Search

Introduction: Initiation and progression of intervertebral disk degeneration are linked to oxidative stress, with reactive oxygen species being a key factor. Therefore, as a potentially novel approach able to regenerate the damaged intervertebral disk, this work aimed to prepare an “active per sé” drug delivery system by combining sericin and crocetin: both are bioactive compounds with antioxidant, anti-inflammatory, immunomodulant and regenerative properties.Methods: In detail, sericin nanoparticles were prepared using crocetin as a cross-linker; then, the nanoparticle dispersions were dried by spray drying as it is (NP), with an excess of sericin (NPS) or crocin/crocetin (NPMix), obtaining three microparticle formulations.Results and Discussion: Before drying, the nanoparticles were nanometric (about 250 nm), with a negative surface charge, and appeared spherical and smooth. Following the drying process, spherical and smooth microparticles were obtained, with a mean diameter of about 1.7–2.30 μm. NPMix was the most active in antioxidant and anti-tyrosinase activities, likely due to the excess of crocin/crocetin, while NPS had the best anti-elastase activity, likely due to sericin in excess. Furthermore, all the formulations could prevent oxidative stress damage on nucleus pulposus cells, with NPMix being the best. Overall, the intrinsic anti-tyrosinase and anti-elastase activities and the ability to protect from oxidative stress-induced damages justify future investigations of these “active per sé” formulations in treating or preventing intervertebral disk degeneration.

Arthrospira platensis is a source of proteins, polysaccharides, polyunsaturated fatty acids (in tiny amounts) and phenolic components that give it different biological properties. The type of nutri...

Recently, we proposed a Good Manufacturing Practice (GMP)-compliant production process for freeze-dried mesenchymal stem cell (MSC)-secretome (lyo-secretome): after serum starvation, the cell supernatant was collected, and the secretome was concentrated by ultrafiltration and freeze-dried, obtaining a standardized ready-to-use and stable powder. In this work, we modified the type of human platelet lysate (HPL) used as an MSC culture supplement during the lyo-secretome production process: the aim was to verify whether this change had an impact on product quality and also whether this new procedure could be validated according to GMP, proving the process robustness. MSCs were cultured with two HPLs: the standard previously validated one (HPL-E) and the new one (HPL-S). From the same pool of platelets, two batches of HPL were obtained: HPL-E (by repeated freezing and thawing cycles) and HPL-S (by adding Ca-gluconate to form a clot and its subsequent mechanical wringing). Bone marrow MSCs from three donors were separately cultured with the two HPLs until the third passage and then employed to produce lyo-secretome. The following indicators were selected to evaluate the process performance: (i) the lyo-secretome quantitative composition (in lipids and proteins), (ii) the EVs size distribution, and (iii) anti-elastase and (iv) immunomodulant activity as potency tests. The new HPL supplementation for MSCs culture induced only a few minimal changes in protein/lipid content and EVs size distribution; despite this, it did not significantly influence biological activity. The donor intrinsic MSCs variability in secretome secretion instead strongly affected the quality of the finished product and could be mitigated by concentrating the final product to reach a determined protein (and lipid) concentration. In conclusion, the modification of the type of HPL in the MSCs culture during lyo-secretome production induces only minimal changes in the composition but not in the potency, and therefore, the new procedure can be validated according to GMP.

Clove oil (CO) is a powerful antioxidant essential oil (EO) with anti-inflammatory, anesthetic, and anti-infective properties. It can be therefore considered a good candidate for wound-healing applications, especially for chronic or diabetic wounds or burns, where the balance of reactive oxygen species (ROS) production and detoxification is altered. However, EOs require suitable formulations to be efficiently administered in moist wound environments. Chitosan hydrophobically modified by an ionic interaction with oleic acid (chitosan oleate, CSO) was used in the present work to stabilize CO nanoemulsions (NEs). The dimensions of the NE were maintained at around 300 nm as the volume distribution for up to six months, and the CO content did not decrease to under 80% over 4 months, confirming the good stabilizing properties of CSO. The antioxidant properties of the CO NE were evaluated in vitro by a 2,2-diphenil-2-picrylhydrazyl hydrate (DPPH) assay, and in fibroblast cell lines by electron paramagnetic resonance (EPR) using α-phenyl-N-tert-butyl nitrone (PBN) as a spin trap; a protective effect was obtained comparable to that obtained with α-tocopherol treatment. In a murine burn model, the ability of CO formulations to favor macroscopic wound closure was evidenced, and a histological analysis revealed a positive effect of the CO NE on the reparation of the lesion after 18 days. Samples of wounds at 7 days were subjected to a histological analysis and parallel dosage of lipid peroxidation by means of a thiobarbituric acid-reactive substances (TBARS) assay, confirming the antioxidant and anti-inflammatory activity of the CO NE.

