Ibrutinib topical delivery for melanoma treatment: The effect of nanostructured lipid carriers' composition on the controlled drug skin deposition.

Melanoma is responsible for more than 80% of deaths related to skin diseases. Ibrutinib (IBR), a Bruton's tyrosine kinase inhibitor, has been proposed to treat this type of tumor. However, its low solubility, extensive first-pass effect, and severe adverse reactions with systemic administration affect therapeutic success. This study proposes developing and comparing the performance of two compositions of nanostructured lipid carriers (NLCs) to load IBR for the topical management of melanomas in their early stages. Initially, the effectiveness of IBR on melanoma proliferation was evaluated in vitro, and the results confirmed that the drug reduces the viability of human melanoma cells by inducing apoptosis at a dose that does not compromise dermal cells. Preformulation tests were then conducted to characterize the physical compatibility between the drug and the selected components used in NLCs preparation. Sequentially, two lipid compositions were used to develop the NLCs. Formulations were then characterized and subjected to in vitro release and permeation tests on porcine skin. The NLCs containing oleic acid effectively controlled IBR release over 24 h compared to the NLCs composed of pomegranate seed oil. Furthermore, the nanoparticles acted as permeation enhancers, increasing the fluidity of the lipids in the stratum corneum, as determined by EPR spectroscopy, which stimulated the IBR penetration more profoundly into the skin. However, the NLCs composition also influenced the permeation promotion factor. Thus, these findings emphasize the importance of the composition of NLCs in controlling and increasing the skin penetration of IBR and pave the way for future advances in melanoma therapy.

Double-blind, vehicle-controlled clinical investigation of peptide OS-01 for skin rejuvenation.

INTRODUCTION: Senescent cells contribute to age-related tissue deterioration, including the skin, which plays important roles in overall health and social interactions. This study aimed to assess the effects of the senotherapeutic peptide, OS-01 (a.k.a. Pep 14), on skin. METHODS: A 12-week split-face, double-blinded, vehicle-controlled study involving 22 participants was conducted. The OS-01-containing formulation was applied to one side of the face, while the other side received an identical control formulation lacking the peptide. Skin characteristics were assessed using instrumental measurements, expert clinical grading, and subjective questionnaires. RESULTS: Results showed that the OS-01 formulation significantly improved one aspect of skin barrier function, as evidenced by reduced trans-epidermal water loss compared to both baseline and vehicle control. Expert grading and Antera 3D image analysis revealed a reduction in wrinkle appearance and indentation in the periorbital area, and improved skin texture and radiance on both sides of the face, with the OS-01-containing formulation demonstrating superior results. Participants also perceived improvements in skin hydration, smoothness, radiance, and overall appearance. CONCLUSION: The findings suggest that the OS-01 formulation promotes skin health by strengthening the skin barrier, protecting against dehydration, reducing the appearance of wrinkles, and improving skin texture and radiance. These effects are likely attributed to the senotherapeutic properties of OS-01 in reducing cellular senescence and its associated detrimental effects.

Senotherapeutic peptide treatment reduces biological age and senescence burden in human skin models.

Cellular senescence is known to play a role in age-related skin function deterioration which potentially influences longevity. Here, a two-step phenotypic screening was performed to identify senotherapeutic peptides, leading to the identification of Peptide (Pep) 14. Pep 14 effectively decreased human dermal fibroblast senescence burden induced by Hutchinson-Gilford Progeria Syndrome (HGPS), chronological aging, ultraviolet-B radiation (UVB), and etoposide treatment, without inducing significant toxicity. Pep 14 functions via modulation of PP2A, an understudied holoenzyme that promotes genomic stability and is involved in DNA repair and senescence pathways. At the single-cell level, Pep 14 modulates genes that prevent senescence progression by arresting the cell cycle and enhancing DNA repair, which consequently reduce the number of cells progressing to late senescence. When applied on aged ex vivo skin, Pep 14 promoted a healthy skin phenotype with structural and molecular resemblance to young ex vivo skin, decreased the expression of senescence markers, including SASP, and reduced the DNA methylation age. In summary, this work shows the safe reduction of the biological age of ex vivo human skins by a senomorphic peptide.

In vitro and in vivo toxicity assessment of the senotherapeutic Peptide 14.

