Aptamer-Mediated Selective Protein Affinity to Improve Scaffold Biocompatibility

Protein adsorption on surfaces occurs shortly after scaffold insertion. This process is of pivotal importance to achieve therapeutic success in tissue engineering (TE), and favorable proteins should be adsorbed at the interface without unfolding to preserve their structure and function. Protein misfolding at the interface is a common phenomenon, which can impair cell adhesion and scaffold colonization. Many efforts have been done to improve scaffold biocompatibility by ameliorating protein adsorption, but with poor results. In the present chapter, we propose the use of a novel class of molecules, aptamers, to improve scaffold biocompatibility. Aptamers are small, single stranded oligonucleotides, which specifically bind to a target molecule: they work as antibodies, but without many of the drawbacks associated to the use of antibodies. We propose to immobilize aptamers on scaffolds to retain specific proteins, acting as docking points to guide cell activity. In particular, we show the results obtained by enriching different polymeric scaffolds with aptamers against human fibronectin, a naturally abundant protein in tissues, which plays a pivotal role in cell adhesion. We demonstrate that scaffold enrichment with aptamers lead to a better colonization of the substrate from cells. The results we obtained pave the way to the possibility of further investigating the role of aptamers as useful molecules to improve scaffold biocompatibility in the contest of tissue engineering

Hyaluronic acid—dexamethasone nanoparticles for local adjunct therapy of lung inflammation

The delivery of a dexamethasone formulation directly into the lung appears as an appro-priate strategy to strengthen the systemic administration, reducing the dosage in the treatment of lung severe inflammations. For this purpose, a hyaluronic acid-dexamethasone formulation was developed, affording an inhalable reconstituted nanosuspension suitable to be aerosolized. The physico-chemical and biopharmaceutical properties of the formulation were tested: size, stability, loading of the spray-dried dry powder, reconstitution capability upon redispersion in aqueous me-dia. Detailed structural insights on nanoparticles after reconstitution were obtained by light and X-ray scattering techniques. (1) The size of the nanoparticles, around 200 nm, is in the proper range for a possible engulfment by macrophages. (2) Their structure is of the core-shell type, hosting dex-amethasone nanocrystals inside and carrying hyaluronic acid chains on the surface. This specific structure allows for nanosuspension stability and provides nanoparticles with muco-inert proper-ties. (3) The nanosuspension can be efficiently aerosolized, allowing for a high drug fraction poten-tially reaching the deep lung. Thus, this formulation represents a promising tool for the lung administration via nebulization directly in the pipe of ventilators, to be used as such or as adjunct therapy for severe lung inflammation.Fil: Cámara, Candelaria Inés. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Físico-química de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Instituto de Investigaciones en Físico-química de Córdoba; ArgentinaFil: Bertocchi, Laura. Departamento de Alimentos y Drogas ; Universita Degli Studi Di Parma;Fil: Ricci, Caterina. Universita Degli Studi Di Milano. Dipartimento Di Beitecnologe Mediche E Medicina Traslazionale.; ItaliaFil: Bassi, Rosaria. Universita Degli Studi Di Milano. Dipartimento Di Beitecnologe Mediche E Medicina Traslazionale.; ItaliaFil: Bianchera, Annalisa. Departamento de Alimentos y Drogas ; Universita Degli Studi Di Parma;Fil: Cantú, Laura F.. Universita Degli Studi Di Milano. Dipartimento Di Beitecnologe Mediche E Medicina Traslazionale.; ItaliaFil: Ruggero, Bettini. Departamento de Alimentos y Drogas ; Universita Degli Studi Di Parma;Fil: Del Favero, Elena. Universita Degli Studi Di Milano. Dipartimento Di Beitecnologe Mediche E Medicina Traslazionale.; Itali

Chitosan Hydrogels for Chondroitin Sulphate Controlled Release: An Analytical Characterization

This paper provides an analytical characterization of chitosan scaffolds obtained by freeze-gelation toward the uptake and the controlled release of chondroitin sulphate (CS), as cartilage repair agent, under different pH conditions. Scanning electron microscopy (SEM), attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR), and liquid chromatography-UV spectrophotometry (LC-UV) techniques were exploited to obtain qualitative and quantitative descriptions of polymer and drug behaviour in the biomaterial. As for morphology, SEM analysis allowed the evaluation of scaffold porosity in terms of pore size and distribution both at the surface (Feret diameter 58±19 μm) and on the cross section (Feret diameter 106±51 μm). LC and ATR-FTIR evidenced a pH-dependent CS loading and release behaviour, strongly highlighting the role of electrostatic forces on chitosan/chondroitin sulphate interactions

