Sandra Simões

Three-dimensional spheroid cell culture of umbilical cord tissue-derived mesenchymal stromal cells leads to enhanced paracrine induction of wound healing

IntroductionThe secretion of trophic factors by mesenchymal stromal cells has gained increased interest given the benefits it may bring to the treatment of a variety of traumatic injuries such as skin wounds. Herein, we report on a three-dimensional culture-based method to improve the paracrine activity of a specific population of umbilical cord tissue-derived mesenchymal stromal cells (UCX®) towards the application of conditioned medium for the treatment of cutaneous wounds.MethodsA UCX® three-dimensional culture model was developed and characterized with respect to spheroid formation, cell phenotype and cell viability. The secretion by UCX® spheroids of extracellular matrix proteins and trophic factors involved in the wound-healing process was analysed. The skin regenerative potential of UCX® three-dimensional culture-derived conditioned medium (CM3D) was also assessed in vitro and in vivo against UCX® two-dimensional culture-derived conditioned medium (CM2D) using scratch and tubulogenesis assays and a rat wound splinting model, respectively.ResultsUCX® spheroids kept in our three-dimensional system remained viable and multipotent and secreted considerable amounts of vascular endothelial growth factor A, which was undetected in two-dimensional cultures, and higher amounts of matrix metalloproteinase-2, matrix metalloproteinase-9, hepatocyte growth factor, transforming growth factor β1, granulocyte-colony stimulating factor, fibroblast growth factor 2 and interleukin-6, when compared to CM2D. Furthermore, CM3D significantly enhanced elastin production and migration of keratinocytes and fibroblasts in vitro. In turn, tubulogenesis assays revealed increased capillary maturation in the presence of CM3D, as seen by a significant increase in capillary thickness and length when compared to CM2D, and increased branching points and capillary number when compared to basal medium. Finally, CM3D-treated wounds presented signs of faster and better resolution when compared to untreated and CM2D-treated wounds in vivo. Although CM2D proved to be beneficial, CM3D-treated wounds revealed a completely regenerated tissue by day 14 after excisions, with a more mature vascular system already showing glands and hair follicles.ConclusionsThis work unravels an important alternative to the use of cells in the final formulation of advanced therapy medicinal products by providing a proof of concept that a reproducible system for the production of UCX®-conditioned medium can be used to prime a secretome for eventual clinical applications.

The role of human umbilical cord tissue-derived mesenchymal stromal cells (UCX®) in the treatment of inflammatory arthritis.

BackgroundECBio has developed proprietary technology to consistently isolate, expand and cryopreserve a well-characterized population of stromal cells from human umbilical cord tissue (UCX® cells). The technology has recently been optimized in order to become compliant with Advanced Medicine Therapeutic Products. In this work we report the immunosuppressive capacity of UCX® cells for treating induced autoimmune inflammatory arthritis.MethodsUCX® cells were isolated using a proprietary method (PCT/IB2008/054067) that yields a well-defined number of cells using a precise proportion between tissue digestion enzyme activity units, tissue mass, digestion solution volume and void volume. The procedure includes three recovery steps to avoid non-conformities related to cell recovery. UCX® surface markers were characterized by flow cytometry and UCX® capacity to expand in vitro and to differentiate into adipocyte, chondrocyte and osteoblast-like cells was evaluated. Mixed Lymphocyte Reaction (MLR) assays were performed to evaluate the effect of UCX® cells on T-cell activation and Treg conversion assays were also performed in vitro. Furthermore, UCX® cells were administered in vivo in both a rat acute carrageenan-induced arthritis model and rat chronic adjuvant induced arthritis model for arthritic inflammation. UCX® anti-inflammatory activity was then monitored over time.ResultsUCX® cells stained positive for CD44, CD73, CD90 and CD105; and negative for CD14, CD19 CD31, CD34, CD45 and HLA-DR; and were capable to differentiate into adipocyte, chondrocyte and osteoblast-like cells. UCX® cells were shown to repress T-cell activation and promote the expansion of Tregs better than bone marrow mesenchymal stem cells (BM-MSCs). Accordingly, xenogeneic UCX® administration in an acute carrageenan-induced arthritis model showed that human UCX® cells can reduce paw edema in vivo more efficiently than BM-MSCs. Finally, in a chronic adjuvant induced arthritis model, animals treated with intra-articular (i.a.) and intra-peritoneal (i.p.) infusions of UCX® cells showed faster remission of local and systemic arthritic manifestations.ConclusionThe results suggest that UCX® cells may be an effective and promising new approach for treating both local and systemic manifestations of inflammatory arthritis.