Senotherapeutic molecules decrease cellular senescence burden, constituting promising approaches to combat the accumulation of senescent cells observed in chronological aging and age-related diseases. Numerous molecules have displayed senotherapeutic potential, but toxicity has been frequently observed. Recently, a new senotherapeutic compound, Peptide 14, was developed to modulate cellular senescence in the skin. In order to assess the potential toxic and genotoxic effects of the peptide, we observed the viability of human primary dermal fibroblasts and epidermal keratinocytes with Peptide 14 treatment, and show that it is mostly non-toxic in concentrations up to 100 µM. Cancer lines were also used to investigate its potential of modulating proliferation. Different concentrations of the peptide promoted a discrete reduction in the proliferation of cancerous cells of the MeWo and HeLa lineages. In full-thickness human skin equivalents, topically formulated Peptide 14 also failed to exert any significant irritation, nor cellular toxicity when added to the culture media. Genotoxic assays including the Ames, micronucleus, and karyotyping tests also indicate the safety of the peptide. Finally, the irritative potential of the peptide was assessed in human subjects in a repeated insult patch test executed using 1 mM peptide. No visible skin reactions were observed in any of the 54 participants. Taken together, the present data support that Peptide 14 is a senotherapeutic molecule with a positive safety profile as tested with cruelty-free models, justifying further studies involving the peptide.

Cardiac differentiation of human pluripotent stem cells using defined extracellular matrix proteins reveals essential role of fibronectin.

Research and therapeutic applications using human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) require robust differentiation strategies. Efforts to improve hPSC-CM differentiation have largely overlooked the role of extracellular matrix (ECM). The present study investigates the ability of defined ECM proteins to promote hPSC cardiac differentiation. Fibronectin (FN), laminin-111, and laminin-521 enabled hPSCs to attach and expand. However, only addition of FN promoted cardiac differentiation in response to growth factors Activin A, BMP4, and bFGF in contrast to the inhibition produced by laminin-111 or laminin-521. hPSCs in culture produced endogenous FN which accumulated in the ECM to a critical level necessary for effective cardiac differentiation. Inducible shRNA knockdown of FN prevented Brachyury+ mesoderm formation and subsequent hPSC-CM generation. Antibodies blocking FN binding integrins α4ß1 or αVß1, but not α5ß1, inhibited cardiac differentiation. Furthermore, inhibition of integrin-linked kinase led to a decrease in phosphorylated AKT, which was associated with increased apoptosis and inhibition of cardiac differentiation. These results provide new insights into defined matrices for culture of hPSCs that enable production of FN-enriched ECM which is essential for mesoderm formation and efficient cardiac differentiation.

Intraovarian injection of mesenchymal stem cells improves oocyte yield and in vitro embryo production in a bovine model of fertility loss.

Valuable female cattle are continuously subject to follicular puncture (ovum pick-up - OPU). This technique is commonly used for in-vitro embryo production, but may result in ovarian lesion. Mesenchymal stem cells (MSC) ameliorate the function of injured tissues, but their use to treat ovarian lesions in cattle has not been established. We investigated whether a local injection of MSC would reduce the negative effects of repeated OPU under acute and chronic scenarios in bovines. First, we performed four OPU sessions and injected 2.5 × 106 MSCs immediately after the 4th OPU procedure (n = 5). The treated organs (right ovary) were compared to their saline-treated counterparts (left), and presented superior production of oocytes and embryos in the three following OPU sessions (P < 0.05). Then, cows with progressive fertility loss went through three OPU sessions. Animals received MSC, saline, or MSC + FSH in both ovaries after the first OPU. In the two following OPU sessions, the MSC and MSC + FSH - treated groups failed to present any significant alteration in the number of oocytes and embryos compared to saline-treated animals. Thus, MSC have beneficial effects on the fertility of OPU-lesioned cows, but not in cows with cystic ovarian disease and chronic ovarian lesions.

Functional cardiac fibroblasts derived from human pluripotent stem cells via second heart field progenitors.

Cardiac fibroblasts (CFs) play critical roles in heart development, homeostasis, and disease. The limited availability of human CFs from native heart impedes investigations of CF biology and their role in disease. Human pluripotent stem cells (hPSCs) provide a highly renewable and genetically defined cell source, but efficient methods to generate CFs from hPSCs have not been described. Here, we show differentiation of hPSCs using sequential modulation of Wnt and FGF signaling to generate second heart field progenitors that efficiently give rise to hPSC-CFs. The hPSC-CFs resemble native heart CFs in cell morphology, proliferation, gene expression, fibroblast marker expression, production of extracellular matrix and myofibroblast transformation induced by TGFß1 and angiotensin II. Furthermore, hPSC-CFs exhibit a more embryonic phenotype when compared to fetal and adult primary human CFs. Co-culture of hPSC-CFs with hPSC-derived cardiomyocytes distinctly alters the electrophysiological properties of the cardiomyocytes compared to co-culture with dermal fibroblasts. The hPSC-CFs provide a powerful cell source for research, drug discovery, precision medicine, and therapeutic applications in cardiac regeneration.

Rare genetic diseases: update on diagnosis, treatment and online resources.

