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Gaia Zucca,1 Barbara Vigani,1 Caterina Valentino,1 Marco Ruggeri,1 Nicoletta Marchesi,1 Alessia Pascale,1 Giulia Giovilli,2 Lorenzo Malavasi,2 Giuseppina Sandri,1 Silvia Rossi1 1Department of Drug Sciences, University of Pavia, Pavia, 27100, Italy; 2Department of Chemistry and INSTM, University of Pavia, Pavia, 27100, ItalyCorrespondence: Silvia Rossi; Barbara Vigani, Department of Drug Sciences, University of Pavia, Via Taramelli 12, Pavia, 27100, Italy, Email silvia.rossi@unipv.it; barbara.vigani@unipv.itPurpose: The main purpose of the study was the formulation development of nanogels (NHs) composed of chondroitin sulfate (CS) and low molecular weight chitosan (lCH), loaded with a naringenin-β-cyclodextrin complex (NAR/β-CD), as a potential treatment for early-stage diabetic retinopathy.Methods: Different formulations of NHs were prepared by varying polymer concentration, lCH ratio, and pH and, then, characterized for particle size, zeta potential, particle concentration (particles/mL) and morphology. Cytotoxicity and internalization were assessed in vitro using Human Umbilical Vein Endothelial Cells (HUVEC). The NAR/β-CD complex was prepared and evaluated for morphology, complexation efficiency, and solubility. Finally, the most promising NH prototype was loaded with NAR/β-CD (NH@NAR/β-CD) and further characterized for encapsulation efficiency, loading capacity, opacity and cytotoxicity on HUVEC; in vitro release test and DPPH assay were performed to investigate NH capability to sustain NAR release and NH@NAR/β-CD antioxidant properties, respectively.Results: NH properties were influenced by polymer concentration, lCH ratio, and pH. N3 (0.5 mg/mL; lCH=1.5:1; pH = 5) and N9 (0.5 mg/mL; lCH=1:1; pH = 5) showed optimal characteristics, including small size (< 350 nm) and positive zeta potential, facilitating cellular uptake. The NAR/β-CD complex showed 71% complexation efficiency and enhanced NAR solubility. Since characterized by superior properties and better in vitro biocompatibility, N3 was loaded with NAR/β-CD. N3@NAR/β-CD capability to sustain in vitro NAR release, radical scavenging activity and in vitro biocompatibility were finally demonstrated.Conclusion: The physico-chemical properties of N3@NAR/β-CD were responsible for their cell uptake, suggesting their potential to target retinal endothelial cells. The high NAR/β-CD complexation efficiency and the sustained NAR release over 72 hours could guarantee the maintenance of an effective drug concentration at the damage site while reducing the injection number. Further studies about the safety and the effectiveness of the intravitreal injection of NHs@NAR/β-CD will be performed on a diabetic animal model. Keywords: polyelectrolyte complexation, cellular uptake, inclusion complex, antioxidant properties, intravitreal administration

Dalila Miele,1 Marco Ruggeri,1 Barbara Vigani,1 Cesar Viseras,2 Francesca Natali,3 Elena Del Favero,4 Silvia Rossi,1 Giuseppina Sandri1 1Department of Drug Sciences, University of Pavia, Pavia, Italy; 2Department of Pharmacy and Pharmaceutical Technology, School of Pharmacy, University of Granada, Granada, Spain; 3Institut Laue-Langevin, Grenoble, France; 4Department of Medical Biotechnology and Translational Medicine, University of Milano, Segrate Milano, ItalyCorrespondence: Giuseppina Sandri, Tel +390382987728, Email giuseppina.sandri@unipv.itBackground: Clay minerals are nanomaterials that have recently been recognized as enabling excipients that can promote cell adhesion, proliferation, and differentiation. When nanoclays are loaded in a 3D polymeric nanostructure, the cell–substrate interaction is enhanced, and other bioactive properties are optimized.Purpose: In this study, hectorite (HEC)- and montmorillonite (MMT)-doped polymeric scaffolds were explored for the treatment of deep and chronic skin lesions.Methods: Scaffolds were manufactured by means of electrospinning and then crosslinked by heating. Physicochemical analyses were correlated with in vitro biopharmaceutical