug delivery vectors.

Lyotropic Liquid Crystalline (LLC) nanoparticles represent an emerging class of smart, biocompatible, and biodegradable systems for the delivery of drugs. Among these, structures with complex 3D architectures such as cubosomes are of particular interest. These are non- lamellar assemblies having hydrophobic and hydrophilic portions able to carry drugs of different nature. They can further be modulated including suitable additives to control the release of the active payload, and to promote an active targeting. Starting from monoolein (GMO) cubic phase, different concentrations of mannose-based esters were added, and the eventual structural modifications were monitored to ascertain the effects of the presence of glycolipids. Moreover, the structural properties of these nanosystems loaded with Dexamethasone (DEX), a very well-known anti-inflammatory steroid, were also studied. Experiments were carried out by synchrotron Small Angle X-ray Scattering (SAXS), Raman Microspectroscopy (RMS) and Attenuated Total Reflectance-Fourier Transform Infrared (ATR-FTIR) measurements. The drug delivery potential (i.e. entrapment efficiency and release properties) of the obtained nanoparticles was evaluated. Finally, in vitro cytocompatibility and anti-inflammatory activity studies of the prepared formulations were carried out. Inclusion of mannose-based surfactants up to 10 mol% influenced the structural parameters of Im3m cubic phase and swollen cubic phases were obtained with the different glycolipids with lattice parameters significantly higher than GMO. A complete cytocompatibility and an increased DEX activity were observed, thus suggesting the possibility to use GMO/glycolipids nanoparticles to formulate innovative drug delivery systems.

Microfluidic manufacturing of tioconazole loaded keratin nanocarriers: Development and optimization by design of experiments.

Fungal infections of the skin, nails, and hair are a common health concern affecting a significant proportion of the population worldwide. The current treatment options include topical and systematic agents which have low permeability and prolonged treatment period, respectively. Consequently, there is a growing need for a permeable, effective, and safe treatment. Keratin nanoparticles are a promising nanoformulation that can improve antifungal agent penetration, providing sustainable targeted drug delivery. In this study, keratin nanoparticles were prepared using a custom-made 3D-printed microfluidic chip and the manufacturing process was optimized using the design of experiments (DoE) approach. The total flow rate (TFR), flow rate ratio (FRR), and keratin concentration were found to be the most influential factors of the size and polydispersity index (PDI) of the nanoparticles. The crosslinking procedure by means of tannic acid as safe and biocompatible compound was also optimized. Keratin nanoparticles loaded with a different amount of tioconazole showed a size lower than 200 nm, a PDI lower than 0.2 and an encapsulation efficiency of 91 ± 1.9 %. Due to their sustained drug release, the formulations showed acceptable in vitro biocompatibility. Furthermore, a significant inhibitory effect compared to the free drug against Microsporum canis.

Hydrogel containing mPEG-PLGA nanoparticles for the vaginal delivery of saquinavir mesylate against HIV infection.

Saquinavir mesylate (SQV) is a protease inhibitor commonly employed for the treatment of human immunodeficiency virus-1 infection. It is generally administered orally as tablets in combination with other antiviral drugs. Another promising route of administration can be represented by the vaginal one through topically applied formulations. This delivery can reduce the first-pass effect in the case of systemic drug adsorption or prevent HIV infection. We propose the formulation of a Carbopol® 974 (C974) hydrogel containing biodegradable mPEG-PL(L)GA nanoparticles (NPs) for the vaginal delivery of SQV, intended both as a prevention and a therapeutic strategy. mPEG-PL(L)GA NPs were incorporated into the C974 polymeric matrix, leading to a reduction of the hydrogel consistency dependent on NPs and C974 concentrations. Despite the moderate drug loading into NPs, the presence of the NPs had an impact on the in vitro release of the drug from the hydrogel at pH 5.5 using immersion cells. A higher amount of the drug was released, probably due to the effect of NPs in promoting the incorporation of the drug into the hydrogel at a high SQV dose. These findings can be useful for the development of topically applied hydrogels for SQV delivery, possibly having improved in vivo therapeutic outcomes.

Synthesis and Properties of Sucrose- and Lactose-Based Aromatic Ester Surfactants as Potential Drugs Permeability Enhancers.

