Preparation and Characterization of Dentin Phosphophoryn-Derived Peptide-Functionalized Lignin Nanoparticles for Enhanced Cellular Uptake.

The surface modification of nanoparticles (NPs) using different ligands is a common strategy to increase NP-cell interactions. Here, dentin phosphophoryn-derived peptide (DSS) lignin nanoparticles (LNPs) are prepared and characterized, the cellular internalization of the DSS-functionalized LNPs (LNPs-DSS) into three different cancer cell lines is evaluated, and their efficacy with the widely used iRGD peptide is compared. It is shown that controlled extent of carboxylation of lignin improves the stability at physiological conditions of LNPs formed upon solvent exchange. Functionalization with DSS and iRGD peptides maintains the spherical morphology and moderate polydispersity of LNPs. The LNPs exhibit good cytocompatibility when cultured with PC3-MM2, MDA-MB-231, and A549 in the conventional 2D model and in the 3D cell spheroid morphology. Importantly, the 3D cell models reveal augmented internalization of peptide-functionalized LNPs and improve antiproliferative effects when the LNPs are loaded with a cytotoxic compound. Overall, LNPs-DSS show equal or even superior cellular internalization than the LNPs-iRGD, suggesting that DSS can also be used to enhance the cellular uptake of NPs into different types of cells, and release different cargos intracellularly.

Sex Differences in Salivary Parameters of Caries Susceptibility in Healthy Individuals.

PURPOSE: To investigate salivary parameters of caries susceptibility in men and women in order to identify potential variations due to sexual dimorphism. MATERIALS AND METHODS: A total of 46 female and 24 male patients, aged 18-40 years, participated in this study. Unstimulated saliva was collected for the evaluation of flow rate, pH, secretory IgA, Snyder test scores, and Streptococcus mutans counts (confirmed by PCR assay). Statistical analysis included the Mann-Whitney, Kruskal-Wallis and Bonferroni tests, one-way ANOVA, and the Spearman correlation at a 5% significance level, followed by a general linear model and multiple linear regressions. RESULTS: Female participants presented lower salivary pH values compared to males (p < 0.05), and different patterns of correlation among salivary parameters were found in men (p < 0.05) and women (p < 0.001). When comparing the variables according to Snyder test scores in men and women, there was a significant difference for S. mutans levels in the male group, and for pH and IgA in the female group (p < 0.05). Gender was found to be a predictor of salivary flow (R2 = 0.05; p < 0.05) and pH (R2 = 0.16; p < 0.001). In the female group, multiple regression showed several predictors for salivary flow rate, pH, IgA and Snyder test scores (p < 0.05), whereas no predictor was found in the male group (p > 0.05). CONCLUSION: Different salivary patterns were observed in men and women, thus the implications of such findings for caries susceptibility require further investigation.

Cytotoxic Orbitide from the Latex of Croton urucurana.

The bioactive ethyl acetate phase obtained from the latex of Croton urucurana Baillon afforded a novel orbitide (1), [1-9-NαC]-crourorb A1, that proved active against NCI-ADR/RES (ovary, multidrug-resistance phenotype) cells with the same potency as doxorubicin (positive control) and inactive up to the highest concentration tested against nontumor NIH/3T3 cells. The structure elucidation was based on 1D and 2D NMR spectroscopy, further supported by HRESIMS data and by application of Marfey's method for determination of the absolute configuration of its amino acid residues. This is the first orbitide obtained from C. urucurana.

Preparation of cetyl palmitate-based PEGylated solid lipid nanoparticles by microfluidic technique.

In recent years, several studies have shown that the use of solid lipid nanoparticles (SLN) as a colloidal drug delivery system was more advantageous than lipid emulsions, liposomes and polymeric nanoparticles. SLNs have numerous advantages of different nanosystems and rule out many of their drawbacks. Despite the numerous advantages of SLNs, translation from the preclinical formulation to the industrial scale-up is limited. In order to provide a reproducible and reliable method of producing nanoparticles, and thus, obtain an industrial scale-up, several methods of synthesis of nanoparticles by microfluidic have been developed. Microfluidic technique allows a good control and a continuous online synthesis of nanosystems compared to synthesis in bulk, leading to a narrow size distribution, high batch-to-batch reproducibility, as well as to the industrial scale-up feasibility. This work described the optimization process to produce SLNs by microfluidics. The SLNs produced by microfluidics were characterized by complementary optical and morphological techniques and compared with those produced by bulk method. SLNs were loaded with paclitaxel and sorafenib, used as model drugs. The anti-cancer efficiency of the SLNs formulation was estimated with 2D and 3D tumour models of two different cell lines, and the cellular uptake was also studied with fluorescence-assisted measurements.