Design and Characterization of Maltoheptaose-b-Polystyrene Nanoparticles, as a Potential New Nanocarrier for Oral Delivery of Tamoxifen

Tamoxifen citrate (TMC), a non-steroidal antiestrogen drug used for the treatment of breast cancer, was loaded in a block copolymer of maltoheptaose-b-polystyrene (MH-b-PS) nanoparticles, a potential drug delivery system to optimize oral chemotherapy. The nanoparticles were obtained from self-assembly of MH-b-PS using the standard and reverse nanoprecipitation methods. The MH-b-PS@TMC nanoparticles were characterized by their physicochemical properties, morphology, drug loading and encapsulation efficiency, and release kinetic profile in simulated intestinal fluid (pH 7.4). Finally, their cytotoxicity towards the human breast carcinoma MCF-7 cell line was assessed. The standard nanoprecipitation method proved to be more efficient than reverse nanoprecipitation to produce nanoparticles with small size and narrow particle size distribution. Moreover, tamoxifen-loaded nanoparticles displayed spherical morphology, a positive zeta potential and high drug content (238.6 ± 6.8 µg mL−1) and encapsulation efficiency (80.9 ± 0.4 %). In vitro drug release kinetics showed a burst release at early time points, followed by a sustained release profile controlled by diffusion. MH-b-PS@TMC nanoparticles showed higher cytotoxicity towards MCF-7 cells than free tamoxifen citrate, confirming their effectiveness as a delivery system for administration of lipophilic anticancer drug

Evaluation of effectiveness of an innovative semen extender (Formula®) comparing with a traditional extender (Lepus®) for artificial insemination in rabbits does

This study aimed to investigate the preservability and viability of the rabbit spermatozoa diluted in a new semen extender Formula® in comparison with Lepus® at 17 °C of storage. The main characteristic of the new extender formulation is the use of an enzymatic agent associated to a polysaccharide as energy source precursor, added with gentamycin. During eight trials, ejaculates from 70 bucks were collected and diluted at 1:10 ratio with both the extenders, after 24 h of storage the semen doses were used for the artificial insemination (AI). Aliquots of the semen doses for each trial were stored at 17 °C, the total and progressive motility were checked at 0, 4, 12, 18, 24, 36, 48, 60, 72, 84, 96, 108 h of storage. A total of 1267 and 1525 does were inseminated, respectively with Formula® and Lepus®. During storage the mean total and progressive motility (77.23% and 72.854%, respectively) were significantly higher for Formula® (p < .01) and the progressive motility at almost 70% was maintained for at least 60 h vs the 24 h of storage for Lepus® with significant differences after 12 h of storage (p < .05). The new extender reported a higher pregnancy rate (p < .05) and an average of 9.25 rabbits born per litter vs 8.83 for the traditional extender (p < .05), while the mean of the newborn alive was 9.08 using Formula® vs 8.51 with Lepus® (p < .05). In conclusion, the use of Formula® is recommended for rabbit semen AI programmes

Nano-adjuvanted dry powder vaccine for the mucosal immunization against airways pathogens