The delivery of therapeutics across biological membranes (e.g., mucosal barriers) by avoiding invasive routes (e.g., injection) remains a challenge in the pharmaceutical field. As such, there is the need to discover new compounds that act as drug permeability enhancers with a favorable toxicological profile. A valid alternative is represented by the class of sugar-based ester surfactants. In this study, sucrose and lactose alkyl aromatic and aromatic ester derivatives have been synthesized with the aim to characterize them in terms of their physicochemical properties, structure-property relationship, and cytotoxicity, and to test their ability as permeability enhancer agents across Calu-3 cells. All of the tested surfactants showed no remarkable cytotoxic effect on Calu-3 cells when applied both below and above their critical micelle concentration. Among the explored molecules, lactose p-biphenyl benzoate (URB1420) and sucrose p-phenyl benzoate (URB1481) cause a reversible ~30% decrease in transepithelial electrical resistance (TEER) with the respect to the basal value. The obtained result matches with the increased in vitro permeability coefficients (Papp) calculated for FTIC-dextran across Calu-3 cells in the presence of 4 mM solutions of these surfactants. Overall, this study proposes sucrose- and lactose-based alkyl aromatic and aromatic ester surfactants as novel potential and safe permeation enhancers for pharmaceutical applications.

Fascinating strategies of marine benthic organisms to cope with emerging pollutant: Titanium dioxide nanoparticles.

Titanium dioxide nanoparticles (NPs) have numerous applications, and their demands have increased as an alternative for banned sunscreen filters. However, the underlying mechanisms of their toxicity, remain largely unknown. Here, we investigate the mechanism of TiO2 NP cytotoxicity and detoxification through time-course experiments (1, 6, and 24 h) based on cellular observations and single-cell transcriptome analyses in a marine benthic foraminifer strain, derived from a common unicellular eukaryotic organism worldwide. After exposure for 1 h, cells enhanced the production of reactive oxygen species (ROS) in acidic endosomes containing TiO2 NPs as well as in mitochondria. In acidic endosomes, ROS were produced through the Fenton reaction on the surface of charged TiO2 NPs. In mitochondria, ROS were associated with porphyrin synthesis that chelated metal ions. Glutathione peroxide and neutral lipids acted as a sink for free radicals, whereas lipid peroxides were excreted to prevent further radical chain reactions. By 24 h, aggregated TiO2 NPs were encapsulated in organic compounds, possibly ceramide, and excreted as mucus, thereby preventing their further uptake. Thus, we reveal that foraminifers can tolerate the toxicity of TiO2 NPs and even prevent their further phagocytosis and uptake by trapping TiO2 NPs inside mucus. This previously unknown strategy could be applied in bioremediation to sequester NPs from the marine environment and can guide management of TiO2 pollution.

3D-printed EVA-based patches manufactured by direct powder extrusion for personalized transdermal therapies.

In recent years, 3D printing has attracted great interest in the pharmaceutical field as a promising tool for the on-demand manufacturing of patient-centered pharmaceutical forms. Among the existing 3D printing techniques, direct powder extrusion (DPE) resulted as the most practical approach thanks to the possibility to directly process excipients and drugs in a single step. The main goal of this work was to determine whether different grades of ethylene vinyl acetate (EVA) copolymer might be employed as new feedstock materials for the DPE technique to manufacture transdermal patches. By selecting two model drugs with different thermal behavior, (i.e., ibuprofen and diclofenac sodium) we also wanted to pay attention to the versatility of EVA excipient in preparing patches for customized transdermal therapies. EVA was combined with 30 % (w/w) of each model drugs. The physicochemical composition of the printed devices was investigated through Fourier-transform infrared spectroscopy, differential scanning calorimetry, and thermogravimetric analyses. FT-IR spectra confirmed that the starting materials were effectively incorporated into the final formulation, and thermal analyses demonstrated that the extrusion process altered the crystalline morphology of the raw polymers inducing the formation of crystals at lower thicknesses. Lastly, the drug release and permeation profile of the printed systems was evaluated for 48 h and showed to be dependent on the VA content of the EVA grade (74.5 % of ibuprofen released from EVA 4030AC matrix and 12.6 % of diclofenac sodium released from EVA1821A matrix). Hence, this study demonstrated that EVA and direct powder extrusion technique could be promising tools for manufacturing transdermal patches. By selecting the EVA grade with the appropriate VA content, drugs with dissimilar melting points could be printed preserving their thermal stability. Moreover, the desired drug release and permeation profile of the drug can be achieved, representing an important advantage in terms of personalized medicine.