characterization to predict the in vivo fate of the clay-doped scaffolds.Results and Discussion: The addition of MMT or HEC to the polymeric scaffold framework modifies the surface arrangement and, consequently, the potential of the scaffolds to interact with biological proteins. The presence of nanoclays alters the nanofiber morphology and size, and MMT doping increases wettability and protein adhesion. This has an impact on fibroblast behavior in a shorter time since scaffold stiffness facilitates cell adhesion and cell proliferation.Conclusion: MMT proved to perform better than HEC, and this could be related to its higher hydrophilicity and protein adhesion. Keywords: nanomaterials, montmorillonite, hectorite, chitosan, nanofibers, electrospinning, tissue engineering

Barbara Vigani, Caterina Valentino, Giuseppina Sandri, Carla Marcella Caramella, Franca Ferrari, Silvia Rossi Department of Drug Sciences, University of Pavia, Pavia, ItalyCorrespondence: Silvia Rossi, Department of Drug Sciences, University of Pavia, Viale Taramelli 12, Pavia, 27100, Italy, Tel +39 0382987357, Fax +39 0382422975, Email silvia.rossi@unipv.itPurpose: Aim of the work was to develop a potential neural scaffold, endowed with neuroprotective and neuroregenerative potential, to be applied at the site of nervous tissue injuries: nanofibers, consisting of gellan gum (GG), spermidine (SP) and gelatin (GL), were prepared via electrospinning. SP was selected for its neuroprotective activity and cationic nature that makes it an ideal GG cross-linking agent. GL was added to improve the scaffold bioactivity.Methods: Mixtures, containing 1.5% w/w GG and increasing SP concentrations (0– 0.125% w/w), were prepared to investigate GG/SP interaction and, thus, to find the best mixture to be electrospun. Mixture rheological and mechanical properties were assessed. The addition of 0.1% w/w GL was also investigated. The most promising GG/SP/GL mixtures were added with poly(ethylene oxide) (PEO) and poloxamer (P407) and, then, electrospun. The resulting fibers were characterized in terms of size and mechanical properties and fiber morphology was observed after soaking in water for 24 hours. Nanofiber biocompatibility was assessed on Schwann cells.Results: More and more structured GG/SP mixtures were obtained by increasing SP concentration, proving its cross-linking potential. After blending with PEO and P407, the mixture consisting of 1.5% w/w GG, 0.05% w/w SP and 0.1% w/w GL was electrospun. The resulting nanofibers appeared homogenous and characterized by a plastic behavior, suggesting a good mechanical resistance when applied at the injury site. Nanofibers were insoluble in aqueous media and able to form a thin gel layer after hydration. GG/SP/GL nanofibers showed a higher compatibility with Schwann cells than GG/SP ones.Conclusion: SP and GL allowed the production of homogenous GG-based nanofibers, which preserved their structure after contact with aqueous media and showed a good compatibility with a neural cell line. After local application at the injury site, nanofibers should support and guide axonal outgrowth, releasing SP in a controlled manner.Graphical Abstract: Keywords: nervous tissue injuries, electrospinning, hydrogel nanofibers, gelatin, Schwann cells

Barbara Vigani,1 Silvia Rossi,1 Giuseppina Sandri,1 Maria Cristina Bonferoni,1 Giulia Milanesi,1 Giovanna Bruni,2 Franca Ferrari1 1Department of Drug Sciences, University of Pavia, Pavia, Italy; 2Department of Chemistry, University of Pavia, Pavia, Italy Aim: The aim of the present work was to develop biodegradable alginate (ALG)-containing fibrous membranes intended for tissue repair, acting as both drug delivery systems and cell growth guidance. Methods: Membranes were prepared by electrospinning. Since ALG can be electrospun only when blended with other spinnable polymers, dextran (DEX) and polyethylene oxide (PEO) were investigated as process adjuvants. ALG/DEX mixtures, characterized by different rheological and conductivity properties, were prepared in phosphate buffer or deionized