Development, characterization, and skin delivery studies of related ultradeformable vesicles: transfersomes, ethosomes, and transethosomes

Ultradeformable vesicles (UDV) have recently become a promising tool for the development of improved and innovative dermal and transdermal therapies. The aim of this work was to study three related UDV: transfersomes, ethosomes, and transethosomes for the incorporation of actives of distinct polarities, namely, vitamin E and caffeine, and to evaluate the effect of the carrier on skin permeation and penetration. These actives were incorporated in UDV formulations further characterized for vesicles imaging by transmission electron microscopy; mean vesicle size and polydispersity index by photon correlation spectroscopy; zeta potential by laser-Doppler anemometry; deformability by pressure-driven transport; and incorporation efficiency (IE) after actives quantification by high-performance liquid chromatography. Topical delivery studies were performed in order to compare UDV formulations regarding the release, skin permeation, and penetration profiles. All UDV formulations showed size values within the expected range, except transethosomes prepared by “transfersomal method”, for which size was smaller than 100 nm in contrast to that obtained for vesicles prepared by “ethosomal method”. Zeta potential was negative and higher for formulations containing sodium cholate. The IE was much higher for vitamin E- than caffeine-loaded UDV as expected. For flux measurements, the following order was obtained: transethosomes (TE) > ethosomes (E) ≥ transfersomes (T). This result was consistent with the release and skin penetration profiles for Vitamin E-loaded UDV. However, the releasing results were totally the opposite for caffeine-loaded UDV, which might be explained by the solubility and thermodynamic activity of this active in each formulation instead of the UDV deformability attending to the higher non-incorporated fraction of caffeine. Anyway, a high skin penetration and permeation for all caffeine-loaded UDV were obtained. Transethosomes were more deformable than ethosomes and transfersomes due to the presence of both ethanol and surfactant in their composition. All these UDV were suitable for a deeper skin penetration, especially transethosomes.

Development of enzyme-loaded nanoparticles: effect of pH

Enzyme superoxide dismutase (SOD) incorporation parameters were evaluated after immobilization in polyisobutylcyanoaclylate (PIBCA) nanoparticles. After initialization of the anionic mechanism of polymerization, pH was increased and its effect on the characteristics of PIBCA nanoparticles analysed. Our goal included optimization of enzyme activity during incorporation into nanoparticles and the influence on size distribution. Unloaded nanoparticles were not significantly affected by the pH increase. At pH 3 the size distribution indicates a bimodal distribution: 58 nm (63%) and 146 nm (37%). When pH was increased to 5 after 1 h of polymerization the size distribution is: 57 nm (70%) and 125 nm (30%). When pH was increased to 5, after 2 h of polymerization, the size distribution is 67 nm (56%) and 160 nm (44%). Meanwhile, the retention of activity of SOD in polymerization medium is 49% at pH 3, and 98% at pH 5. The effect of pH increase from 3 to 5, after 1 h of polymerization, on the characteristics of loaded nanoparticles is an increase of retention of enzyme activity (18 to 30%); and the evidence of a pH-dependent smaller size population of loaded nanoparticles. In fact at pH 3 the size distribution is 83 nm (15%), 195 nm (15%), 440 nm (70%) and when pH is increased from 3 to 5 the size distribution becomes 55 nm (30%); 170 nm (30%); 430 nm (40%).

What Makes Umbilical Cord Tissue-Derived Mesenchymal Stromal Cells Superior Immunomodulators When Compared to Bone Marrow Derived Mesenchymal Stromal Cells?