Microfluidics assembly of inhalable liposomal ciprofloxacin characterised by an innovative in vitro pulmonary model.

Respiratory tract infections (RTIs) are reported to be the leading cause of death worldwide. Delivery of liposomal antibiotic nano-systems via the inhalation route has drawn significant interest in RTIs treatment as it can directly target the site of infection and reduces the risk of systemic exposure and side effects. Moreover, this formulation system can improve pharmacokinetics and biodistribution and enhance the activity against intracellular pathogens. Microfluidics is an innovative manufacturing technology that can produce nanomedicines in a homogenous and scalable way. The objective of this study was to evaluate the antibiofilm efficacy of two liposomal ciprofloxacin formulations with different vesicle sizes manufactured by using a 3D-printed microfluidic chip. Each formulation was characterised in terms of size, polydispersity index, charge and encapsulation. Moreover, the aerosolisation characteristics of the liposomal formulations were investigated and compared with free ciprofloxacin solution using laser diffraction and cascade impaction methods. The in vitro drug release was tested using the dialysis bag method. Furthermore, the drug transport and drug release studies were conducted using the alveolar epithelial H441 cell line integrated next-generation impactor in vitro model. Finally, the biofilm eradication efficacy was evaluated using a dual-chamber microfluidic in vitro model. Results showed that both liposomal-loaded ciprofloxacin formulations and free ciprofloxacin solution had comparable aerosolisation characteristics and biofilm-killing efficacy. The liposomal ciprofloxacin formulation of smaller vesicle size showed significantly slower drug release in the dialysis bag technique compared to the free ciprofloxacin solution. Interestingly, liposomal ciprofloxacin formulations successfully controlled the release of the drug in the epithelial cell model and showed different drug transport profiles on H441 cell lines compared to the free ciprofloxacin solution, supporting the potential for inhaled liposomal ciprofloxacin to provide a promising treatment for respiratory infections.

Microfluidic assembly of "Turtle-Like" shaped solid lipid nanoparticles for lysozyme delivery.

After two decades of research in the field of nanomedicine, nanoscale delivery systems for biologicals are becoming clinically relevant tools. Microfluidic-based fabrication processes are replacing conventional techniques based on precipitation, emulsion, and homogenization. Here, the focus is on solid lipid nanoparticles (SLNs) for the encapsulation and delivery of lysozyme (LZ) as a model biologic. A thorough analysis was conducted to compare conventional versus microfluidic-based production techniques, using a 3D-printed device. The efficiency of the microfluidic technique in producing LZ-loaded SLNs (LZ SLNs) was demonstrated: LZ SLNs were found to have a lower size (158.05 ± 4.86 nm vs 180.21 ± 7.46 nm) and higher encapsulation efficacy (70.15 ± 1.65 % vs 53.58 ± 1.13 %) as compared to particles obtained with conventional methods. Cryo-EM studies highlighted a peculiar turtle-like structure on the surface of LZ SLNs. In vitro studies demonstrated that LZ SLNs were suitable to achieve a sustained release over time (7 days). Enzymatic activity of LZ entrapped into SLNs was challenged on Micrococcus lysodeikticus cultures, confirming the stability and potency of the biologic. This systematic analysis demonstrates that microfluidic production of SLNs can be efficiently used for encapsulation and delivery of complex biological molecules.

A microfluidic approach to fabricate sucrose decorated liposomes with increased uptake in breast cancer cells.

