Modulation of Serotonin-Related Genes by Extracellular Vesicles of the Probiotic Escherichia coli Nissle 1917 in the Interleukin-1ß-Induced Inflammation Model of Intestinal Epithelial Cells.

Inflammatory bowel disease (IBD) is a chronic inflammatory condition involving dysregulated immune responses and imbalances in the gut microbiota in genetically susceptible individuals. Current therapies for IBD often have significant side-effects and limited success, prompting the search for novel therapeutic strategies. Microbiome-based approaches aim to restore the gut microbiota balance towards anti-inflammatory and mucosa-healing profiles. Extracellular vesicles (EVs) from beneficial gut microbes are emerging as potential postbiotics. Serotonin plays a crucial role in intestinal homeostasis, and its dysregulation is associated with IBD severity. Our study investigated the impact of EVs from the probiotic Nissle 1917 (EcN) and commensal E. coli on intestinal serotonin metabolism under inflammatory conditions using an IL-1ß-induced inflammation model in Caco-2 cells. We found strain-specific effects. Specifically, EcN EVs reduced free serotonin levels by upregulating SERT expression through the downregulation of miR-24, miR-200a, TLR4, and NOD1. Additionally, EcN EVs mitigated IL-1ß-induced changes in tight junction proteins and oxidative stress markers. These findings underscore the potential of postbiotic interventions as a therapeutic approach for IBD and related pathologies, with EcN EVs exhibiting promise in modulating serotonin metabolism and preserving intestinal barrier integrity. This study is the first to demonstrate the regulation of miR-24 and miR-200a by probiotic-derived EVs.

Microbiota-Derived Extracellular Vesicles as a Postbiotic Strategy to Alleviate Diarrhea and Enhance Immunity in Rotavirus-Infected Neonatal Rats.

Rotavirus (RV) infection is a major cause of acute gastroenteritis in children under 5 years old, resulting in elevated mortality rates in low-income countries. The efficacy of anti-RV vaccines is limited in underdeveloped countries, emphasizing the need for novel strategies to boost immunity and alleviate RV-induced diarrhea. This study explores the effectiveness of interventions involving extracellular vesicles (EVs) from probiotic and commensal E. coli in mitigating diarrhea and enhancing immunity in a preclinical model of RV infection in suckling rats. On days 8 and 16 of life, variables related to humoral and cellular immunity and intestinal function/architecture were assessed. Both interventions enhanced humoral (serum immunoglobulins) and cellular (splenic natural killer (NK), cytotoxic T (Tc) and positive T-cell receptor γδ (TCRγδ) cells) immunity against viral infections and downregulated the intestinal serotonin receptor-3 (HTR3). However, certain effects were strain-specific. EcoR12 EVs activated intestinal CD68, TLR2 and IL-12 expression, whereas EcN EVs improved intestinal maturation, barrier properties (goblet cell numbers/mucin 2 expression) and absorptive function (villus length). In conclusion, interventions involving probiotic/microbiota EVs may serve as a safe postbiotic strategy to improve clinical symptoms and immune responses during RV infection in the neonatal period. Furthermore, they could be used as adjuvants to enhance the immunogenicity and efficacy of anti-RV vaccines.

Therapeutic Applications of Essential Oils from Native and Cultivated Ecuadorian Plants: Cutaneous Candidiasis and Dermal Anti-Inflammatory Activity.