LinTT1 peptide-functionalized liposomes for targeted breast cancer therapy.

Breast cancer, with around 2 million new cases in 2019, is the second most common cancer worldwide and the second leading cause of cancer death among females. The aim of this work is to prepare a targeting nanoparticle through the conjugation of LinTT1 peptide, a specific molecule targeting p32 protein overexpressed by breast cancer and cancer associated cells, on liposomes' surface. This approach increases the cytotoxic effects of doxorubicin (DOX) and sorafenib (SRF) co-loaded in therapeutic liposomes on both 2D and 3D breast cancer cellular models. The liposome functionalization leads to a higher interaction with 3D breast cancer spheroids than bare ones. Moreover, interaction studies between LinTT1-functionalized liposomes and M2 primary human macrophages show an internalization of 50% of the total nanovesicles that interact with these cells, while the other 50% results only associated to cell surface. This finding suggests the possibility to use the amount of associated liposomes to enrich the hypoxic tumor area, exploiting the ability of M2 macrophages to accumulate in the central core of tumor mass. These promising results highlight the potential use of DOX and SRF co-loaded LinTT1-functionalized liposomes as nanomedicines for the treatment of breast cancer, especially in triple negative cancer cells.

Salivary biomarkers for caries susceptibility and mental stress in individuals with facial pain.

Objective: To assess salivary biomarkers for dental caries susceptibility and mental stress in young adults with perceived facial pain.Methods: Males and females who reported facial pain and pain-free controls participated in this study. Facial pain was investigated using the RDC/TMD. Unstimulated saliva was then collected for the evaluation of salivary flow rate (SFR), pH, Streptococcus mutans counts, morning cortisol, and S-IgA.Results: Women with facial pain had significantly lower SFR values, and the facial pain group showed different correlations among biomarkers for caries susceptibility and cortisol levels when compared to controls. Notably, higher SFR values were associated with a lower likelihood of having facial pain.Conclusion: Differences in SFR values, particularly in women, and markedly distinct interactions among the salivary biomarkers analyzed were observed between individuals with facial pain and pain-free controls. Hence, a connection between the dynamics of saliva, stress response, and facial pain perception might exist.

Dual-Crosslinked Dynamic Hydrogel Incorporating {Mo154 } with pH and NIR Responsiveness for Chemo-Photothermal Therapy.

Polyoxometalates are an emerging class of molecular clusters, with well-defined structures and chemical compositions that are produced through simple, low-cost, and highly reproducible methods. In particular, the wheel-shaped cluster {Mo154 } is a promising photothermal agent due to its intervalence charge transfer transitions. However, its toxicity hinders its systemic administration, being the development of a localized delivery system still incipient. Herein, an injectable and self-healing hydrogel of easy preparation and administration is developed, incorporating both {Mo154 } and doxorubicin for synergistic photothermal and chemotherapy applications. The hydrogel is composed of benzylaldehyde functionalized polyethylene glycol, poly(N-isopropylacrylamide) functionalized chitosan and {Mo154 }. The gelation occurs within 60 s at room temperature, and the dual crosslinking by Schiff base and electrostatic interactions generates a dynamic network, which enables self-healing after injection. Moreover, the hydrogel delivers chemotherapeutic drugs, with a release triggered by dual near infra-red (NIR) radiation and pH changes. This stimuli-responsive release system along with the photothermal conversion ability of the hydrogel allows the simultaneous combination of photothermal and chemotherapy. This synergic system efficiently ablates the cancer tumor in vivo with no systemic toxicity. Overall, this work paves the way for the development of novel {Mo154 }-based systems, incorporated in self-healing and injectable hydrogels for dual chemo-photothermal therapy.

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.

Systematic in vitro biocompatibility studies of multimodal cellulose nanocrystal and lignin nanoparticles.

