Polyphenols-Loaded Sericin Self-Assembling Nanoparticles: A Slow-Release for Regeneration by Tissue-Resident Mesenchymal Stem/Stromal Cells.

Mesenchymal stem/stromal cells (MSCs) are a therapeutic target to promote tissue regeneration, mainly when oxidative stress-mediated damage is involved in disease pathogenesis. Here, slow-release silk sericin nanoparticles (SNPs) loaded with natural antioxidant polyphenols were developed to sustain regeneration by tissue-resident MSCs. SNPs were prepared by exploiting a self-assembly method with poloxamer and were loaded with proanthocyanidins (P), quercetin (Q) or epigallocatechin gallate (E). SNPs, with a diameter less than 150 nm, were able to encapsulate both hydrophilic (P and E) and hydrophobic (Q) drugs. A slow and controlled release was obtained from SNPs for all the actives in PBS, while in EtOH, Q and E showed a burst release but P did not. Kinetic models revealed lower diffusion of P than other biomolecules, probably due to the higher steric hindrance of P. The in vitro anti-oxidant, anti-elastase and anti-tyrosinase properties of SNPs were assessed: loading the P and E into SNPs preserved the in vitro biological activities whereas for Q, the anti-elastase activity was strongly improved. Moreover, all formulations promoted MSC metabolic activity over 72 h. Finally, SNPs exhibited a strong ability to protect MSCs from oxidative stress, which supports their potential use for regenerative purposes mediated by tissue-resident MSCs.

Silk fibroin nanoparticles for celecoxib and curcumin delivery: ROS-scavenging and anti-inflammatory activities in an in vitro model of osteoarthritis.

This paper aims at demonstrating silk fibroin nanoparticles (SFNs) promote anti-inflammatory properties of celecoxib (CXB) or curcumin (CUR), and could be exploited for osteoarthritis (OA) treatment. Nanoparticles were prepared by desolvation method and physico-chemically characterized (FT-IR, DSC, TGA, SEM, size distribution and drug release); empty and drug loaded nanoparticles were tested for their ROS-scavenging activity, hemolytic properties, cytotoxicity, and anti-inflammatory potency in an OA in vitro model. Results indicate that a controlled drug release has been achieved by varying the drug loading. Curcumin plus SFNs exhibited a synergistic antioxidant effect, while CXB was, in some manner, inhibitory. Both free drugs resulted highly cytotoxic while cell viability reached high values when encapsulated in SFNs. No appreciable differences in anti-inflammatory activity was evidenced between CUR loaded SFNs and CXB. In conclusion, SFNs is an optimal carrier to improve cyto- and hemo-compatibility of both CUR and CXB.

Association of silk sericin and platelet lysate: Premises for the formulation of wound healing active medications.

Silk sericin (SS), a globular and water-soluble protein, showed to in vitro sustain the proliferation of different mammalian cell lines, and to in vitro protect them from the oxidative stress because of its free-radical-scavenging activity. Also, platelet-derived products, such as platelet gel, platelet rich plasma or platelet lysate (PL) are used in cell cultures as a serum substitute due to their great content of growth factors. In the clinical practice, both SS and PL were employed for various reparative treatments and showed to sustain the wound repair process regulating fibroblast recruitment, migration, proliferation and differentiation. For these reasons, the combination of PL and SS could lead to effective and innovative products, either for the ex vivo cell expansion, or for the production of active wound healing advanced medications.

In vitro efficacy of silk sericin microparticles and platelet lysate for intervertebral disk regeneration.

Intervertebral disk degeneration is an oxidative and inflammatory pathological condition that induces viability and functionality reduction of Nucleus Pulposus cells (NPs). Cellular therapies were previously proposed to repair and substitute the herniated disk but low proliferative index and pathological conditions of NPs dramatically reduced the efficacy of this approach. To overcome these problems we proposed, for the first time, a therapeutic system based on the association of silk sericin microparticles and platelet-derived products. Silk sericin (SS) is a bioactive protein with marked antioxidant properties, while platelet lysate (PL) and platelet poor plasma (PPP) represent a source of growth factors able to support cell viability and to promote tissue regeneration. We demonstrated that the mixture PL + PPP promoted NPs proliferation with a significant reduction of cellular doubling time. SS microparticles, alone or in combination with PPP, presented the higher ROS-scavenging activity while, SS microparticles and PL resulted as the best association able to protect NPs against oxidative stress induce by hydroxide peroxide. Based on these results, the authors are confident that, with the ever increasing need of efficacious tools for regenerative medicine purposes, SS microparticles and PL + PPP association could represent an effective approach for the development of low impact and non-invasive therapies.

Fabrication of Innovative Silk/Alginate Microcarriers for Mesenchymal Stem Cell Delivery and Tissue Regeneration.

The aim of this study was to exploit silk fibroin's properties to develop innovative composite microcarriers for mesenchymal stem cell (MSCs) adhesion and proliferation. Alginate microcarriers were prepared, added to silk fibroin solution, and then treated with ethanol to induce silk conformational transition. Microcarriers were characterized for size distribution, coating stability and homogeneity. Finally, in vitro cytocompatibility and suitability as delivery systems for MSCs were investigated. Results indicated that our manufacturing process is consistent and reproducible: silk/alginate microcarriers were stable, with spherical geometry, about 400 µm in average diameter, and fibroin homogeneously coated the surface. MSCs were able to adhere rapidly onto the microcarrier surface and to cover the surface of the microcarrier within three days of culture; moreover, on this innovative 3D culture system, stem cells preserved their metabolic activity and their multi-lineage differentiation potential. In conclusion, silk/alginate microcarriers represent a suitable support for MSCs culture and expansion. Since it is able to preserve MSCs multipotency, the developed 3D system can be intended for cell delivery, for advanced therapy and regenerative medicine applications.