Essential oils are a complex mixture of aromatic substances whose pharmacological actions, including antimicrobial, antioxidant, anticancer, and anti-inflammatory activities, have been widely reported. This study aimed to evaluate the anti-Candida and dermal anti-inflammatory activity of essential oils from native and cultivated Ecuadorian plants. Essential oils from Bursera graveolens, Dacryodes peruviana, Mespilodaphne quixos, and Melaleuca armillaris were isolated by hydrodistillation and were characterized physically and chemically. Its tolerance was analyzed by in vitro and in vivo studies. The antifungal activity was studied against Candida albicans, Candida glabrata, and Candida parapsilosis, whereas the anti-inflammatory effect was evaluated by a mouse ear edema model. The main compounds were limonene, α-phellandrene, (E)-methyl cinnamate, and 1,8-cineole, respectively. All essential oils showed high tolerability for skin application, antifungal activity against the three Candida strains, and anti-inflammatory efficacy by decreasing edema and overexpression of pro-inflammatory cytokines. Dacryodes peruviana essential oil showed the highest antifungal activity. On the other hand, Dacryodes peruviana and Melaleuca armillaris showed the greatest anti-inflammatory potential, decreasing edema by 53.3% and 65.25%, respectively, and inhibiting the overexpression of TNF-α, IL-8, IL-17A, and IL-23. The results suggest that these essential oils could be used as alternative therapies in the treatment of both cutaneous candidiasis and dermal inflammation.

Thymol-loaded PLGA nanoparticles: an efficient approach for acne treatment.

BACKGROUND: Acne is a common skin disorder that involves an infection inside the hair follicle, which is usually treated with antibiotics, resulting in unbalanced skin microbiota and microbial resistance. For this reason, we developed polymeric nanoparticles encapsulating thymol, a natural active compound with antimicrobial and antioxidant properties. In this work, optimization physicochemical characterization, biopharmaceutical behavior and therapeutic efficacy of this novel nanostructured system were assessed. RESULTS: Thymol NPs (TH-NP) resulted on suitable average particle size below 200 nm with a surface charge around - 28 mV and high encapsulation efficiency (80%). TH-NP released TH in a sustained manner and provide a slow-rate penetration into the hair follicle, being highly retained inside the skin. TH-NP possess a potent antimicrobial activity against Cutibacterium acnes and minor effect towards Staphylococcus epidermis, the major resident of the healthy skin microbiota. Additionally, the stability and sterility of developed NPs were maintained along storage. CONCLUSION: TH-NP showed a promising and efficient alternative for the treatment of skin acne infection, avoiding antibiotic administration, reducing side effects, and preventing microbial drug resistance, without altering the healthy skin microbiota. Additionally, TH-NP enhanced TH antioxidant activity, constituting a natural, preservative-free, approach for acne treatment.

Cell-to-Cell Communication by Host-Released Extracellular Vesicles in the Gut: Implications in Health and Disease.

Communication between cells is crucial to preserve body homeostasis and health. Tightly controlled intercellular dialog is particularly relevant in the gut, where cells of the intestinal mucosa are constantly exposed to millions of microbes that have great impact on intestinal homeostasis by controlling barrier and immune functions. Recent knowledge involves extracellular vesicles (EVs) as mediators of such communication by transferring messenger bioactive molecules including proteins, lipids, and miRNAs between cells and tissues. The specific functions of EVs principally depend on the internal cargo, which upon delivery to target cells trigger signal events that modulate cellular functions. The vesicular cargo is greatly influenced by genetic, pathological, and environmental factors. This finding provides the basis for investigating potential clinical applications of EVs as therapeutic targets or diagnostic biomarkers. Here, we review current knowledge on the biogenesis and cargo composition of EVs in general terms. We then focus the attention to EVs released by cells of the intestinal mucosa and their impact on intestinal homeostasis in health and disease. We specifically highlight their role on epithelial barrier integrity, wound healing of epithelial cells, immunity, and microbiota shaping. Microbiota-derived EVs are not reviewed here.

Screening Anti-Inflammatory Effects of Flavanones Solutions.