BackgroundThe current challenge for immunotherapies is to generate effective antitumor immunity. Since tumor immune escape mechanisms do not impact pre-existing and consolidated immune responses, we tested the hypothesis of redirecting a pregenerated immunity to cancer: to recall a non-tumor antigen response against the tumor, silk fibroin nanoparticles (SFNs) have been selected as ‘Trojan-horse’ carriers, promoting the antigen uptake by the tumor cells.MethodsSFNs have been loaded with either ovalbumin (OVA) or CpG oligonucleotide (CpG) as antigen or adjuvant, respectively. In vitro uptake of SFNs by tumor (B16/F10 melanoma and MB49 bladder cancer) or dendritic cells, as well as the presence of OVA-specific T cells in splenic and tumor-infiltrating lymphocytes, were assessed by cytometric analyses. Proof-of-concept of in vivo efficacy was achieved in an OVA-hyperimmune B16/F10 murine melanoma model: SFNs-OVA or SFNs-CpG were injected, separately or in association, into the subcutaneous peritumoral area. Cancer dimensions/survival time were monitored, while, at the molecular level, system biology approaches based on graph theory and experimental proteomic data were performed.ResultsSFNs were efficiently in vitro uptaken by cancer and dendritic cells. In vivo peritumor administration of SFNs-OVA redirected OVA-specific cytotoxic T cells intratumorally. Proteomics and systems biology showed that peritumoral treatment with either SFNs-OVA or SFNs-CpG dramatically modified tumor microenvironment with respect to the control (CTR), mainly involving functional modules and hubs related to angiogenesis, inflammatory mediators, immune function, T complex and serpins expression, redox homeostasis, and energetic metabolism. Both SFNs-OVA and SFNs-CpG significantly delayed melanoma growth/survival time, and their effect was additive.ConclusionsBoth SFNs-OVA and SFNs-CpG induce effective anticancer response through complementary mechanisms and show the efficacy of an innovative active immunotherapy approach based on the redirection of pre-existing immunity against cancer cells. This approach could be universally applied for solid cancer treatments if translated into the clinic using re-call antigens of childhood vaccination.

Simple Summary Recently, mesenchymal stromal cells have been proposed as a valid approach for treating osteoarthritis diseases, mainly due to the secretion of soluble factors and extracellular vesicles known as secretome. In this paper, an injectable freeze-dried pharmaceutical powder containing canine mesenchymal stromal cells-secretome (Lyosecretome) has been formulated, and in vitro potency experiments were conducted. Furthermore, the Lyosecretome was applied in dogs affected by osteoarthritis to assess its safety. Results indicated that intra-articular injection of allogeneic Lyosecretome is safe and does not induce an adverse response, thus paving the way for future studies regarding the efficacy. Abstract In recent years, mesenchymal stromal cells (MSCs) have shown promise as a therapy in treating musculoskeletal diseases, and it is currently believed that their therapeutic effect is mainly related to the release of proteins and extracellular vesicles (EVs), known as secretome. In this work, three batches of canine MSC-secretome were prepared by standardized processes according to the current standard ISO9001 and formulated as a freeze-dried powder named Lyosecretome. The final products were characterized in protein and lipid content, EV size distribution and tested to ensure the microbiological safety required for intraarticular injection. Lyosecretome induced the proliferation of adipose tissue-derived canine MSCs, tenocytes, and chondrocytes in a dose-dependent manner and showed anti-elastase activity, reaching 85% of inhibitory activity at a 20 mg/mL concentration. Finally, to evaluate the safety of the preparation, three patients affected by bilateral knee or elbow osteoarthritis were treated with two intra-articular injections (t = 0 and t = 40 days) of the allogeneic Lyosecretome (20 mg corresponding 2 × 106 cell equivalents) resuspended in hyaluronic acid in one joint and placebo (mannitol resuspended in hyaluronic acid) in the other joint. To establish the safety of the treatment, the follow-up included a questionnaire addressed to the owner and orthopaedic examinations to assess lameness grade, pain score, functional disability score and range of motion up to day 80 post-treatment. Overall, the collected data suggest that intra-articular injection of allogeneic Lyosecretome is safe and does not induce a clinically significant local or systemic adverse response.

Aim: To validate the use of ultrafiltration (UF) as an alternative applicable industrial method to replace ultracentrifugation (UC) in the purification of mesenchymal stromal cell (MSC)-secretome. Materials & methods: Pharmaceutical formulations containing secretome and/or extracellular vesicles were extracted from adipose-MSCs and bone marrow-MSCs by combining UF or UC with lyophilization. Results & conclusion: UF led to higher protein, lipid, cytokine and exosomes yields compared with UC. The isolation procedure and cell source influenced immunomodulatory activity, which was in vitro evaluated by inhibition of phytohemagglutinin-activated peripheral blood mononuclear cell proliferation, and by modulation of IL-10, IFN-γ and IL-6. A secretome dosage was identified to obtain the same immunomodulatory activity of MSCs, paving the way for cell-free therapy.