Developing targeted drug delivery systems is an urgent need to decrease the side effects and increase the drug's efficiency. Most cancer cells show an increased sugar consumption compared to healthy cells due to the deregulation of sugar transporters. Consequently, liposomes, as a biocompatible nanocarrier, could be surface decorated by sugars to enhance drug targeting into cancer cells. Our work outlines a new strategy to easily manufacture sucrose decorated liposomes using sucrose stearate, a biocompatible and biodegradable non-ionic surfactant, with a scalable microfluidic approach. Sucrose decorated liposomes were loaded with berberine hydrochloride, a well-known phytochemical compound to investigate its effects on triple-negative breast cancer cells (MDA-MB-231). Using the microfluidic manufacturing system, we prepared berberine-loaded liposomes using a mixture of phosphatidylcholine and cholesterol with and without sucrose stearate with a size up to 140 nm and narrow polydispersity. Stability was confirmed for 90 days, and the in vitro release profile was evaluated. The formulations showed acceptable in vitro biocompatibility and significantly higher anti-proliferative effect on MDA-MB-231 cell line. These results have been confirmed by an increased uptake evaluated by flow cytometry and confocal microscopy. Taken together, our findings represent an innovative, easy, and scalable approach to obtain sugar decorated liposomal formulations without any surface-chemistry reactions. They can be potentially used as an anticancer targeted drug delivery system.

Poly(3-hydroxybutyrate): A potential biodegradable excipient for direct 3D printing of pharmaceuticals.

During the past decades, 3D printing has revolutionised different areas of research. Despite the considerable progress achieved in 3D printing of pharmaceuticals, the limited choice of suitable materials remains a challenge to overcome. The growing search for sustainable excipients has led to an increasing interest in biopolymers. Poly(3-hydroxybutyrate) (PHB) is a biocompatible and biodegradable biopolymer obtained from bacteria that could be efficiently employed in the pharmaceutical field. Here we aimed to demonstrate its potential application as a thermoplastic material for personalised medicine through 3D printing. More specifically, we processed PHB by using direct powder extrusion, a one-step additive manufacturing technique. To assess and denote the feasibility and versatility of the process, a 3D square model was manufactured in different dimensions (sidexheight: 12x2 mm; 18x2 mm; 24x2 mm) and loaded with increasing percentages of a model drug (up to 30% w/w). The manufacturing process was influenced by the drug content, and indeed, an increase in the amount of the drug determined a reduction in the printing temperature, without affecting the other parameters (such as the layer height). The composition of the model squares was investigated using Fourier-transform infrared spectroscopy, the resulting spectra confirmed that the starting materials were successfully incorporated into the final formulations. The thermal behaviour of the printed systems was characterized by differential scanning calorimetry, and thermal gravimetric analysis. Moreover, the sustained drug release profile of the formulations was performed over 21 days and showed to be dependent on the dimensions of the printed object and on the amount of loaded drug. Indeed, the formulation with 30% w/w in the dimension 24x2 mm released the highest amount of drug. Hence, the results suggested that PHB and direct powder extrusion technique could be promising tools for the manufacturing of prolonged release and personalised drug delivery forms.

Comparative Analysis of the Antimicrobial Activity of Essential Oils and Their Formulated Microemulsions against Foodborne Pathogens and Spoilage Bacteria.

The antimicrobial activity of several essential oils (EOs) and their related microemulsions (MEs) was investigated. EOs were obtained from Cannabis sativa L. cv CS (C. sativa), Carum carvi L. (C. carvi), Crithmum maritimum L. (C. maritimum), Cuminum cyminum L. (C. cyminum), x Cupressocyparis leylandii A.B. Jacks & Dallim. (C. leylandii), Cupressus arizonica Greene (C. arizonica), Ferula assa-foetida L. (F. assa-foetida)., Ferula gummosa Boiss. (F. gummosa), Juniperus communis L. (J. communis), Juniperus x pfitzeriana (Spath) P.A. Schmidt (J. pfitzeriana), Pimpinella anisum L (P. anisum). Preliminary screening revealed that Cuminum cyminum, Crithmum maritimum, and Pimpinella anisum (10% v/v) were effective against all tested microorganisms (Escherichia coli ATCC 35218, Listeria monocytogenes ATCC 7644, Staphylococcus aureus ATCC 29213, Pseudomonas fluorescens DSM 4358, and Candida albicans ATCC 10231), with growth inhibition diameter from 10 to 25 mm. These EOs were used to formulate the MEs with an average size < 50 nm and a good stability over 30 days. EOs' antimicrobial activity was further enhanced in the MEs, with a generalized lowering of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values. C. cyminum-ME reached, in most cases, MIC two times lower (0.312%) than the corresponding EO (0.625%) and even eight times lower against S. aureus (0.156 vs. 1.25%). A more remarkable microbicide effect was noted for C. cyminum-ME, with MBC values eight times lower (from 0.312 to 0.625%) than the corresponding EO (from 2.5 to 5%). Overall, MEs resulted in an efficient system for EOs encapsulation, enhancing solubility and lowering concentration to exert antimicrobial efficacy.