There are a large number of remedies in traditional medicine focused on relieving pain and inflammation. Flavanones have been a potential source in the search for leading compounds and biologically active components, and they have been the focus of much research and development in recent years. Eysenhardtia platycarpa is used in traditional medicine for the treatment of kidney diseases, bladder infections, and diabetes mellitus. Many compounds have been isolated from this plant, such as flavones, flavanones, phenolic compounds, triterpenoid acids, chalcones, sugars, and fatty acids, among others. In this paper, natural flavanone 1 (extracted from Eysenhardtia platycarpa) as lead compound and flavanones 1a-1d as its structural analogues were screened for anti-inflammatory activity using Molinspiration® and PASS Online in a computational study. The hydro alcoholic solutions (FS) of flavanones 1, 1a-1d (FS1, FS1a-FS1d) were also assayed to investigate their in vivo anti-inflammatory cutaneous effect using two experimental models, a rat ear edema induced by arachidonic acid (AA) and a mouse ear edema induced by 12-O-tetradecanoylphorbol acetate (TPA). Histological studies and analysis of pro-inflammatory cytokines TNF-α, IL-1ß, and IL-6 were also assessed in AA-inflamed rat ear tissue. The results showed that the flavanone hydro alcoholic solutions (FS) caused edema inhibition in both evaluated models. This study suggests that the evaluated flavanones will be effective when used in the future in skin pathologies with inflammation, with the results showing 1b and 1d to be the best.

Luminescent PtII and PtIV Platinacycles with Anticancer Activity Against Multiplatinum-Resistant Metastatic CRC and CRPC Cell Models.

Platinum-based chemotherapy persists to be the only effective therapeutic option against a wide variety of tumours. Nevertheless, the acquisition of platinum resistance is utterly common, ultimately cornering conventional platinum drugs to only palliative in many patients. Thus, encountering alternatives that are both effective and non-cross-resistant is urgent. In this work, we report the synthesis, reduction studies, and luminescent properties of a series of cyclometallated (C,N,N')PtIV compounds derived from amine-imine ligands, and their remarkable efficacy at the high nanomolar range and complete lack of cross-resistance, as an intrinsic property of the platinacycle, against multiplatinum-resistant colorectal cancer (CRC) and castration-resistant prostate cancer (CRPC) metastatic cell lines generated for this work. We have also determined that the compounds are effective and selective for a broader cancer panel, including breast and lung cancer. Additionally, selected compounds have been further evaluated, finding a shift in their antiproliferative mechanism towards more cytotoxic and less cytostatic than cisplatin against cancer cells, being also able to oxidize cysteine residues and inhibit topoisomerase II, thereby holding great promise as future improved alternatives to conventional platinum drugs.

Extracellular vesicles and soluble factors secreted by Escherichia coli Nissle 1917 and ECOR63 protect against enteropathogenic E. coli-induced intestinal epithelial barrier dysfunction.

BACKGROUND: Enteric pathogens have developed mechanisms to disrupt tight junctions and increase gut permeability. Many studies have analysed the ability of live probiotics to protect intestinal epithelial cells against tight junction damage caused by bacterial pathogens. Escherichia coli Nissle 1917 (EcN) is among the probiotics that positively modulates the intestinal epithelial barrier by regulating expression and distribution of tight junction proteins. We previously reported that regulation of ZO-1, claudin-14 and claudin-2 is mediated by EcN secreted factors, either free-released or associated with outer membrane vesicles (OMVs). Factors secreted by commensal ECOR63 elicited comparable effects in intact epithelial T-84 and Caco-2 cell monolayers. RESULTS: Here we analyse the ability of OMVs and soluble secreted factors to protect epithelial barrier function in polarized T-84 and Caco-2 cells infected with enteropathogenic Escherichia coli (EPEC). Transepithelial electrical resistance, paracellular permeability, mRNA levels and subcellular distribution of tight junction proteins were monitored in the absence or presence of EcN and ECOR63 extracellular fractions. EPEC downregulated expression of ZO-1 ZO-2, occludin and claudin-14 and altered the subcellular localization of ZO-1, occludin and F-actin cytoskeleton. OMVs and soluble factors secreted by EcN and ECOR63 counteracted EPEC-altered transepithelial resistance and paracellular permeability, preserved occludin and claudin-14 mRNA levels, retained ZO-1 and occludin at tight junctions in the cell boundaries and ameliorated F-actin disorganization. Redistribution of ZO-1 was not accompanied by changes at mRNA level. CONCLUSION: This study provides new insights on the role of microbiota secreted factors on the modulation of intestinal tight junctions, expanding their barrier-protective effects against pathogen-induced disruption.