Natural biopolymer nanoparticles (NPs), including nanocrystalline cellulose (CNC) and lignin, have shown potential as scaffolds for targeted drug delivery systems due to their wide availability, cost-efficient preparation, and anticipated biocompatibility. As both CNC and lignin can potentially cause complications in cell viability assays because of their ability to scatter the emitted light and absorb the assay reagents, we investigated the response of bioluminescent (CellTiter-Glo®), colorimetric (MTT® and AlamarBlue®), and fluorometric (LIVE/DEAD®) assays for the determination of the biocompatibility of the multimodal CNC and lignin constructs in murine RAW 264.7 macrophages and 4T1 breast adenocarcinoma cell lines. Here, we have developed multimodal CNC and lignin NPs harboring the radiometal chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid and the fluorescent dye cyanine 5 for the investigation of nanomaterial biodistribution in vivo with nuclear and optical imaging, which were then used as the model CNC and lignin nanosystems in the cell viability assay comparison. CellTiter-Glo® based on the detection of ATP-dependent luminescence in viable cells revealed to be the best assay for both nanoconstructs for its robust linear response to increasing NP concentration and lack of interference from either of the NP types. Both multimodal CNC and lignin NPs displayed low cytotoxicity and favorable interactions with the cell lines, suggesting that they are good candidates for nanosystem development for targeted drug delivery in breast cancer and for theranostic applications. Our results provide useful guidance for cell viability assay compatibility for CNC and lignin NPs and facilitate the future translation of the materials for in vivo applications.

All-in-one microfluidic assembly of insulin-loaded pH-responsive nano-in-microparticles for oral insulin delivery.

Here, a continuous two-step glass-capillary microfluidic technique to produce a multistage oral delivery system is reported. Insulin is successfully encapsulated into liposomes, which are coated with chitosan to improve their mucoadhesion. The encapsulation in an enteric polymer offers protection from the harsh gastric conditions. Insulin permeability is enhanced across an intestinal monolayer.

Dual-peptide functionalized acetalated dextran-based nanoparticles for sequential targeting of macrophages during myocardial infarction.

The advent of nanomedicine has recently started to innovate the treatment of cardiovascular diseases, in particular myocardial infarction. Although current approaches are very promising, there is still an urgent need for advanced targeting strategies. In this work, the exploitation of macrophage recruitment is proposed as a novel and synergistic approach to improve the addressability of the infarcted myocardium achieved by current peptide-based heart targeting strategies. For this purpose, an acetalated dextran-based nanosystem is designed and successfully functionalized with two different peptides, atrial natriuretic peptide (ANP) and linTT1, which target, respectively, cardiac cells and macrophages associated with atherosclerotic plaques. The biocompatibility of the nanocarrier is screened on both macrophage cell lines and primary macrophages, showing high safety, in particular after functionalization of the nanoparticles' surface. Furthermore, the system shows higher association versus uptake ratio towards M2-like macrophages (approximately 2-fold and 6-fold increase in murine and human primary M2-like macrophages, respectively, compared to M1-like). Overall, the results demonstrate that the nanosystem has potential to exploit the "hitchhike" effect on M2-like macrophages and potentially improve, in a dual targeting strategy, the ability of the ANP peptide to target infarcted heart.

Utilization of green formulation technique and efficacy estimation on cell line studies for dual anticancer drug therapy with niosomes.

The aim of the present study was to prepare niosome formulations for the simultaneous encapsulation, dual drug therapy, of two anticancer drugs by the ecological probe sonication method. Poloxamer and sorbitan monostearate were used as surface active agents in niosomes, and the water soluble doxorubicin and poorly-water soluble paclitaxel were used as anticancer drugs. Thorough physicochemical analysis were performed for the niosomes, and their cytotoxicity and activity were evaluated on MCF-7 and PC3-MM2 cancer cell lines. Prepared niosomes were small in size with sizes ranging from 137 nm to 893 nm, and entrapment efficiencies were high, ranging from 91.24% to 99.99%. During the four weeks stability testing, the particle size remained stable. The niosomal formulations showed in vitro sustained drug release profiles for doxorubicin and clearly increased the dissolution rate of poorly water soluble paclitaxel. The incorporation of both the drugs into niosomes improved cell penetration and antiproliferative activity of the drugs PC3-MM2 cell lines. As a conclusion, doxorubicin and paclitaxel loaded niosome formulations resulted in relatively stable, small sized niosomes with improved drug release profiles, low toxicity, better cell penetration and antiproliferative activity. The niosomes showed synergistic effect due to the presence of both drugs, which can overcome multidrug resistance.