Nasal vaccination has been shown to provide optimal protection against respiratory pathogens. However, mucosal vaccination requires the implementation of specific immunization strategies to improve its effectiveness. Nanotechnology appears a key approach to improve the effectiveness of mucosal vaccines, since several nanomaterials provide mucoadhesion, enhance mucosal permeability, control antigen release and possess adjuvant properties. Mycoplasma hyopneumoniae is the main causative agent of enzootic pneumonia in pigs, a respiratory disease responsible for considerable economic losses in the pig farming worldwide. The present work developed, characterized, and tested in vivo an innovative dry powder nasal vaccine, obtained from the deposition on a solid carrier of an inactivated antigen and a chitosan-coated nanoemulsion, as an adjuvant. The nanoemulsion was obtained through a low-energy emulsification technique, a method that allowed to achieve nano droplets in the order of 200 nm. The oil phase selected was alpha-tocopherol, sunflower oil, and poly(ethylene glycol) hydroxystearate used as non-ionic tensioactive. The aqueous phase contained chitosan, which provides a positive charge to the emulsion, conferring mucoadhesive properties and favoring interactions with inactivated M. hyopneumoniae. Finally, the nanoemulsion was layered with a mild and scalable process onto a suitable solid carrier (i.e., lactose, mannitol, or calcium carbonate) to be transformed into a solid dosage form for administration as dry powder. In the experimental study, the nasal vaccine formulation with calcium carbonate was administered to piglets and compared to intramuscular administration of a commercial vaccine and of the dry powder without antigen, aimed at evaluating the ability of IN vaccination to elicit an in vivo local immune response and a systemic immune response. Intranasal vaccination was characterized by a significantly higher immune response in the nasal mucosa at 7 days post-vaccination, elicited comparable levels of Mycoplasma-specific IFN-gamma secreting cells and comparable, if not higher, responsiveness of B cells expressing IgA and IgG in peripheral blood mononuclear cells, with those detected upon a conventional intramuscular immunization. In conclusion, this study illustrates a simple and effective strategy for the development of a dry powder vaccine formulation for nasal administration which could be used as alternative to current parenteral commercial vaccines

Chitosan-Coated Nanoparticles: Effect of Chitosan Molecular Weight on Nasal Transmucosal Delivery

Drug delivery to the brain represents a challenge, especially in the therapy of central nervous system malignancies. Simvastatin (SVT), as with other statins, has shown potential anticancer properties that are difficult to exploit in the central nervous system (CNS). In the present work the physico⁻chemical, mucoadhesive, and permeability-enhancing properties of simvastatin-loaded poly-ε-caprolactone nanocapsules coated with chitosan for nose-to-brain administration were investigated. Lipid-core nanocapsules coated with chitosan (LNCchit) of different molecular weight (MW) were prepared by a novel one-pot technique, and characterized for particle size, surface charge, particle number density, morphology, drug encapsulation efficiency, interaction between surface nanocapsules with mucin, drug release, and permeability across two nasal mucosa models. Results show that all formulations presented adequate particle sizes (below 220 nm), positive surface charge, narrow droplet size distribution (PDI < 0.2), and high encapsulation efficiency. Nanocapsules presented controlled drug release and mucoadhesive properties that are dependent on the MW of the coating chitosan. The results of permeation across the RPMI 2650 human nasal cell line evidenced that LNCchit increased the permeation of SVT. In particular, the amount of SVT that permeated after 4 hr for nanocapsules coated with low-MW chitosan, high-MW chitosan, and control SVT was 13.9 ± 0.8 μg, 9.2 ± 1.2 µg, and 1.4 ± 0.2 µg, respectively. These results were confirmed by SVT ex vivo permeation across rabbit nasal mucosa. This study highlighted the suitability of LNCchit as a promising strategy for the administration of simvastatin for a nose-to-brain approach for the therapy of brain tumors

Alla ricerca della fonte miracolosa della rigenerazione tissutale: isolamento, caratterizzazione e differenziamento in tessuti duri di cellule multipotenti da placenta umana