A combination of sugar esters and chitosan to promote in vivo wound care.

In recent years, researchers are exploring innovative green materials fabricated from renewable natural substances to meet formulation needs. Among them, biopolymers like chitosans and biosurfactants such as sugar fatty acid esters are of potential interest due to their biocompatibility, biodegradability, functionality, and cost-effectiveness. Both classes of biocompounds possess the ability to be efficiently employed in wound dressing to help physiological wound healing, which is a bioprocess involving uncontrolled oxidative damage and inflammation, with an associated high risk of infection. In this work, we synthesized two different sugar esters (i.e., lactose linoleate and lactose linolenate) that, in combination with chitosan and sucrose laurate, were evaluated in vitro for their cytocompatibility, anti-inflammatory, antioxidant, and antibacterial activities and in vivo as wound care agents. Emphasis on Wnt/ß-catenin associated machineries was also set. The newly designed lactose esters, sucrose ester, and chitosan possessed sole biological attributes, entailing considerable blending for convenient formulation of wound care products. In particular, the mixture composed of sucrose laurate (200 µM), lactose linoleate (100 µM), and chitosan (1%) assured its superiority in terms of efficient wound healing prospects in vivo together with the restoring of the Wnt/ß-catenin signaling pathway, compared with the marketed wound healing product (Healosol®), and single components as well. This innovative combination of biomaterials applied as wound dressing could effectively break new ground in skin wound care.

Factors affecting the rheological behaviour of carbomer dispersions in hydroalcoholic medium: Towards the optimization of hand sanitiser gel formulations.

Hand sanitizers represent a primary measure for the prevention of transmissible infections, whose use has been greatly increased during CoViD-19 pandemic. Most of the commercially available products are hydrogels, employing carbomers as thickening agents. However, few information is still available regarding performances of carbomers in hydroalcoholic media containing a percentage of alcohols ≥ 60% v/v as recommended for disinfection. The aim of this study was to investigate the colloidal behaviour of carbomer 974 and carbomer 980 in hydroalcoholic media containing from 50 to 80% w/w of alcohol (ethanol or isopropanol) and neutralised with triethanolamine or aminomethyl propanol. Both carbomers provide transparent hydrogels in water, but carbomer 980 should be preferred for the formulation of hydrogel with a percentage of alcohol ≥ 50% w/w for its better solvation. The critical alcohol concentration (CAlC), above which polymer precipitation occurs, depends on the type of alcohol and base used. Carbomer dispersions with a higher content of alcohol can be prepared using aminomethyl propanol rather than triethanolamine. The choice of the more suitable components is fundamental for the isopropanol-based dispersions since the CAlC is closer to the recommended concentrations for disinfection. Overall, these results provide helpful insights for the correct preparation of alcohol-based hand sanitizers using carbomers.

Nasal vaccination against SARS-CoV-2: Synergistic or alternative to intramuscular vaccines?

It is striking that all marketed SARS-CoV-2 vaccines are developed for intramuscular administration designed to produce humoral and cell mediated immune responses, preventing viremia and the COVID-19 syndrome. They have a high degree of efficacy in humans (70-95%) depending on the type of vaccine. However, little protection is provided against viral replication and shedding in the upper airways due to the lack of a local sIgA immune response, indicating a risk of transmission of virus from vaccinated individuals. A range of novel nasal COVID-19 vaccines are in development and preclinical results in non-human primates have shown a promising prevention of replication and shedding of virus due to the induction of mucosal immune response (sIgA) in upper and lower respiratory tracts as well as robust systemic and humoral immune responses. Whether these results will translate to humans remains to be clarified. An IM prime followed by an IN booster vaccination would likely result in a better well-rounded immune response, including prevention (or strong reduction) in viral replication in the upper and lower respiratory tracts.

Permeability-enhancing effects of three laurate-disaccharide monoesters across isolated rat intestinal mucosae.