Functionalization of carboxylated lignin nanoparticles for targeted and pH-responsive delivery of anticancer drugs.

AIM: To carboxylate kraft lignin toward the functionalization of carboxylated lignin nanoparticles (CLNPs) with a block copolymer made of PEG, poly(histidine) and a cell-penetrating peptide and then evaluate the chemotherapeutic potential of the innovative nanoparticles. MATERIALS & METHODS: The produced nanoparticles were characterized and evaluated in vitro for stability and biocompatibility and the drug release profiles and antiproliferative effect were also assessed. RESULTS: The prepared CLNPs showed spherical shape and good size distribution, good stability in physiological media and low cytotoxicity in all the tested cell lines. A poorly water-soluble cytotoxic agent was successfully loaded into the CLNPs, improving its release profiles in a pH-sensitive manner and showing an enhanced antiproliferative effect in the different cancer cells compared with a normal endothelial cell line. CONCLUSION: The resulting CLNPs are promising candidates for anticancer therapy.

[1-9-NαC]-crourorb A1 isolated from Croton urucurana latex induces G2/M cell cycle arrest and apoptosis in human hepatocarcinoma cells.

[1-9-NαC]-crourorb A1 is a cyclic peptide isolated from Croton urucurana Baillon latex, found in midwestern Brazil, that has been shown to exert cytotoxic effects against a panel of cancer cell lines. However, the underlying mechanisms responsible for the crourorb A1-induced cytotoxicity in cancer cells remain unknown. In this study, the effects of crourorb A1 on the viability, apoptosis, cell cycle and migration of Huh-7 (human hepatocarcinoma) cells were investigated. We evaluated the viability of Huh-7 cells treated with crourorb A1 in 2D and 3D collagen cultures and found that cells in 3D culture exhibited increased resistance to crourorb A1 compared to cells in 2D culture (IC50: 62µg/ml versus 35.75µg/ml). Crourorb A1 treatment decreases the viability of Huh-7 cells in a dose- and time-dependent manner and is associated with the induction of apoptosis, in the absence of necrotic cells, through the activation of caspase-3/7 and increased expression of the pro-apoptotic proteins Bak, Bid, Bax, Puma, Bim, and Bad. The effects of crourorb A1 are also associated with G2/M phase cell cycle arrest and increases in cyclin-dependent kinase (CDK1) and cyclin B1 expression. A significant reduction in Huh-7 cell migration induced by crourorb A1 was also observed in the presence of mitomycin C. Finally, we showed that the JNK/MAP pathway, but not ERK signaling, is involved in crourorb A1-induced hepatocarcinoma cell mortality.

In vitro evaluation of biodegradable lignin-based nanoparticles for drug delivery and enhanced antiproliferation effect in cancer cells.

Currently, nanosystems have been developed and applied as promising vehicles for different biomedical applications. We have developed three lignin nanoparticles (LNPs): pure lignin nanoparticles (pLNPs), iron(III)-complexed lignin nanoparticles (Fe-LNPs), and Fe3O4-infused lignin nanoparticles (Fe3O4-LNPs) with round shape, narrow size distribution, reduced polydispersity and good stability at pH 7.4. The LNPs showed low cytotoxicity in all the tested cell lines and hemolytic rates below 12% after 12 h of incubation. Additionally, they induced hydrogen peroxide production in a small extent and time-dependent manner, and the interaction with the cells increased over time, exhibiting a dose-dependent cell uptake. Concerning the drug loading, pLNPs showed the capacity to efficiently load poorly water-soluble drugs and other cytotoxic agents, e.g. sorafenib and benzazulene (BZL), and improve their release profiles at pH 5.5 and 7.4 in a sustained manner. Furthermore, the BZL-pLNPs presented an enhanced antiproliferation effect in different cells compared to the pure BZL and showed a maximal inhibitory concentration ranging from 0.64 to 12.4 µM after 24 h incubation. Overall, LNPs are promising candidates for drug delivery applications, and the superparamagnetic behavior of Fe3O4-LNPs makes them promising for cancer therapy and diagnosis, such as magnetic targeting and magnetic resonance imaging.