Multifunctional Serine Protease Inhibitor-Coated Water-Soluble Gold Nanoparticles as a Novel Targeted Approach for the Treatment of Inflammatory Skin Diseases.

The overexpression and increased activity of the serine protease Kallikrein 5 (KLK5) is characteristic of inflammatory skin diseases such as Rosacea. The use of inhibitors of this enzyme-such as 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF·HCl) or the anti-human recombinant Kallikrein 5 (anti-KLK5) antibody-in the treatment of the disease has been limited due to their low bioavailability, for which their immobilization in drug delivery agents can contribute to making serine protease inhibitors clinically useful. In this work, we synthesized gold nanoparticles (GNP) coated with a mixture of hydroxyl- and carboxyl-terminated thiolates (GNP.OH/COOH), whose carboxyl groups were used to further functionalize the nanoparticles with the serine protease inhibitor AEBSF·HCl either electrostatically or covalently (GNP.COOH AEBSF and GNP.AEBSF, respectively), or with the anti-KLK5 antibody (GNP.antiKLK5). The synthesized and functionalized GNP were highly water-soluble, and they were extensively characterized using UV-vis absorption spectroscopy, Transmission Electron Microscopy (TEM), Dynamic Light Scattering (DLS), and Thermogravimetric Analysis (TGA). GNP.OH/COOH and their subsequent functionalizations effectively inhibited KLK5 in vitro. Internalization of fluorophore-coated GNP.OH/COOH in human keratinocytes (HaCaT cells) was proven using confocal fluorescence microscopy. Cell viability assays revealed that the cytotoxicity of free AEBSF is importantly decreased when it is incorporated in the nanoparticles, either ionically (GNP.COOH AEBSF) or, most importantly, covalently (GNP.AEBSF). The functionalized nanoparticles GNP.AEBSF and GNP.antiKLK5 inhibited intracellular KLK5 activity in HaCaT cells and diminished secretion of IL-8 under inflammatory conditions triggered by TLR-2 ligands. This study points to the great potential of these GNP as a new intracellular delivery strategy for both small drugs and antibodies in the treatment of skin diseases such as Rosacea.

On the stability and biological behavior of cyclometallated Pt(IV) complexes with halido and aryl ligands in the axial positions.

A series of cyclometallated platinum(IV) compounds (3a, 3a' and 3b') with a meridional [C,N,N'] terdentate ligand, featuring an halido and an aryl group in the axial positions has been evaluated for electrochemical reduction and preliminary biological behavior against a panel of human adenocarcinoma (A-549 lung, HCT-116 colon, and MCF-7 breast) cell lines and the normal bronquial epithelial BEAS-2B cells. Cathodic reduction potentials (shifting from -1.463 to -1.570V) reveal that the platinum(IV) compounds under study would be highly reluctant to be reduced in a biological environment. Actually ascorbic acid was not able to reduce complex 3a', the most prone to be reduced according its reduction potential, over a period of one week. These results suggest an intrinsic activity for the investigated platinum(IV) complexes (3a, 3a' and 3b'), which exhibit a remarkable cytotoxicity effectiveness (with IC50 values in the low micromolar range), even greater than that of cisplatin. The IC50 for A-549 lung cells and clog P values were found to follow the same trend: 3b'>3a'>3a. However, no correlation was observed between reduction potential and in vitro activity. As a representative example, cyclometallated platinum(IV) compound 3a', exercise its antiproliferative activity directly over non-microcytic A-549 lung cancer cells through a mixture of cell cycle arrest (13% arrest at G1 phase and 46% arrest at G2 phase) and apoptosis induction (increase of early apoptosis by 30 times with regard to control). To gain further insights into the mode of action of the investigated platinum(IV) complexes, drug uptake, cathepsin B inhibition and ROS generation were also evaluated. Interestingly an increased ROS generation could be related with the antiproliferative activity of the cyclometallated platinum(IV) series under study in the cisplatin-resistant A-549 lung and HCT-116 cancer cell lines.

The secreted autotransporter toxin (Sat) does not act as a virulence factor in the probiotic Escherichia coli strain Nissle 1917.