Laurate (C12)-sucrose esters are established intestinal epithelial permeation enhancers (PEs) with potential for use in oral delivery. Most studies have examined blends of ester rather than specific monoesters, with little variation on the sugar moiety. To investigate the influence of varying the sugar moiety on monoester performance, we compared three monoesters: C12-sucrose, C12-lactose, and C12-trehalose. The assays were: critical micellar concentration (CMC) in Krebs-Henseleit buffer, MTS and lactate dehydrogenase assays in Caco-2 cells, transepithelial electrical resistance (TEER) and apparent permeability coefficient (Papp) of [14C] mannitol across isolated rat intestinal mucosae, and tissue histology. For CMC, the rank order was C12-trehalose (0.21 mM) < C12-sucrose (0.34 mM) < C12-lactose (0.43 mM). Exposure to Caco-2 cells for 120 min produced TC50 values in the MTS assay from 0.1 to 0.4 mM. Each ester produced a concentration-dependent decrease in TEER across rat mucosae with 80% reduction seen with 8 mM in 5 min, but C12-trehalose was less potent. C12-sucrose and C12-lactose increased the Papp of [14C] mannitol across mucosae with similar potency and efficacy, whereas C12-trehalose was not as potent or efficacious, even though it still increased flux. In the presence of the three esters, gross intestinal histology was unaffected except at 8 mM for C12-sucrose and C12-lactose. In conclusion, the three esters enhanced permeability likely via tight junction modulation in rat intestinal tissue. C12-trehalose was not quite as efficacious, but neither did it damage tissue to the same extent. All three can be considered as potential PEs to be included in oral formulations.

Microfluidics for nanomedicines manufacturing: An affordable and low-cost 3D printing approach.

During the last decade, an innovative lab on a chip technology known as microfluidics became popular in the pharmaceutical field to produce nanomedicines in a scalable way. Nevertheless, the predominant barriers for new microfluidics users are access to expensive equipment and device fabrication expertise. 3D printing technology promises to be an enabling new field that helps to overcome these drawbacks expanding the realm of microfluidics. Among 3D printing techniques, fused deposition modeling allows the production of devices with relatively inexpensive materials and printers. In this work, we developed two different microfluidic chips designed to obtain a passive micromixing by a "zigzag" bas-relief and by the presence of "split and recombine" channels. Computational fluid dynamics studies improved the evaluation of the mixing potential. A fused deposition modeling 3D printer was used to print the developed devices with polypropylene as manufacturing material. Then, two different model nanocarriers (i.e., polymeric nanoparticles and liposomes), loading cannabidiol as model drug, were formulated evaluating the influence of manufacturing parameters on the final nanocarrier characteristics with a design of experiments approach (2-level full factorial design). Both the chips showed an effective production of nanocarriers with tunable characteristics and with an efficient drug loading. These polypropylene-based microfluidic chips could represent an affordable and low-cost alternative to common microfluidic devices for the effective manufacturing of nanomedicines (both polymer- and lipid-based) after appropriate tuning of manufacturing parameters.

Anti-SASP and anti-inflammatory activity of resveratrol, curcumin and ß-caryophyllene association on human endothelial and monocytic cells.

A challenging and promising new branch of aging-related research fields is the identification of natural compounds able to modulate the senescence-associated secretory phenotype (SASP), which characterizes senescent cells and can contribute to fuel the inflammaging. We investigated both the anti-SASP and anti-inflammatory activities of a nutritional supplement, namely Fenoxidol™, composed of turmeric extract bioCurcumin (bCUR), Polydatin (the natural glycosylated precursor of Resveratrol-RSV), and liposomal ß-caryophyllene (BCP), in two human cellular models, such as the primary endothelial cell line, HUVECs and the monocytic cell line, THP-1. Replicative and Doxorubicin-induced senescent HUVECs, both chosen as cellular models of SASP, and lipopolysaccharides (LPS)-stimulated THP-1, selected as a model of the inflammatory response, were treated with the three single natural compounds or with a combination of them (MIX). In both senescent HUVEC models, MIX treatment significantly reduced IL-1ß and IL-6 expression levels and p16ink4a protein, and also increased SIRT1 protein level, as well as downregulated miR-146a and miR-21 expression, two of the so-called inflamma-miRNAs, more effectively than the single compounds. In THP-1 cells stimulated with LPS, the MIX showed a significant effect in decreasing IL-1ß, IL-6, TNF-α, and miR-146a expression levels and Caspase-1 activation, in association with an up-regulation of SIRT1 protein, compared to the single compounds. Overall, our results suggest that the three analysed compounds can have a combined effect in restraining SASP in senescent HUVECs as well as the inflammatory response in LPS-stimulated THP-1 cells.