This chapter focuses on silk fibroin micro-drug delivery systems, with particular attention to microparticles/microspheres, microcapsules, microneedles and microfibers. Their production by different techniques, properties and applications, both in vitro and in vivo, are described. Moreover, their more recent employment for the delivery of drugs, and tissue engineering and regenerative medicine purposes is reported.

Regenerative medicine aims to restore the normal function of diseased or damaged cells, tissues, and organs using a set of different approaches, including cell-based therapies. In the veterinary field, regenerative medicine is strongly related to the use of mesenchymal stromal cells (MSCs), which belong to the body repair system and are defined as multipotent progenitor cells, able to self-replicate and to differentiate into different cell types. This review aims to take stock of what is known about the MSCs and their use in the veterinary medicine focusing on clinical reports on dogs and horses in musculoskeletal diseases, a research field extensively reported in the literature data. Finally, a perspective regarding the use of the secretome and/or extracellular vesicles (EVs) in the veterinary field to replace parental MSCs is provided. The pharmaceuticalization of EVs is wished due to the realization of a Good Manufacturing Practice (GMP product suitable for clinical trials.

Recently, 3D-printed scaffolds for the controlled release of mesenchymal stem cell (MSC) freeze-dried secretome (Lyosecretome) have been proposed to enhance scaffold osteoinduction and osteoconduction; coprinting of poly(ε-caprolactone) (PCL) with alginate hydrogels allows adequate mechanical strength to be combined with the modulable kinetics of the active principle release. This study represents the feasibility study for the sterile production of coprinted scaffolds and the proof of concept for their in vitro biological efficacy. Sterile scaffolds were obtained, and Lyosecretome enhanced their colonization by MSCs, sustaining differentiation towards the bone line in an osteogenic medium. Indeed, after 14 days, the amount of mineralized matrix detected by alizarin red was significantly higher for the Lyosecretome scaffolds. The amount of osteocalcin, a specific bone matrix protein, was significantly higher at all the times considered (14 and 28 days) for the Lyosecretome scaffolds. Confocal microscopy further confirmed such results, demonstrating improved osteogenesis with the Lyosecretome scaffolds after 14 and 28 days. Overall, these results prove the role of MSC secretome, coprinted in PCL/alginate scaffolds, in inducing bone regeneration; sterile scaffolds containing MSC secretome are now available for in vivo pre-clinical tests of bone regeneration.

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.

Silk proteins have been studied and employed for the production of drug delivery (nano)systems. They show excellent biocompatibility, controllable biodegradability and non-immunogenicity and, if needed, their properties can be modulated by blending with other polymers. Silk fibroin (SF), which forms the inner core of silk, is a (bio)material officially recognized by the Food and Drug Administration for human applications. Conversely, the potential of silk sericin (SS), which forms the external shell of silk, could still be considered under evaluation. At the best of our knowledge, nanoparticles based on silk sericin "alone" cannot be produced, due to its physicochemical instability influenced by extreme pH, high water solubility and temperature; for these reasons, it almost always needs to be combined with other polymers for the development of drug delivery systems. In this review, we focused on silk proteins as bioactive natural carriers, since they show not only optimal features as inert excipients, but also remarkable intrinsic biological activities. SF has anti-inflammatory properties, while SS presents antioxidant, anti-tyrosine, anti-aging, anti-elastase and anti-bacterial features. Here, we give an overview on SF or SS silk-based nanosystems, with particular attention on the production techniques.

Simple Summary Electroporation-based therapies (reversible electroporation, irreversible electroporation, electrochemotherapy) are used for the selective treatment of deep-seated tumors. The combination of the structural modifications of the lipid bilayer of cell membranes, due to the application of electrical pulses in the targeted tissue, with the concomitant systemic (intravenous) administration of drugs can be considered as a sort of bridge between local-regional and systemic treatments. A possible further application of these techniques can be envisaged in their use as enhancers of the so-called “enhanced permeability and retention” effect. The intratumoral uptake of drug-loaded nanocarriers concomitant with the application of electric pulses in the target tumor is a new scenario worthy of attention and can represent a potential new frontier for drug delivery in oncology. Abstract Surgical resection is the gold standard for the treatment of many kinds of tumor, but its success depends on the early diagnosis and the absence of metastases. However, many deep-seated tumors (liver, pancreas, for example) are often unresectable at the time of diagnosis. Chemotherapies and radiotherapies are a second line for cancer treatment. The “enhanced permeability and retention” (EPR) effect is believed to play a fundamental role in the passive uptake of drug-loaded nanocarriers, for example polymeric nanoparticles, in deep-seated tumors. However, criticisms of the EPR effect were recently raised, particularly in advanced human cancers: obstructed blood vessels and suppressed blood flow determine a heterogeneity of the EPR effect, with negative consequences on nanocarrier accumulation, retention, and intratumoral distribution. Therefore, to improve the nanomedicine uptake, there is a strong need for “EPR enhancers”. Electrochemotherapy represents an important tool for the treatment of deep-seated tumors, usually combined with the systemic (intravenous) administration of anticancer drugs, such as bleomycin or cisplatin. A possible new strategy, worthy of investigation, could be the use of this technique as an “EPR enhancer” of a target tumor, combined with the intratumoral administration of drug-loaded nanoparticles. This is a general overview of the rational basis for which EP could be envisaged as an “EPR enhancer” in nanomedicine.