Angiopep2-functionalized polymersomes for targeted doxorubicin delivery to glioblastoma cells.

A targeted drug delivery nanosystem for glioblastoma multiforme (GBM) based on polymersomes (Ps) made of poly(dimethylsiloxane)-poly(2-methyloxazoline) (PDMS-PMOXA) diblock copolymers was developed to evaluate their potential to actively target brain cancer cells and deliver anticancer drugs. Angiopep2 was conjugated to the surface of preformed Ps to target the low density lipoprotein receptor-related protein 1 that are overexpressed in blood brain barrier (BBB) and glioma cells. The conjugation efficiency yield for angiopep2 was estimated to be 24%. The angiopep2-functionalized Ps showed no cellular toxicity after 24h and enhanced the cellular uptake around 5 times more in U87MG glioblastoma cells compared to the non-targeted Ps. The encapsulation efficiency of doxorubicin (DOX) in Ps was 13% by co-solvent method, compared to a film rehydration method (4%). The release profiles of the DOX from Ps showed a release of 42% at pH 5.5 and 40% at pH 7.4 after 24h, indicating that Ps can efficiently retain the DOX with a slow release rate. Furthermore, the in vitro antiproliferative activity of DOX-loaded Ps-Angiopep2 showed enhanced toxicity to U87MG glioblastoma cells, compared to non-targeted Ps. Overall, our in vitro results suggested that angiopep2-conjugated Ps can be used as nanocarriers for efficient targeted DOX delivery to glioblastoma cells.

Caryocar coriaceum Wittm. (Pequi) fixed oil presents hypolipemic and anti-inflammatory effects in vivo and in vitro.

Caryocar coriaceum Wittm. (Pequi) is found in southern Ceará, where the fruit is used as food and in folk medicine as an anti-inflammatory, and to promote healing. However, little is known about the effects of repeated administration of its oil on the biochemical parameters of the blood. This work aimed to evaluate the effects Caryocar coriaceum fixed oil (OFCC); on the lipid profiles of healthy mice, on dyslipidemia induced by tyloxapol, and to study its anti-inflammatory effect both in vivo and in vitro. The results revealed significant reduction in total serum cholesterol and triglycerides, and an increase in HDL-C. The paw edema (induced by carrageenan) and myeloperoxidase (MPO), in polymorphonuclear culture cells, was reduced at all dose levels. Results demonstrated that Caryocar coriaceum's fix oil present anti-inflammatory activity and, for the first time describe the hypolipidemic effects, supporting its traditional use and suggest that present a potential cardioprotective effect.

Prevalência e produção de exoenzimas por espécies de Candida provenientes da mucosa bucal de pacientes com AIDS e indivíduos hígidos / Prevalence and exoenzyme production by Candida species from the oral mucosa of AIDS patients and healthy individuals

Infecções por leveduras do gênero Candida têm gerado elevados índices de morbidade e mortalidade, longo período de permanência em hospitais, dificuldade e alto custo de tratamento.Indivíduos imunodeprimidos como os portadores da Síndrome da Imunodeficiência Adquirida (Aids) apresentam grande suscetibilidade a desenvolver essas infecções em razão do baixo número de linfócitos T-CD4, menor que 200 cel/mm3. Candida albicans é a espécie mais estudadae está relacionada com os processos de colonização e patogenicidade na boca do homem. Essa característica é decorrente, entre outros fatores, da produção de exoenzimas facilitadoras dainteração do fungo com as células do hospedeiro. Este estudo verificou a produção das exoenzimas proteinase, fosfolipase, gelatinase e hemolisina de amostras bucais de Candida isoladas de 49 pacientes com Aids (grupo teste) e de 26 indivíduos hígidos (grupo controle). C. albicans foi a espécie mais prevalente no grupo teste (59,2por cento) e Candida parapsilosis (53,8por cento) no grupo controle. C. albicans apresentou resultados significativos para a produção de proteinase (ambos os grupos) e fosfolipase no grupo teste. Já as espécies de Candida não albicans (CNA) apresentaram resultadosaltamente significativos para fosfolipase no grupo controle. Em relação às enzimas gelatinase e hemolisina, não foram encontradas diferenças significantes entre C. albicans e espécies CNA. Por fim, não foi encontrada diferença estatística na produção de exoenzimas quando foi comparado o grupo teste com o grupo controle.