Rare genetic diseases collectively impact a significant portion of the world's population. For many diseases there is limited information available, and clinicians can find difficulty in differentiating between clinically similar conditions. This leads to problems in genetic counseling and patient treatment. The biomedical market is affected because pharmaceutical and biotechnology industries do not see advantages in addressing rare disease treatments, or because the cost of the treatments is too high. By contrast, technological advances including DNA sequencing and analysis, together with computer-aided tools and online resources, are allowing a more thorough understanding of rare disorders. Here, we discuss how the collection of various types of information together with the use of new technologies is facilitating diagnosis and, consequently, treatment of rare diseases.

The In Vitro and In Vivo Antiangiogenic Effects of Flavokawain B.

Angiogenesis is implicated in the development of a variety of pathological processes, most commonly cancer. It is essential for tumor growth and metastasis, making it an important cancer therapeutic target. Naturally occurring substances have led to the discovery of anticancer agents. Flavokawain B (FKB), a chalcone isolated from the root extracts of kava-kava plant, inhibits proliferation and causes apoptosis in vitro and in vivo of various cancer cell lines. The antimetastatic potential of FKB has also been suggested. In our study, we confirm the antiangiogenic action of FKB in vitro and, for the first time, demonstrate its strong antiangiogenic activity in vivo, using a zebrafish model. Our data show that FKB inhibits human brain endothelial cell (HUVEC) migration and tube formation even at very low and non-toxic concentrations. Moreover, FKB blocks angiogenesis process in zebrafish, with a dramatic reduction of subintestinal vein formation in a dose-dependent manner. Flavokawain B at the concentration of 2.5 µg/mL did not exhibit any toxic effects in zebrafish larvae and caused a markedly or complete obliteration of subintestinal vein formation. Our findings along with previously published data confirm that FKB may form the basis for creating an additional tool in the treatment of cancer and other neovascularization-related diseases. Copyright © 2017 John Wiley & Sons, Ltd.

Mesenchymal stem cells engrafted in a fibrin scaffold stimulate Schwann cell reactivity and axonal regeneration following sciatic nerve tubulization.

The present study investigated the effectiveness of mesenchymal stem cells (MSCs) associated with a fibrin scaffold (FS) for the peripheral regenerative process after nerve tubulization. Adult female Lewis rats received a unilateral sciatic nerve transection followed by repair with a polycaprolactone (PCL)-based tubular prosthesis. Sixty days after injury, the regenerated nerves were studied by immunohistochemistry. Anti-p75NTR immunostaining was used to investigate the reactivity of the MSCs. Basal labeling, which was upregulated during the regenerative process, was detected in uninjured nerves and was significantly greater in the MSC-treated group. The presence of GFP-positive MSCs was detected in the nerves, indicating the long term survival of such cells. Moreover, there was co-localization between MSCs and BNDF immunoreactivity, showing a possible mechanism by which MSCs improve the reactivity of SCs. Myelinated axon counting and morphometric analyses showed that MSC engrafting led to a higher degree of fiber compaction combined with a trend of increased myelin sheath thickness, when compared with other groups. The functional result of MSC engrafting was that the animals showed higher motor function recovery at the seventh and eighth week after lesion. The findings herein show that MSC+FS therapy improves the nerve regeneration process by positively modulating the reactivity of SCs.

Priming mesenchymal stem cells boosts stem cell therapy to treat myocardial infarction.

Cardiovascular diseases are the number one cause of death globally and are projected to remain the single leading cause of death. Treatment options abounds, although efficacy is limited. Recent studies attribute discrete and ephemeral benefits to adult stem cell therapies, indicating the urge to improve stem cell based-therapy. In this study, we show that priming mesenchymal stem cells (MSC) towards cardiomyogenic lineage enhances their beneficial effects in vivo as treatment option for acute phase myocardial infarction. MSC were primed using cardiomyogenic media for 4 days, after which peak expression of key cardiomyogenic genes are reached and protein expression of Cx-43 and sarcomeric α-actinin are observed. MSC and primed MSC (pMSC) were characterized in vitro and used to treat infarcted rats immediately after left anterior descending (LAD) occlusion. Echocardiography analysis indicated that MSC-treated myocardium presented discrete improvement in function, but it also showed that pMSC treatment lead to superior beneficial results, compared with undifferentiated MSC. Seven days after cell injection, MSC and pMSC could still be detected in the myocardium. Connexin-43 expression was quantified through immunoblotting, and was superior in pMSC, indicating that this could be a possible explanation for the superior performance of pMSC therapy.

Doxorubicin Cardiotoxicity and Cardiac Function Improvement After Stem Cell Therapy Diagnosed by Strain Echocardiography.