It has been demonstrated that the biological effector of mesenchymal stem/stromal cells (MSCs) is their secretome, which is composed of a heterogeneous pool of bioactive molecules, partially enclosed in extracellular vesicles (EVs). Therefore, the MSC secretome (including EVs) has been recently proposed as possible alternative to MSC therapy. The secretome can be considered as a protein-based biotechnological product, it is probably safer compared with living/cycling cells, it presents virtually lower tumorigenic risk, and it can be handled, stored and sterilized as an Active Pharmaceutical/Principle Ingredient (API). EVs retain some structural and technological analogies with synthetic drug delivery systems (DDS), even if their potential clinical application is also limited by the absence of reproducible/scalable isolation methods and Good Manufacturing Practice (GMP)-compliant procedures. Notably, EVs secreted by MSCs preserve some of their parental cell features such as homing, immunomodulatory and regenerative potential. This review focuses on MSCs and their EVs as APIs, as well as DDS, considering their ability to reach inflamed and damaged tissues and to prolong the release of encapsulated drugs. Special attention is devoted to the illustration of innovative therapeutic approaches in which nanomedicine is successfully combined with stem cell therapy, thus creating a novel class of “next generation drug delivery systems.”

Silk sericin (SS) is, together with silk fibroin (SF), one of the two proteins forming the silkworm cocoon. SS is ideal ingredient for cosmetic applications in the formulation of specific products for skin care and hair due to its peculiar physical-chemical composition. SS also showed a great potential in different pharmacological and biotechnological applications, as anticancer drug, anticoagulant, cell culture additive, wound healing agent and drug delivery carrier. Reasons for SS use in biomedical applications derive from its physical-chemical composition. As a consequence, a detailed characterization of SS in terms of average molecular weight, molecular weight distribution and hydro/lipophilic character is crucial to properly address and assess its quality, cosmetic or pharmacological use. In this study, the application of different and complementary chromatographic modes allows a detailed investigation of SS protein isolated from wastewater using two diverse extraction methods. Hydrophilic interaction liquid chromatography (HILIC using an AdvanceBio Glycan Map column) and reverse phase (RP using Symmetry300 C18 column) were applied to intact protein characterization to derive data on protein hydrophilicity and on hydrophobic components of the two SS preparations (SS#1 and SS#2). A higher hydrophilic character of SS#1 was observed by HILIC trace, coherently with the preparation method used, while no significant differences in hydrophobicity were detectable in the RPLC separations. Size distribution was also defined by using a SEC-UV-MS method (using TSKgel SuperSW2000 column) properly optimized to maximize both the size selectivity and the method sensitivity. Taken together, the chromatographic data allowed to better characterize the SS samples obtained by different extraction methods, and the structural properties were correlated to their biological activities.

Pulmonary acute and chronic diseases, such as chronic obstructive pulmonary disease, pulmonary fibrosis and pulmonary hypertension, are considered to be major health issues worldwide. Cellular therapies with Mesenchymal Stem Cells (MSCs) offer a new therapeutic approach for chronic and acute lung diseases related to their anti-inflammatory, immunomodulatory, regenerative, pro-angiogenic and anti-fibrotic properties. Such therapeutic effects can be attributed to MSC-secretome, made of free soluble proteins and extracellular vesicles (EVs). This review summarizes the recent findings related to the efficacy and safety of MSC-derived products in pre-clinical models of lung diseases, pointing out the biologically active substances contained into MSC-secretome and their mechanisms involved in tissue regeneration. A perspective view is then provided about the missing steps required for the secretome "pharmaceuticalization" into a high quality, safe and effective medicinal product, as well as the formulation strategies required for EV non-invasive route of administration, such as inhalation.

Tissue repair and regeneration is an interdisciplinary field focusing on developing bioactive substitutes aimed at restoring pristine functions of damaged, diseased tissues. Biomaterials, intended as those materials compatible with living tissues after in vivo administration, play a pivotal role in this area and they have been successfully studied and developed for several years. Namely, the researches focus on improving bio-inert biomaterials that well integrate in living tissues with no or minimal tissue response, or bioactive materials that influence biological response, stimulating new tissue re-growth. This review aims to gather and introduce, in the context of Italian scientific community, cutting-edge advancements in biomaterial science applied to tissue repair and regeneration. After introducing tissue repair and regeneration, the review focuses on biodegradable and biocompatible biomaterials such as collagen, polysaccharides, silk proteins, polyesters and their derivatives, characterized by the most promising outputs in biomedical science. Attention is pointed out also to those biomaterials exerting peculiar activities, e.g., antibacterial. The regulatory frame applied to pre-clinical and early clinical studies is also outlined by distinguishing between Advanced Therapy Medicinal Products and Medical Devices.