A dry powder formulation from silk fibroin microspheres as a topical auto-gelling device.

With the aim of establishing the formulation of a new hydrophilic auto-gelling medical device for biomedical applications, fibroin-based microspheres were prepared. The proposed microspheres were produced by a cost-effective and industrially scalable technique, such as the spray-drying. Spray-dried silk fibroin microspheres were obtained and the effects of different hydrophilic polymer on the process yield, microsphere morphology and conformation transition of fibroin were evaluated. The final auto-gelling formulations were obtained by adding calcium gluconate (as a calcium source for alginate crosslinking) to the prepared microspheres and tested by an in vitro gelling test. This study showed that the combination of fibroin with sodium alginate and poloxamer produced the most promising auto-gelling formulation for specific biomedical applications, such as the treatment of pressure ulcers.

TNF-α blocker effect of naringenin-loaded sericin microparticles that are potentially useful in the treatment of psoriasis.

This study aims to evaluate the effect of combined use of the racemic flavanone Naringenin (NRG) and the protein sericin as TNF-α blockers. Sericin (SMs) and (R/S) NRG-loaded Sericin (SNRGMs) microparticles were prepared by spray-drying, characterized in terms of morphology and particle size distribution, and encapsulation efficiency was determined. Concerning morphology and particle size distribution of microparticles, results indicated that they were not affected by the presence of NRG. The encapsulation efficiency was almost quantitative (93%), thus proving that sericin can be advantageously loaded with (R/S) NRG. Biological evaluation of (R/S) NRG, SMs and SNRGMs was then performed in lipopolysaccharide (LPS)-stimulated human peripheral blood mononuclear cells (hPBMC). SNRGMs resulted cytotoxic at the higher dose used (200 µg/mL) and the effect was greater than (R/S) NRG alone. Moreover, even if sericin alone was not effective in suppressing LPS-induced serum TNF-α levels, SNRGMs loaded with 9.3% of (R/S) NRG were significantly more potent than (R/S) NRG alone. In summary, this study provides the proof of concept that sericin-based microspheres loaded with TNF-α-blockers could contribute to the down regulation of the cytokine and represents the starting point for the development of new topical formulations for the treatment of middle-stage psoriasis.

Formulation of microspheres containing Crataegus monogyna Jacq. extract with free radical scavenging activity.

Extracts of Crataegus monogyna Jacq. (hawthorn) show an interesting free radical scavenging (FRS) effect, related to their flavonoids content. Unfortunately, their oral administration is affected by their low bioavailability. The aim of this work is to obtain a multiparticulate drug delivery system for hawthorn extracts for oral administration. The extracts from flowering tops (FL) or fruits (FR) of hawthorn were obtained with maceration, using ethanol as an extraction solvent, and their antioxidant activity was evaluated. FL extract showed the highest FRS activity (EC50 3.72 ± 1.21 µg/ml), so it was selected to prepare microparticulate systems by a spray-drying technique, which were characterized by granulometric analysis, scanning electron microscopy-energy dispersive X-ray spectroscopy, confocal fluorescence microscopy and hyperoside content. Antioxidant activity was evaluated before and after gastrointestinal transit in vitro simulation. Results indicate that the microparticulate systems maintained the antioxidant activity of hawthorn also after gastrointestinal transit in vitro simulation, exhibiting properties suitable for oral administration.

Sericins exhibit ROS-scavenging, anti-tyrosinase, anti-elastase, and in vitro immunomodulatory activities.

Some biological properties of Bombyx mori sericins from twenty strains were investigated, fourteen fed with artificial diet, two with fresh mulberry leaves and four with both diets. Sericin exhibited ROS-scavenging, anti-tyrosinase and anti-elastase properties, the strain significantly influenced these properties, while diet only influenced the anti-tyrosinase activity. Sericins were clustered into 5 groups and one sericin from each group was further studied: sericins showed anti-proliferative activity on in vitro stimulated peripheral blood mononuclear cells; some strains decreased in vitro secretion of IFNγ, while no effects were observed on TNFα and IL10 release. Therefore, a mixture of sericins extracted from the most promising strains may be useful for dermatological and cosmetic use.

Regenerated silk fibroin scaffold and infrapatellar adipose stromal vascular fraction as feeder-layer: a new product for cartilage advanced therapy.

Articular cartilage has limited repair and regeneration potential, and the scarcity of treatment modalities has motivated attempts to engineer cartilage tissue constructs. The use of chondrocytes in cartilage tissue engineering has been restricted by the limited availability of these cells, their intrinsic tendency to lose their phenotype during the expansion, as well as the difficulties during the first cell adhesion to the scaffold. Aim of this work was to evaluate the intra-articular adipose stromal vascular fraction attachment on silk fibroin scaffold to promote chondrocytes adhesion and proliferation. Physicochemical characterization has demonstrated that three-dimensionally organized silk fibroin scaffold is an ideal biopolymer for cartilage tissue engineering; it allows cell attachment, scaffold colonization, and physically cell holding in the area that must be repaired; the use of adipose-derived stem cells is a promising strategy to promote adhesion and proliferation of chondrocytes to the scaffold as an autologous human feeder layer.