Doxorubicin (Dox) is one of the most effective chemotherapeutic agents; however, it causes dose-dependent cardiotoxicity. Evaluation of left ventricular function relies on measurements based on M-mode echocardiography. A new technique based on quantification of myocardial motion and deformation, strain echocardiography, has been showed promising profile for early detection of cardiac dysfunction. Different therapy strategies, such as flavonoid plant extracts and stem cells, have been investigated to improve heart function in toxic cardiomyopathy. This work aimed to assess early cardiac function improvement after treatments with either flavonoid extract from Camellia sinensis or mesenchymal stem cells in Dox cardiotoxicity using strain echocardiography. Twenty Wistar rats were randomly assigned to four groups. They received water (control, Dox, Dox + stem cells) or 100 mg/kg C. sinensis extract (Dox + C. sinensis) via gavage, daily, for four weeks. Animals also received saline (control) or 5 mg/kg doxorubicin (Dox, Dox + C. sinensis, Dox + stem cells) via intraperitoneal injection, weekly, for four weeks. Stem cells were injected (3 × 106 cells) through tail vein prior the beginning of the experiment (Dox + stem cells). Animals were evaluated by hematological, electrocardiography, echocardiography, and histopathological examinations. Dox cardiotoxicity was only diagnosed with strain echocardiography, detecting a decrease in ventricular function. C. sinensis extract did not prevent ventricular dysfunction induced by Dox. However, strain echocardiography examination revealed that Dox cardiotoxicity was significantly suppressed in rats treated with stem cells. In conclusion, strain echocardiography was able to detect precocity signs of heart failure and stem cell therapy showed cardioprotection effect against Dox cardiotoxicity.

Characterization of Decellularized Heart Matrices as Biomaterials for Regular and Whole Organ Tissue Engineering and Initial In-vitro Recellularization with Ips Cells.

Tissue engineering strategies, based on solid/porous scaffolds, suffer from several limitations, such as ineffective vascularization, poor cell distribution and organization within scaffold, in addition to low final cell density, among others. Therefore, the search for other tissue engineering approaches constitutes an active area of investigation. Decellularized matrices (DM) present major advantages compared to solid scaffolds, such as ideal chemical composition, the preservation of vascularization structure and perfect three-dimensional structure. In the present study, we aimed to characterize and investigate murine heart decellularized matrices as biomaterials for regular and whole organ tissue engineering. Heart decellularized matrices were characterized according to: 1. DNA content, through DNA quantificationo and PCR of isolated genomic DNA; 2. Histological structure, assessed after Hematoxylin and Eosin, as well as Masson's Trichrome stainings; 3. Surface nanostructure analysis, performed, using SEM. Those essays allowed us to conclude that DM was indeed decellularized, with preserved extracellular matrix structure. Following characterization, decellularized heart slices were seeded with induced Pluripotent Stem cells (iPS). As expected, but - to the best of our knowledge - never shown before, decellularization of murine heart matrices maintained matrix biocompatibility, as iPS cells rapidly attached to the surface of the material and proliferated. Strikingly though, heart DM presented a differentiation induction effect over those cells, which lost their pluripotency markers after 7 days of culture in the DM. Such loss of differentiation markers was observed, even though bFGF containing media mTSR was used during such period. Gene expression of iPS cells cultured on DM will be further analyzed, in order to assess the effects of culturing pluripotent stem cells in decellularized heart matrices.

Time-dependent migration of systemically delivered bone marrow mesenchymal stem cells to the infarcted heart.

In this study the time course of homing and the body distribution of systemically delivered bone marrow mesenchymal stem cells (BM-MSCs) after myocardial infarction (MI) were evaluated. BM-MSCs were isolated from Wistar rats, expanded in vitro, and their phenotypical characterization was performed by flow cytometer. Rats were randomly divided into three groups: control, sham MI, and MI. BM-MSCs (5 x 10(6)) were labeled with (99m)Tc-HMPAO and injected through the tail vein 7 days after MI. Gamma camera imaging was performed at 5, 15, 30, and 60 min after cell inoculation. Due to the (99m)Tc short half-life, cell migration and location were also evaluated in heart sections using DAPI-labeled cells 7 days after transplantation. Phenotypical characterization showed that BM-MSCs were CD90(+), CD73(+), CD54(+), and CD45(-). Five minutes after (99m)Tc-HMPAO-labeled cell injection, they were detected in various tissues. The cells migrated mainly to the lungs (approximately 70%) and, in small amounts, to the heart, kidneys, spleen, and bladder. The number of cells in the heart and lungs decreased after 60 min. MI markedly increased the amount of cells in the heart, but not in the lungs, during the period of observation (4.55 +/- 0.32 vs. 6.34 +/- 0.67% of uptake in infarcted hearts). No significant differences were observed between control and sham groups. Additionally, 7 days after DAPI-labeled cells injection, they were still detected in the heart but only in infarcted areas. These results suggest that the migration of systemically delivered BM-MSCs to the heart is time dependent and MI specifically increases BM-MSCs homing to injured hearts. However, the systemic delivery is limited by cell entrapment in the lungs.