From the end of 2019, the world population has been faced the spread of the novel coronavirus SARS-CoV-2 responsible for COVID-19 infection. In approximately 14% of the patients affected by the novel coronavirus, the infection progresses with the development of pneumonia that requires mechanical ventilation. At the moment, there is no specific antiviral treatment recommended for the COVID-19 pandemic and the therapeutic strategies to deal with the infection are only supportive. In our opinion, mesenchymal stem cell secretome could offer a new therapeutic approach in treating COVID-19 pneumonia, due to the broad pharmacological effects it shows, including anti-inflammatory, immunomodulatory, regenerative, pro-angiogenic and anti-fibrotic properties.

This work aims at designing a drug delivery system for rifampicin (RIF) to be used for the therapy of infections from mycobacterium tuberculosis or other lung-colonizing bacteria. We are proposing, in particular, the delivery of RIF by micelles based on inulin functionalized with vitamin E (INVITE). We previously demonstrated that INVITE micelles are formed from the self-assembling sustained by the interaction, within the hydrophobic core, of aromatic groups belonging to vitamin E. It points on the effectiveness of these biocompatible systems in incorporating aromatic-group-bearing hydrophobic drug such as RIF. The succinilated derivative of INVITE, namely INVITESA, was further studied. Other than a full physicochemical characterization, the obtained micelles containing RIF were tested for their antibacterial activity against Gram- or Gram+bacteria including mycobacterium smegmatis. Furthermore, uptake studies on human alveolar macrophages and MTT studies were performed.

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.

(1) Objective: to obtain a reproducible, robust, well-defined, and cost-affordable in vitro model of human cartilage degeneration, suitable for drug screening, (2) Methods: we proposed 3D models of engineered cartilage, considering two human chondrocyte sources (articular/nasal) and five culture methods (pellet, alginate beads, silk/alginate microcarriers, and decellularized cartilage). Engineered cartilages were treated with pro-inflammatory cytokine IL-1&beta, to promote cartilage degradation, (3) Results: articular chondrocytes have been rejected since they exhibit low cellular doubling with respect to nasal cells, with longer culture time for cell expansion, furthermore, pellet and alginate bead cultures lead to insufficient cartilage matrix production. Decellularized cartilage resulted as good support for degeneration model, but long culture time and high cell amount are required to obtain the adequate scaffold colonization. Here, we proposed, for the first time, the combined use of decellularized cartilage, as aggrecanase substrate, with pellet, alginate beads, or silk/alginate microcarriers, as polymeric scaffolds for chondrocyte cultures. This approach enables the development of suitable models of cartilaginous pathology. The results obtained after cryopreservation also demonstrated that beads and microcarriers are able to preserve chondrocyte functionality and metabolic activity, (4) Conclusions: alginate and silk/alginate-based scaffolds can be easily produced and cryopreserved to obtain a cost-affordable and ready-to-use polymer-based product for the subsequent screening of anti-inflammatory drugs for cartilage diseases.

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.

The aim of this work is to propose a keratinocytes (KC) culture method for clinical practice with irradiated adipose-derived mesenchymal stromal cells (ASCs) as human feeder layer, avoiding murine immortalized fibroblasts, commonly request for producing skin substitutes. ASCs were isolated, expanded, irradiated, and co-cultured with autologous or allogeneic KC. All experiments were performed using murine fibroblasts as control. Cell counts, flow cytometric analysis and ELISA were carried out, in order to define cell yield, viability and cytokine secretion. Results indicate that the optimal X-ray dose for ASCs is 120 Gy and the optimal seeding density is 625 cells/cm2; moreover, flow cytometric analysis shows that the percentage of feeder layer cells reaches values lower than 1%, within 8 days of co-culture. KC reach confluence in 6.9 days on ASCs substrate and, after confluence, the number of live cells increases again in a multilayered structure. Moreover, results show higher levels of interleukin (IL)-1a in co-culture with ASCs compared with 3T3, while no differences were observed for IL-6 and IL-8. Therefore, human ASCs enable to obtain effectively in vitro expanded KC and represent a viable alternative to murine fibroblasts for the production of clinical use skin substitutes.