BACKGROUND: Escherichia coli Nissle 1917 (EcN) is a probiotic used in the treatment of intestinal diseases. Although it is considered safe, EcN is closely related to the uropathogenic E. coli strain CFT073 and contains many of its predicted virulence elements. Thus, it is relevant to assess whether virulence-associated genes are functional in EcN. One of these genes encodes the secreted autotransporter toxin (Sat), a member of the serine protease autotransporters of Enterobacteriaceae (SPATEs) that are secreted following the type V autotransporter pathway. Sat is highly prevalent in certain E. coli pathogenic groups responsible for urinary and intestinal infections. In these pathogens Sat promotes cytotoxic effects in several lines of undifferentiated epithelial cells, but not in differentiated Caco-2 cells. RESULTS: Here we provide evidence that sat is expressed by EcN during the colonization of mouse intestine. The EcN protein is secreted as an active serine protease, with its 107 kDa-passenger domain released into the medium as a soluble protein. Expression of recombinant EcN Sat protein in strain HB101 increases paracellular permeability to mannitol in polarized Caco-2 monolayers. This effect, also reported for the Sat protein of diffusely adherent E. coli, is not observed when this protein is expressed in the EcN background. In addition, we show that EcN supernatants confer protection against Sat-mediated effects on paracellular permeability, thus indicating that other secreted EcN factors are able to prevent barrier disruption caused by pathogen-related factors. Sat is not required for intestinal colonization, but the EcNsat::cat mutant outcompetes wild-type EcN in the streptomycin-treated mouse model. Analysis of the presence of sat in 29 strains of the ECOR collection isolated from stools of healthy humans shows 34.8 % positives, with high prevalence of strains of the phylogenetic groups D and B2, related with extra-intestinal infections. CONCLUSIONS: Sat does not act as a virulence factor in EcN. The role of Sat in intestinal pathogenesis relies on other genetic determinants responsible for the bacterial pathotype.

Proteomic analysis of outer membrane vesicles from the probiotic strain Escherichia coli Nissle 1917.

Escherichia coli Nissle 1917 (EcN) is a probiotic used for the treatment of intestinal disorders. EcN improves gastrointestinal homeostasis and microbiota balance; however, little is known about how this probiotic delivers effector molecules to the host. Outer membrane vesicles (OMVs) are constitutively produced by Gram-negative bacteria and have a relevant role in bacteria-host interactions. Using 1D SDS-PAGE and highly sensitive LC-MS/MS analysis we identified in this study 192 EcN vesicular proteins with high confidence in three independent biological replicates. Of these proteins, 18 were encoded by strain-linked genes and 57 were common to pathogen-derived OMVs. These proteins may contribute to the ability of this probiotic to colonize the human gut as they fulfil functions related to adhesion, immune modulation or bacterial survival in host niches. This study describes the first global OMV proteome of a probiotic strain and provides evidence that probiotic-derived OMVs contain proteins that can target these vesicles to the host and mediate their beneficial effects on intestinal function. All MS data have been deposited in the ProteomeXchange with identifier PXD000367 (http://proteomecentral.proteomexchange.org/dataset/PXD000367).

Extracellular Vesicles of the Probiotic Escherichia coli Nissle 1917 Reduce PepT1 Levels in IL-1ß-Treated Caco-2 Cells via Upregulation of miR-193a-3p.