MSCs derived from the umbilical cord tissue, termed UCX, were investigated for their immunomodulatory properties and compared to bone marrow-derived MSCs (BM-MSCs), the gold-standard in immunotherapy. Immunogenicity and immunosuppression were assessed by mixed lymphocyte reactions, suppression of lymphocyte proliferation and induction of regulatory T cells. Results showed that UCX were less immunogenic and showed higher immunosuppression activity than BM-MSCs. Further, UCX did not need prior activation or priming to exert their immunomodulatory effects. This was further corroborated in vivo in a model of acute inflammation. To elucidate the potency differences observed between UCX and BM-MSCs, gene expression related to immune modulation was analysed in both cell types. Several gene expression profile differences were found between UCX and BM-MSCs, namely decreased expression of HLA-DRA, HO-1, IGFBP1, 4 and 6, ILR1, IL6R and PTGES and increased expression of CD200, CD273, CD274, IL1B, IL-8, LIF and TGFB2. The latter were confirmed at the protein expression level. Overall, these results show that UCX seem to be naturally more potent immunosuppressors and less immunogenic than BM-MSCs. We propose that these differences may be due to increased levels of immunomodulatory surface proteins such as CD200, CD273, CD274 and cytokines such as IL1β, IL-8, LIF and TGFβ2.

Novel tretinoin formulations: a drug-in-cyclodextrin-in-liposome approach

Abstract Purpose: The aims of this experimental work were the incorporation and full characterization of the system Tretinoin-in-dimethyl-beta-cyclodextrin-in-ultradeformable vesicles (Tretinoin-CyD-UDV) and Tretinoin-in-ultradeformable vesicles (Tretinoin-UDV). Methods: The Tretinoin-CyD complex was prepared by kneading and the UDV by adding soybean phosphatidylcholine (SPC) to Tween® 80 followed by an appropriate volume of sodium phosphate buffer solution to make a 10%–20% lipid suspension. The resulting suspension was brought to the final mean vesicles size, of approximately 150 nm, by sequential filtration. The physicochemical characterization was based on: the evaluation of mean particle size and polydispersity index (PI) measured by photon correlation spectroscopy (PCS) and atomic force microscopy (AFM) topographic imaging; zeta potential (ζ-potential) and the SPC concentration determined by Laser–Doppler anemometry and an enzymatic-colorimetric test, respectively. The quantification of the incorporated Tretinoin and its chemical stability (during preparation and storage) was assayed by a HPLC at 342 nm. Results: It was possible to obtain the system Tretinoin-CyD-UDV. The mean vesicle size was the most stable parameter during experiments time course. AFM showed that Tretinoin-CyD-UDV samples were very heterogeneous in size, having three distinct subpopulations, while Tretinoin-UDV samples had only one homogeneous size population. The results of the ζ-potential measurements have shown that vesicle surface charge was low, as expected, presenting negative values. The incorporation efficiency was high, and no significant differences between Tretinoin-CyD-UDV and Tretinoin-UDV were observed. However, only Tretinoin-UDV with 20% lipid concentration formulation remained chemically stable during the evaluation period. Conclusion: According to our results, Tretinoin-UDV with 20% lipid concentration seems to be a better approach than Tretinoin-CyD-UDV, attending to the higher chemical stability.

Lycopene from Tomatoes: Vesicular Nanocarrier Formulations for Dermal Delivery

This experimental work aimed to develop a simple, fast, economic, and environmentally friendly process for the extraction of lycopene from tomato and incorporate this lycopene-rich extract into ultradeformable vesicular nanocarriers suitable for topical application. Lycopene extraction was conducted without a cosolvent for 30 min. The extracts were analyzed and incorporated in transfersomes and ethosomes. These formulations were characterized, and the cellular uptake was observed by confocal microscopy. Dermal delivery of lycopene formulations was tested under in vitro and in vivo conditions. Lycopene extraction proved to be quite safe and selective. The vesicular formulation was taken up by the cells, being more concentrated around the nucleus. Epicutaneous application of lycopene formulations decreased the level of anthralin-induced ear swelling by 97 and 87%, in a manner nonstatistically different from the positive control. These results support the idea that the lycopene-rich extract may be a good alternative to the expensive commercial lycopene for incorporation into advanced topical delivery systems.