3D printed clotrimazole intravaginal ring for the treatment of recurrent vaginal candidiasis.

Vulvovaginal candidiasis is a vaginal infection caused by the fungal pathogen Candida albicans that, most commonly, affects women of reproductive age. Its first-line treatment consists in topical applications of conventional drug formulations (e.g., creams, gels, tablets) containing imidazole drugs. The treatment involves single or multiple daily applications and, in the case of recurrences, daily administration of oral antifungal drugs for up to one month. Intravaginal rings are flexible, biocompatible medical devices that, compared to conventional drug formulations, offer the possibility of a controlled vaginal drug delivery over a determined period with a single application, thus increasing patient compliance. Among innovative manufacturing techniques, in recent years, fused deposition modeling 3D printing has emerged in the pharmaceutical field to produce different therapeutics combining drugs and polymers. This technique allows to print objects layer by layer with many different thermoplastic materials after a computer-assisted design. Thermoplastic polyurethanes are flexible and biocompatible materials that can be efficiently employed for the manufacturing of drug release systems, already utilized to prepare vaginal devices. In this work, we produced a clotrimazole-loaded intravaginal ring by fused deposition modeling 3D printing combining the drug with thermoplastic polyurethane using hot melt extrusion. The rings were computer-designed and then printed with two different drug concentrations (i.e., 2% and 10% w/w). The intravaginal rings were first tested in an agar-diffusion test to evaluate their effectiveness against C. albicans; and the 10% loaded ring was selected for further studies. Drug release was evaluated in two different media (i.e., 50% ethanol and vaginal fluid simulant) showing a sustained release over a period of seven days. Next, in vitro time-kill analysis against C. albicans in simulated vaginal fluid was performed and displayed a complete growth inhibition after 5 days, compared to the control. These results suggest a potential application of these 3D printed intravaginal rings for the treatment of vulvovaginal candidiasis and for the long-time treatment of recurrences.

Peptide-guided resiquimod-loaded lignin nanoparticles convert tumor-associated macrophages from M2 to M1 phenotype for enhanced chemotherapy.

Nanomedicines represent innovative and promising alternative technologies to improve the therapeutic effects of different drugs for cancer ablation. Targeting M2-like tumor-associated macrophages (TAMs) has emerged as a favorable therapeutic approach to fight against cancer through the modulation of the tumor microenvironment. However, the immunomodulatory molecules used for this purpose present side effects upon systemic administration, which limits their clinical translation. Here, the biocompatible lignin polymer is used to prepare lignin nanoparticles (LNPs) that carry a dual agonist of the toll-like receptors TLR7/8 (resiquimod, R848). These LNPs are targeted to the CD206-positive M2-like TAMs using the "mUNO" peptide, in order to revert their pro-tumor phenotype into anti-tumor M1-like macrophages in the tumor microenvironment of an aggressive triple-negative in vivo model of breast cancer. Overall, we show that targeting the resiquimod (R848)-loaded LNPs to the M2-like macrophages, using very low doses of R848, induces a profound shift in the immune cells in the tumor microenvironment towards an anti-tumor immune state, by increasing the representation of M1-like macrophages, cytotoxic T cells, and activated dendritic cells. This effect consequently enhances the anticancer effect of the vinblastine (Vin) when co-administered with R848-loaded LNPs. STATEMENT OF SIGNIFICANCE: Lignin-based nanoparticles (LNPs) were successfully developed to target a potent TLR7/8 agonist (R848) of the tumor microenvironment (TME). This was achieved by targeting the mannose receptor (CD206) on the tumor supportive (M2-like) macrophages with the "mUNO" peptide, to reprogram them into an anti-tumor (M1-like) phenotype for enhanced chemotherapy. LNPs modified the biodistribution of the R848, and enhanced its accumulation and efficacy in shifting the immunological profile of the cells in the TME, which was not achieved by systemic administration of free R848. Moreover, a reduction in the tumor volumes was observed at lower equivalent doses of R848 compared with other studies. Therefore, the co-administration of R848@LNPs is a promising chemotherapeutic application in aggressive tumors, such as the triple-negative breast cancer.