PepT1, a proton-coupled oligopeptide transporter, is crucial for intestinal homeostasis. It is mainly expressed in small intestine enterocytes, facilitating the absorption of di/tri-peptides from dietary proteins. In the colon, PepT1 expression is minimal to prevent excessive responses to proinflammatory peptides from the gut microbiota. However, increased colonic PepT1 is linked to chronic inflammatory diseases and colitis-associated cancer. Despite promising results from animal studies on the benefits of extracellular vesicles (EVs) from beneficial gut commensals in treating IBD, applying probiotic EVs as a postbiotic strategy in humans requires a thorough understanding of their mechanisms. Here, we investigate the potential of EVs of the probiotic Nissle 1917 (EcN) and the commensal EcoR12 in preventing altered PepT1 expression under inflammatory conditions, using an interleukin (IL)-1-induced inflammation model in Caco-2 cells. The effects are evaluated by analyzing the expression of PepT1 (mRNA and protein) and miR-193a-3p and miR-92b, which regulate, respectively, PepT1 mRNA translation and degradation. The influence of microbiota EVs on PepT1 expression is also analyzed in the presence of bacterial peptides that are natural substrates of colonic PepT1 to clarify how the regulatory mechanisms function under both physiological and pathological conditions. The main finding is that EcN EVs significantly decreases PepT1 protein via upregulation of miR-193a-3p. Importantly, this regulatory effect is strain-specific and only activates in cells exposed to IL-1ß, suggesting that EcN EVs does not control PepT1 expression under basal conditions but can play a pivotal role in response to inflammation as a stressor. By this mechanism, EcN EVs may reduce inflammation in response to microbiota in chronic intestinal disorders by limiting the uptake of bacterial proinflammatory peptides.

Exploring the effect of the axial ligands on the anticancer activity of [C,N,N'] Pt(IV) cyclometallated compounds.

The synthesis of three novel [C,N,N'] Pt(IV) cyclometallated compounds containing hydroxo, dichloroacetato or trifluoroacetato axial ligands is reported. Compound [PtCl(OH)2{(CH3)2N(CH2)2NCH(4-FC6H3)}] (3) was prepared by the oxidative addition of hydrogen peroxide to [C,N,N'] Pt(II) cyclometallated compound [PtCl{(CH3)2N(CH2)2NCH(4-FC6H3)}] (1) and further the reaction of compound 3 with dichloroacetate or trifluoroacetate anhydrides led to the formation of the corresponding compounds [PtCl(CHCl2COO)2{(CH3)2N(CH2)2NCH(4-FC6H3)}] (4) and [PtCl(CF3COO)2{(CH3)2N(CH2)2NCH(4-FC6H3)}] (5). The properties of the new compounds along with those of the compound [PtCl3{(CH3)2N(CH2)2NCH(4-FC6H3)}] (2), including stability in aqueous media, reduction potential using cyclic voltammetry, cytotoxic activity against the HCT116 CRC cell line, DNA interaction, topoisomerase I and cathepsin inhibition, and computational studies involving reduction of the Pt(IV) compounds and molecular docking studies, are presented. Interestingly, the antiproliferative activity of these compounds against the HCT116 CRC cell line, which is in all cases higher than that of cisplatin, follows the same trend as the reduction potentials so that the most easily reduced compound 2 is the most potent. In contrast, according to the electrophoretic mobility and molecular docking studies, the efficacy of these compounds in binding to DNA is not related to their cytotoxicity. The most active compound 2 does not modify the DNA electrophoretic mobility while the less potent compound 3 is the most efficient in binding to DNA. Although compounds 2 and 3 have only a slight effect on cell cycle distribution and apoptosis induction, generation of ROS to a higher extent for the most easily reduced compound 2 was observed.

In vitro and in vivo studies of ocular topically administered NLC for the treatment of uveal melanoma.

Uveal melanoma is one of the most common and aggressive intraocular malignancies, and, due to its great capability of metastasize, it constitutes the most incident intraocular tumor in adults. However, to date there is no effective treatment since achieving the inner ocular tissues still constitutes one of the greatest challenges in actual medicine, because of the complex structure and barriers. Uncoated and PEGylated nanostructured lipid carriers were developed to achieve physico-chemical properties (mean particle size, homogeneity, zeta potential, pH and osmolality) compatible for the ophthalmic administration of (S)-(-)-MRJF22, a new custom-synthetized prodrug for the potential treatment of uveal melanoma. The colloidal physical stability was investigated at different temperatures by Turbiscan® Ageing Station. Morphology analysis and mucoadhesive studies highlighted the presence of small particles suitable to be topically administered on the ocular surface. In vitro release studies performed using Franz diffusion cells demonstrated that the systems were able to provide a slow and prolonged prodrug release. In vitro cytotoxicity test on Human Corneal Epithelium and Human Uveal Melanoma cell lines and Hen's egg-chorioallantoic membrane test showed a dose-dependent cytotoxic effect of the free prodrug on corneal cells, whose cytocompatibility improved when encapsulated into nanoparticles, as also confirmed by in vivo studies on New Zealand albino rabbits. Antiangiogenic capability and preventive anti-inflammatory properties were also investigated on embryonated eggs and rabbits, respectively. Furthermore, preliminary in vivo biodistribution images of fluorescent nanoparticles after topical instillation in rabbits' eyes, suggested their ability to reach the posterior segment of the eye, as a promising strategy for the treatment of choroidal uveal melanoma.