Pickering emulsions: challenges and opportunities in topical delivery

ABSTRACT Introduction: Topical drug delivery is a challenging area with many advantages such as avoidance of first passage effect, stabilization of blood concentrations and attainment of local therapeutic effect with fewer side effects. Despite all these advantages, topical drug delivery remains limited to few molecules, since skin acts as a barrier to the delivery of many therapeutic molecules. To overcome this obstacle, a favored strategy relies on selecting suitable vehicles for dermatologic therapy, such as emulsions, gels and, more recently, nanoparticulate systems. Areas covered: Particle-stabilized emulsions, also known as Pickering emulsions, have garnered interest in recent years. Although most of the investigation on Pickering emulsions has been based on model systems with inorganic or organic solid particles, recent advances have been made regarding the application of nanocarriers, protein-based particles or cyclodextrins for this purpose. This review reports the latest advances in Pickering emulsions technical challenges, and discusses the potential benefits and drawbacks of using these formulations for topical pharmaceutical and cosmetic applications as an alternative to conventional surfactant-based systems. Expert opinion: Pickering emulsions appear as a multifunctional dosage form with endless advantages. A great deal of progress is expected in this area, which might represent a renewed vision for the pharmaceutical and cosmetic industry.

In vitro and in vivo topical delivery studies of tretinoin-loaded ultradeformable vesicles

INTRODUCTION Ultradeformable vesicles are highly promising tools to enhance the percutaneous transport of different drugs such as tretinoin across the skin barrier and also to increase the formulation stability at absorption site and reduce the drug induced irritation. METHODS Topical delivery of tretinoin-loaded ultradeformable vesicles (tretinoin-UDV) was evaluated concerning different studies, such as: the release and permeation profiles (tape stripping); skin penetration (fluorescence analysis); induced electrical changes in skin barrier properties; cytotoxicity (Trypan Blue assay) and skin irritation in in vivo conditions (Draize test). The novel formulation performance was also compared to a commercial tretinoin formulation regarding in vivo studies. RESULTS It was obtained a sustained and controlled drug release, as expected for UDV formulation. In addition, a dermal delivery was observed regarding the permeation study since it was not detected any drug amount in the receptor phase after 24h. Nile Red-UDV stained intensively mostly in the stratum corneum, corroborating the tape stripping results. Tretinoin-UDV decreased skin resistance, suggesting its ability to induce skin barrier disruption. Finally, the formulation vehicle (empty UDV) and tretinoin-UDV were not toxic under in vitro and in vivo conditions, at least, at 5×10(-3)mg/mL and 0.5mg/mL of tretinoin, respectively. CONCLUSION Tretinoin-UDV is a promising delivery system for tretinoin dermal delivery without promoting skin irritation (unlike other commercial formulations), which is quite advantageous for therapeutic purpose.

Topical Delivery of Antioxidants

Reactive oxygen species (ROS) and free radicals have been implicated in a number of diseases and disorders, and the skin, for its localization, is exposed to a large number of environmental threats. Free radical scavengers and antioxidants have thus been proposed as protective or therapeutic agents against ROS-mediated injuries. Oral treatment with several antioxidants has been reported to provide skin protection against deleterious effects of ultraviolet radiation. Topical delivery of antioxidants has increasingly gained interest and development, especially by offering better targeting to the upper skin layer. However, the topical delivery of antioxidants for dermal action is a challenging research field since the molecules are, in general, susceptible to degradation. The search for a new delivery system that, simultaneously, preserves the antioxidant stability and enhances its deposition on the skin, opened a new chapter in drug delivery design. Nanocarriers have been successful in enhancing the clinical efficiency of several drugs. More recent approaches in modulating through the skin delivery led to the development of specialized nanoparticulated systems. The first part of this article presents a review of the potential of antioxidants as pharmacological agents in ROS related diseases, with a special focus on oxidative stress implicated skin pathologies: ROS formation and natural protection against ROS toxicity, ROS-mediated skin damage and skin protection by antioxidants. In the second part of this work, we present reported formulation strategies for dermal delivery of antioxidants focusing on the nanoparticulated systems developed in recent years.