water; surfactants were added to modulate polymer solution surface tension. The Design of Experiments (DoE) approach (full factorial design) was used to investigate the role of polymer solution features (rheological properties, surface tension, and conductivity) on electrospun fiber morphology. A high viscosity at 1,000 s-1 (1.3–1.9 Pa.s) or a high pseudoplasticity index (≥1.7), combined with a low surface tension (30–32 mN/m) and a low conductivity (800–1,000 µS/cm), was responsible for the production of ALG/DEX homogeneous fibers. Such ranges were successfully employed for the preparation of ALG-containing fibers, using PEO, instead of DEX, as process adjuvant. ALG/DEX and ALG/PEO fibers were subsequently subjected to cross-linking/coating processes to make them slowly biodegradable in aqueous medium. In particular, ALG/PEO fibers were cross-linked and coated with CaCl2/chitosan solutions in water/ethanol mixtures. Due to DEX high content, ALG/DEX fibers were soaked in a polylactide-co-glycolide (PLGA) solution in ethyl acetate. Results: Both cross-linking and coating processes made fibers insoluble in physiological medium and produced an increase in their mechanical resistance, assessed by means of a tensile test. PLGA-coated ALG/DEX and chitosan-coated ALG/PEO fibers were biocompatible and able to support fibroblast adhesion. Conclusion: The DoE approach allowed to draw up guidelines useful for the preparation of homogeneous fibers, starting from mixtures of ALG and non-ionic polymers. Such fibers, upon coating, resulted to be good cell substrates, allowing cell adhesion and growth. Keywords: DoE approach, ALG-based fibers, rheological properties, conductivity, surface tension, chitosan, PLGA

Francesca Saporito,1 Giuseppina Sandri,1 Maria Cristina Bonferoni,1 Silvia Rossi,1 Cinzia Boselli,1 Antonia Icaro Cornaglia,2 Barbara Mannucci,3 Pietro Grisoli,1 Barbara Vigani,1 Franca Ferrari1 1Department of Drug Sciences, University of Pavia, Pavia, 2Department of Public Health, Experimental and Forensic Medicine, University of Pavia, Pavia, 3Centro Grandi Strumenti, University of Pavia, Pavia, Italy Abstract: Chronic wounds and severe burns are diseases responsible for severe morbidity and even death. Wound repair is a crucial process and tissue regeneration enhancement and infection prevention are key factors to minimize pain, discomfort, and scar formation. The aim of this work was the development of lipid nanoparticles (solid lipid nanoparticles and nanostructured lipid carriers [NLC]), to be loaded with eucalyptus or rosemary essential oils and to be used, as medical devices, to enhance healing of skin wounds. Lipid nanoparticles were based on natural lipids: cocoa butter, as solid lipid, and olive oil or sesame oil, as liquid lipids. Lecithin was chosen as surfactant to stabilize nanoparticles and to prevent their aggregation. The systems were prepared by high shear homogenization followed by ultrasound application. Nanoparticles were characterized for physical–chemical properties, bioadhesion, cytocompatibility, in vitro proliferation enhancement, and wound healing properties toward normal human dermal fibroblasts. Antimicrobial activity of nanoparticles was evaluated against two reference microbial strains, one of Staphylococcus aureus, the other of Streptococcus pyogenes. Finally, the capability of nanoparticles to promote wound healing in vivo was evaluated on a rat burn model. NLC based on olive oil and loaded with eucalyptus oil showed appropriate physical–chemical properties, good bioadhesion, cytocompatibility, in vitro proliferation enhancement, and wound healing properties toward fibroblasts, associated to antimicrobial properties. Moreover, the in vivo results evidenced the capability of these NLC to enhance the healing process. Olive oil, which is characterized by a high content of oleic acid, proved to exert a synergic effect with eucalyptus oil with respect to antimicrobial activity and wound repair promotion. Keywords: lipid nanoparticles, eucalyptus essential oil, antimicrobial properties, wound healing