Gel-Dispersed Nanostructured Lipid Carriers Loading Thymol Designed for Dermal Pathologies.

Purpose: Acne vulgaris is one of the most prevalent dermal disorders affecting skin health and appearance. To date, there is no effective cure for this pathology, and the majority of marketed formulations eliminate both healthy and pathological microbiota. Therefore, hereby we propose the encapsulation of an antimicrobial natural compound (thymol) loaded into lipid nanostructured systems to be topically used against acne. Methods: To address this issue, nanostructured lipid carriers (NLC) capable of encapsulating thymol, a natural compound used for the treatment of acne vulgaris, were developed either using ultrasonication probe or high-pressure homogenization and optimized using 22-star factorial design by analyzing the effect of NLC composition on their physicochemical parameters. These NLC were optimized using a design of experiments approach and were characterized using different physicochemical techniques. Moreover, short-term stability and cell viability using HaCat cells were assessed. Antimicrobial efficacy of the developed NLC was assessed in vitro and ex vivo. Results: NLC encapsulating thymol were developed and optimized and demonstrated a prolonged thymol release. The formulation was dispersed in gels and a screening of several gels was carried out by studying their rheological properties and their skin retention abilities. From them, carbomer demonstrated the capacity to be highly retained in skin tissues, specifically in the epidermis and dermis layers. Moreover, antimicrobial assays against healthy and pathological skin pathogens demonstrated the therapeutic efficacy of thymol-loaded NLC gelling systems since NLC are more efficient in slowly reducing C. acnes viability, but they possess lower antimicrobial activity against S. epidermidis, compared to free thymol. Conclusion: Thymol was successfully loaded into NLC and dispersed in gelling systems, demonstrating that it is a suitable candidate for topical administration against acne vulgaris by eradicating pathogenic bacteria while preserving the healthy skin microbiome.

Semi-solid functionalized nanostructured lipid carriers loading thymol for skin disorders.

Acne constitutes one of the most prevalent skin disorder affecting both skin and mental health of patients. However, no cure has been developed so far. In this area, Thymol constitutes a potential candidate since it is able to restore the healthy microbiota of the skin. However, its permeation properties cause its fast elimination and, to avoid this problem, thymol has been loaded into nanostructured lipid carriers (TH-NLCs). Moreover, to increase the suitability of these systems for skin applications, several surface functionalization strategies of TH-NLCs had been assessed. Among the different molecules, phosphatidylcholine-TH-NLCs demonstrated to be safe as well as to provide high antioxidant activity in cellular studies. Therefore, to administer these systems to the skin, functionalized TH-NLCs were dispersed into a carbomer gel developing semi-solid formulations. Rheological properties, porosity and extensibility of TH dispersed in carbomer as well as phosphatidylcholine-TH-NLCs were assessed demonstrating suitable properties for dermal applications. Moreover, both formulations were applied in healthy volunteers demonstrating that gel-phosphatidylcholine-TH-NLCs were able to increase in skin hydration, decrease water loss and reduce skin sebum. Therefore, gel-phosphatidylcholine-TH-NLCs proved to be a suitable system for skin pathologies linked with high sebum generation, loss of hydration and high oxidation, such as acne vulgaris.

Colloidal hydrogel systems of thymol-loaded PLGA nanoparticles designed for acne treatment.