Although sorted semen is experimentally used for artificial, intrauterine, and intratubal insemination and in vitro fertilization, its commercial application in swine species is still far from a reality. This is because of the low sort rate and the large number of sperm required for routine artificial insemination in the pig, compared with other production animals, and the greater susceptibility of porcine spermatozoa to stress induced by the different sex sorting steps and the postsorting handling protocols. The encapsulation technology could overcome this limitation in vivo, protecting and allowing the slow release of low-dose sorted semen. The aim of this work was to evaluate the impact of the encapsulation process on viability, acrosome integrity, and on the in vitro fertilizing potential of sorted boar semen. Our results indicate that the encapsulation technique does not damage boar sorted semen; in fact, during a 72-hour storage, no differences were observed between liquid-stored sorted semen and encapsulated sorted semen in terms of plasma membrane (39.98 ± 14.38% vs. 44.32 ± 11.72%, respectively) and acrosome integrity (74.32 ± 12.17% vs. 66.07 ± 10.83%, respectively). Encapsulated sorted spermatozoa presented a lower penetration potential than nonencapsulated ones (47.02% vs. 24.57%, respectively, P < 0.0001), and a significant reduction of polyspermic fertilization (60.76% vs. 36.43%, respectively, polyspermic ova/total ova; P < 0.0001). However, no difference (P > 0.05) was observed in terms of total efficiency of fertilization expressed as normospermic oocytes/total oocytes (18.45% vs. 15.43% for sorted diluted and sorted encapsulated semen, respectively). The encapsulation could be an alternative method of storing of pig sex sorted spermatozoa and is potentially a promising technique in order to optimize the use of low dose of sexed spermatozoa in vivo.

In the last 30 years, encapsulation technology has been applied to different species to minimize the loss of spermatozoa after artificial insemination. In particular, the vehiculation of boar sperm cells in barium alginate membrane has proved a valid strategy to reduce the risk of polyspermy and optimize in vivo fertilizing yields. Controlled release of male gametes into the female genital tract has reduced the minimum fertilizing dose of spermatozoa. Notwithstanding these results, encapsulation has not yet reached commercial application, largely due to the additional costs of production. However, encapsulation could be useful in advanced reproductive technology, such as sex sorting, to store sorted boar semen. The controlled release of flow cytometrically sorted spermatozoa could be a promising strategy to reduce the number of cells necessary for each insemination and hence allow the widescale use of sex sorting in this species.

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.

This work reports on the formation of a carrier-in-carrier device for the systemic delivery and targeting of hydrophobic drugs mediated by micelle-loaded mesenchymal stromal cells (MSCs) (carrier-in-carrier) to be administered by intravenous injection. The innate ability of MSCs to reach injured tissues such as the central nervous system or other damaged tissues, is the key for the second order delivery and first order targeting. Inulin- d -alfa-tocopherol succinate micelles (INVITE M) are able to incorporate highly hydrophobic drugs and, due to their dimensions (≈7 nm diameter), to penetrate the cell membrane easily and quickly. This study demonstrates that the curcumin loaded micelles (INVITE MC), sterilized by filtration, reached the maximum loading in MSCs in few minutes and that the loading was concentration-dependent. When “naked” curcumin was used, an evident cytotoxicity on MSCs was detected, while INVITE micelles protected them from this effect. Moreover, MSCs loaded with INVITE MC are able to release the entrapped drug. This study strongly supports the feasibility of the carrier-in-carrier approach for the therapy of selected diseases, i.e., this innovative drug delivery system will be proposed for the treatment of the amyotrophic lateral sclerosis (ALS).

The paper focuses on the preparation of polyacrylate based biomaterials designed as patches for dermal/transdermal drug delivery using materials obtained by the high internal phase emulsion (HIPE) technique. In particular, butyl acrylate and glycidyl methacrylate were selected, respectively, as backbone and functional monomer while two different crosslinkers, bifunctional or trifunctional, were used to form the covalent network. The influence of PEG on the main properties of the materials was also investigated. The obtained materials show a characteristic and interconnected internal structure as confirmed by SEM studies. By an industrial point of view, an interesting feature of this system is that it can be shaped as needed, in any form and thickness. The physiochemically characterized materials showed a tailorable curcumin (model of hydrophobic drugs) drug release, effective mechanical properties and cell viability and resulted neither pro nor anti-angiogenic as demonstrated in vivo by the chick embryo choriallantoic membrane (CAM) assay. Based on these results, the obtained polyHIPEs could be proposed as devices for dermal/transdermal drug delivery and/or for the direct application on wounded skin.

The aim of this work was to load an anticancer drug, paclitaxel (PTX), on Silk Fibroin Nanoparticles (SFNs) by using an exogenous approach. SFNs were produced, freeze-dried and then loaded with PTX. An exogenous method allowed us to reduce both drug loss and environmental impact. In order to quantify PTX loaded in SFNs, a simple and reliable method using reversed phase liquid chromatography coupled to tandem mass spectrometry (rp-UHPLC-MS/MS) was developed. This methodology was validated by the determination of spiked QC samples in three consecutive days. Good accuracy and precision of the method were obtained, while the intra-day and inter-day precisions were less than 10.3%. For PTX, the limit of quantitation (LOQ) was 5.0 ng/mL. Recovery from the matrix (SFNs-PTX pellets) was calculated (81.2% at LOQ value) as PTX was entrapped in a new matrix like the polymer silk fibroin-based. This method was successfully applied to determine the encapsulation efficiency (1.00 ± 0.19%) and the nanoparticle loading (0.12 ± 0.02% w/w). The in vitro anticancer activity of SFNs-PTX was tested against CFPAC-1 cancer cells, results demonstrated a very high cytotoxic activity of SFNs-PTX, with a dose dependent inhibition of CFPAC-1 proliferation, confirmed by the IC50 value of 3450 ± 750 ng/mL.