Thymol-loaded PLGA nanoparticles (TH-NPs) were incorporated into different semi-solid formulations using variable gelling agents (carbomer, polysaccharide and poloxamer). The formulations were physicochemically characterized in terms of size, polydispersity index and zeta potential. Moreover, stability studies were performed by analyzing the backscattering profile showing that the gels were able to increase the nanoparticles stability at 4 °C. Moreover, rheological properties showed that all gels were able to increase the viscosity of TH-NPs with the carbomer gels showing the highest values. Moreover, the observation of carbomer dispersed TH-NPs under electron microscopical techniques showed 3D nanometric cross-linked filaments with the NPs found embedded in the threads. In addition, cytotoxicity studies showed that keratinocyte cells in contact with the formulations obtained cell viability values higher than 70 %. Furthermore, antimicrobial efficacy was assessed against C. acnes and S. epidermidis showing that the formulations eliminated the pathogenic C. acnes but preserved the resident S. epidermidis which contributes towards a healthy skin microbiota. Finally, biomechanical properties of TH-NPs dispersed in carbomer gels in contact with healthy human skin were studied showing that they did not alter skin properties and were able to reduce sebum which is increased in acne vulgaris. As a conclusion, TH-NPs dispersed in semi-solid formulations and, especially in carbomer gels, may constitute a suitable solution for the treatment of acne vulgaris.

Hydrogel of Thyme-Oil-PLGA Nanoparticles Designed for Skin Inflammation Treatment.

Thyme oil (THO) possesses excellent antibacterial and antioxidant properties which are suitable for skin inflammatory disorders such as acne vulgaris. However, THO is insoluble in water and its components are highly volatile. Therefore, these drawbacks may be overcome by its encapsulation in biodegradable PLGA nanoparticles (THO-NPs) that had been functionalized using several strategies. Moreover, cell viability was studied in HaCat cells, confirming their safety. In order to assess therapeutic efficacy against acne, bacterial reduction capacity and antioxidant properties were assessed. Moreover, the anti-inflammatory and wound-healing abilities of THO-NPs were also confirmed. Additionally, ex vivo antioxidant assessment was carried out using pig skin, demonstrating the suitable antioxidant properties of THO-NPs. Moreover, THO and THO-NPs were dispersed in a gelling system, and stability, rheological properties, and extensibility were assessed. Finally, the biomechanical properties of THO-hydrogel and THO-NP-hydrogel were studied in human volunteers, confirming the suitable activity for the treatment of acne. As a conclusion, THO has been encapsulated into PLGA NPs, and in vitro, ex vivo, and in vivo assessments had been carried out, demonstrating excellent properties for the treatment of inflammatory skin disorders.

Pt(II) complexes with (N,N') or (C,N,E)(-) (E=N,S) ligands: cytotoxic studies, effect on DNA tertiary structure and structure-activity relationships.

The cytotoxic activity of two series of platinum(II) complexes containing the polyfunctional imines R(1)-CHN-R(2) [R(1)=phenyl or ferrocenyl unit and R(2)=(CH2)n-CH2-NMe2 where n=1 or 2) (1 and 2) or C6H4-2-SMe (3)] acting as a bidentate (N,N') (4-7) or terdentate [C(phenyl or ferrocenyl),N,N'](-) (8-10) or [C(ferrocenyl),N,S](-) ligand (11) in front of A549 lung, MDA-MB231 breast and HCT116 colon human adenocarcinoma cell lines is reported. The results reveal that most of the platinum(II) complexes are active against the three assayed lines and compounds 6, 7 and the platinacycles 10 and 11 exhibit a remarkable antiproliferative activity, even greater than cisplatin itself, in the cisplatin resistant HCT116 human cancer cell line. Electrophoretic DNA migration studies showed that most of them modify the DNA tertiary structure in a similar way as the reference cisplatin. Solution studies of a selection of the most relevant complexes have also been performed in order to test: (a) their stability in the aqueous biological medium and/or the formation of biologically active species and (b) their proclivity to react with 9-methylguanine (9-MeG), as a model nucleobase. Computational studies at DFT level have also been performed in order to explain the different solution behaviour of the complexes and their proclivity to react with the nucleobase.