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.

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.

The aim of this work was to develop a novel carrier-in-carrier system based on stem cell-extracellular vesicles loaded of silk/curcumin nanoparticles by endogenous technique. Silk nanoparticles were produced by desolvation method and curcumin has been selected as drug model because of its limited water solubility and poor bioavailability. Nanoparticles were stable, with spherical geometry, 100 nm in average diameter and the drug content reached about 30%. Cellular uptake studies, performed on mesenchymal stem cells (MSCs), showed the accumulation of nanoparticles in the cytosol around the nuclear membrane, without cytotoxic effects. Finally, MSCs were able to release extracellular vesicles entrapping silk/curcumin nanoparticles. This combined biological-technological approach represents a novel class of nanosystems, combining beneficial effects of both regenerative cell therapies and pharmaceutical nanomedicine, avoiding the use of viable replicating stem cells.

Hydrogels are among the most common materials used in drug delivery, as polymeric micelles are too. They, preferentially, load hydrophilic and hydrophobic drugs, respectively. In this paper, we thought to combine the favorable behaviors of both hydrogels and polymeric micelles with the specific aim of delivering hydrophilic and hydrophobic drugs for dual delivery in combination therapy, in particular for colon drug delivery. Thus, we developed a hydrogel by UV crosslinking of a methacrylated (MA) amphiphilic derivative from inulin (INU) (as known INU is specifically degraded into the colon) and vitamin E (VITE), called INVITEMA. The methacrylated micelles were physicochemically characterized and subjected to UV irradiation to form what we called the &ldquo, nanogrids&rdquo, The INVITEMA nanogrids were characterized by DSC, SEM, TEM, water uptake and beclomethasone dipropionate (BDP) release. In particular, the release of the hydrophobic drug was specifically assessed to verify that it can spread along the hydrophilic portions and, therefore, effectively released. These systems can open new pharmaceutical applications for known hydrogels or micelle systems, considering that in literature only few examples are present.

The use of artificial insemination (AI) in buffalo (Bubalus bubalis) is limited by poor ovarian activity during the hot season, seasonal qualitative patterns in semen, low resistance of sperm cells in the female tract, difficulties in estrus detection, and variable estrus duration. Although AI procedures are commonly used in bovine, use of AI has been limited in buffalo. In the zootechnical field, different studies have been conducted to develop techniques for improvement of fertilizing ability of buffalo spermatozoa after AI. In this study, for the first time, the use of alginate encapsulation and cryopreservation of buffalo spermatozoa is described, and the same procedure was performed with Holstein Friesian (Bos taurus) semen. Results obtained from in vitro analyses indicate that the encapsulation process does not have detrimental effects (compared to controls) on quality parameters (membrane integrity, progressive motility, path average velocity) in either species. Similarly, there were no detrimental effects after cryopreservation in either species. The fertilizing potential of encapsulated and cryopreserved semen was evaluated after AI in 25 buffalo and 113 bovine females. Pregnancy rates were not affected in either species. The results of this study show proof of concept for the use of frozen semen controlled-release devices in buffalo.

The aim of this study is to assess whether stromal vascular fraction (SVF)-soaked silk fibroin nonwoven mats (silk-SVF) can preserve the functionality of encapsulated pancreatic endocrine cells (alginate-PECs) after transplantation in the subcutaneous tissue of diabetic mice. Silk scaffolds are selected to create an effective 3D microenvironment for SVF delivery in the subcutaneous tissue before diabetes induction: silk-SVF is subcutaneously implanted in the dorsal area of five healthy animals; after 15 d, mice are treated with streptozotocin to induce diabetes and then alginate-PECs are implanted on the silk-SVF. All animals appear in good health, increasing weight during time, and among them, one presents euglycemia until the end of experiments. On the contrary, when PECs are simultaneously implanted with SVF after diabetes induction, mice are euthanized due to suffering. This work clearly demonstrates that silk-SVF creates a functional niche in subcutaneous tissue and preserves endocrine cell survival and engraftment.

Mare milk is taken into account as a substitute for cow milk in case of allergy, especially in infant nutrition due to a composition similar to human milk. The aim of the work is to define some mare milk characteristics during lactation, from foaling to the 4 th month of lactation. All the parameters considered to describe fat globule dimensions vary significantly during the lactation period. Fatty acid composition generally changes within the first days of lactation and after 42-56 days, with different behavior when considering medium or higher carbon chains. The ROS-scavenging activity expresses a positive trend and reaches the zenith at day 84. Finally there is a number of correlations between fat globules dimensional parameters and fatty acid composition and between long chain unsaturated fatty acids and ROS-scavenging activity, while dimensional parameters and ROS-scavenging activity are not statistically related. Results could be useful in veterinary and human field as milk substitutes.

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.

A routine use of boar-sexed semen is limited by the long sorting time necessary to obtain an adequate number of sexed spermatozoa for artificial insemination and by the high susceptibility of spermatozoa of this species to damages induced by sorting procedure and subsequent cryopreservation. The aim of this work was to study the impact of encapsulation in barium alginate membrane on sorted boar spermatozoa by evaluating membrane integrity, chlortetracycline staining patterns, protein tyrosine phosphorylation, and Hsp70 immunolocalization during storage over 72 hours in liquid or encapsulated form. The encapsulation procedure significantly (P < 0.05) decreased the overall membrane integrity of control unsorted semen (81.8 vs. 57.4, CTR vs. CPS), but did not negatively affect the overall viability and the chlortetracycline staining patterns of sorted encapsulated cells. Moreover, encapsulation significantly decreased (P < 0.05) the overall phosphotyrosin A pattern cell percentage in unsorted (98.4 vs. 92.6, CTR vs. CPS) but not in sorted semen (64.0 vs. 74.2; SORT CTR vs. SORT CPS). As for Hsp70, the overall percentage of cells displaying the different patterns was significantly influenced (P < 0.05) by treatment but not by storage time. The sorting procedure seems to induce the major changes, whereas encapsulation tends to exert a protective effect on sorted semen by increasing the percentage of spermatozoa displaying the T pattern (2.8 vs. 24.3; SORT CTR vs. SORT CPS). In conclusion, our data confirm that the damaging impact of the encapsulation in barium alginate capsules seems to be limited when compared with that of the sorting procedure and, moreover, the association of the two procedures does not result in an algebraic sum of the negative effects. These results suggest the possibility of a future utilization of the encapsulation technology in order to store sorted spermatozoa and permit their controlled release in the female genital tract.

The use of artificial insemination (AI) in buffalo (Bubalus bubalis) is limited by poor ovarian activity during the hot season, seasonal qualitative patterns in semen, low resistance of sperm cells in the female tract, difficulties in estrus detection, and variable estrus duration. Although AI procedures are commonly used in bovine, use of AI has been limited in buffalo. In the zootechnical field, different studies have been conducted to develop techniques for improvement of fertilizing ability of buffalo spermatozoa after AI. In this study, for the first time, the use of alginate encapsulation and cryopreservation of buffalo spermatozoa is described, and the same procedure was performed with Holstein Friesian (Bos taurus) semen. Results obtained from in vitro analyses indicate that the encapsulation process does not have detrimental effects (compared to controls) on quality parameters (membrane integrity, progressive motility, path average velocity) in either species. Similarly, there were no detrimental effects after cryopreservation in either species. The fertilizing potential of encapsulated and cryopreserved semen was evaluated after AI in 25 buffalo and 113 bovine females. Pregnancy rates were not affected in either species. The results of this study show proof of concept for the use of frozen semen controlled-release devices in buffalo.

Skin substitutes are epidermal, dermal or complete bilayered constructs, composed by natural or synthetic scaffolds and by adherent cells such as fibroblasts, keratinocytes or mesenchymal stem cells. Silk fibroin is a promising polymer to realize scaffolds, since it is biocompatible, biodegradable, and exhibits excellent mechanical properties in terms of tensile strength. Moreover, fibroin can be added of others components in order to modify the biomaterial properties for the purpose. The aim of this work is to prepare silk fibroin films for adipose-derived stem cell (ADSCs) culture as a novel feeder layer for skin tissue engineering. Pectin has been added to promote the protein conformational transition and construct strength, while glycerol as plasticizer, providing biomaterial flexibility. Eighteen formulations were prepared by casting method using fibroin, pectin (range 1-10% w/w), and glycerol (range 0-20% w/w); films were characterized by Fourier transform infrared spectroscopy and differential scanning calorimetry assay, to select the optimal composition. A stable fibroin conformation was obtained using 6% w/w pectin, and the best mechanical properties were obtained using 12% w/w glycerol. Films were sterilized, and human ADSCs were seeded and cultured for 15 days. Cells adhere to the support assuming a fibroblastic-like shape and reaching confluence. The ultrastructural analysis evidences typical active-cell features and adhesion structures that promote cell anchorage to the film, thus developing a multilayered cell structure. This construct could be advantageously employed in cutaneous wound healing or where the use of ADSCs scaffold is indicated either in human or veterinary field.