The improvement of life quality and lifespan lengthening in Western countries leads as a consequence a more and more urgent need for organs and tissues replacement. As organs and tissues derived from heterologous donors are not easily available and, moreover, oblige to a life-spanning use of immunosuppressive drugs, effective alternatives are needed. The aim of this work was the investigation of the regenerative potential nested in placenta, as an ethically acceptable and promising source of multipotent cells for tissue repair. A protocol is reported that allows the isolation from placenta of a homogeneous population of human chorionic multipotent stromal cells (hCMSCs) by the combination of plastic adherence and subsequent cell sorting of CD34neg cells. This population displays the typical characteristics of mesenchymal stem cells and exhibits clonal properties, as testified by the analysis of surface markers and doubling time. As for the differentiation potential, evidence was provided on the ability of hCMSCs to differentiate into osteoblastic cells as well as into adipoblasts and chondroblasts. In particular, concerning chondrogenic differentiation, culture protocols were useful to get the induction of the expression in hCMSCs of typical chondrogenic genes, but some work still has to be done in order to obtain a clear hyaline cartilage to satisfy the target of articular cartilage regeneration. Moreover hCMSCs possess an osteogenic potential if cultivated in appropriate conditions. When a canonical osteoinductive medium was used, differentiation towards the osteogenic lineage was scanty and only small calcified nodules were visualized. In order to improve these results, cells were grown in the presence of collagen or Bio-oss® as substrates. Collagen didn’t influence gene expression of hCSMCs, while the presence of Bio-oss®, especially if combined with an osteogenic medium, allowed better differentiation and maturation of cells into osteoblasts or osteocytes. In experiments performed on a rat model of critical size defect, hCMSCs confirmed their ability to settle and proliferate on Bio-oss® scaffolds. Moreover those cells were able to fill the created defect by forming a soft compact tissue. On the whole, data collected in this PhD thesis suggest that placenta could be an effective and abundant source of cells for tissue regeneration.Il miglioramento della qualità e l’allungamento dell’aspettativa di vita nei Paesi occidentali comportano una sempre più urgente necessità di sostituire organi e tessuti. Dato che le donazioni di organi sono assolutamente insufficienti e che, inoltre, il trapianto eterologo obbliga all’assunzione per tutta la vita di farmaci immunosoppressori, appare evidente la necessità di fonti alternative di tessuti. Lo scopo dello studio era l’analisi del potenziale rigenerativo insito nella placenta umana, considerata una fonte eticamente accettabile ed estremamente promettente di cellule multipotenti per la rigenerazione tissutale. La tesi riporta un protocollo che consente l’isolamento dalla placenta di una popolazione omogenea di cellule multipotenti stromali (hCMSCs) grazie alla combinazione di adesione su plastica e successiva selezione immunomagnetica delle cellule CD34-. Questa popolazione mostra le caratteristiche tipiche delle cellule mesenchimali e possiede proprietà clonali, come testimoniato dall’analisi dei markers superficiali e dai tempi di duplicazione. Per quanto riguarda il potenziale differenziativo, si dimostra che le hCMSCs sono in grado di differenziare in adipoblasti, condroblasti e osteoblasti. In particolare, a proposito del differenziamento cartilagineo, sono stati adottati protocolli di coltura utili all’ottenimento dell’induzione dell’espressione da parte delle hCMSCs di geni tipici del tessuto cartilagineo. Tuttavia, il tessuto ottenuto mostra caratteristiche fibrocartilaginee, che suggeriscono la necessità di migliorare ulteriormente le condizioni di differenziamento al fine di ottenere un sostituto funzionale per la cartilagine ialina articolare. Inoltre le hCMSCs sono in grado di differenziare in tessuto osseo, se opportunamente trattate. L’utilizzo di un medium osteoinduttivo classico produce solo un modesto differenziamento e una scarsa deposizione di noduli calcificati. Per migliorare tali risultati le cellule sono state coltivate in presenza di collagene o Bio-oss® come substrati. Il collagene non influisce sull’espressione genica delle hCMSCs, mentre la presenza Bio-oss®, specialmente se associata ad un medium osteogenico adeguato, consente un differenzamento più completo ed una migliore maturazione delle cellule in osteoblasti e osteociti. Negli esperimenti condotti in vivo su un modello murino di difetto critico le hCMSCs hanno confermato la capacità di colonizzare gli scaffold di Bio-oss® e di proliferare su di essi. Inoltre tali cellule sono state in grado di colmare il difetto creato originando un tessuto molle compatto. Nel complesso i dati raccolti in questo lavoro di tesi suggeriscono che la placenta può costituire effettivamente una fonte utile ed abbondante di cellule per la rigenerazione tissutale

Highly Polymorphic Materials and Dissolution Behaviour: The Peculiar Case of Rifaximin

Rifaximin is a locally acting antibiotic practically insoluble in water. It presents several crystal phases characterized by different degrees of hydration. The aim of this work is to investigate the dissolution behaviour of rifaximin &alpha;, &beta;, and amorphous forms in relation to their relative thermodynamic stability to contribute to clarifying possible solvent- or humidity-mediated conversion patterns. Kinetic and intrinsic solubility were investigated along with particle size distribution, specific surface area, and external morphology. The solution and moisture mediated conversion from metastable &alpha; and amorphous forms to stable &beta; form were elucidated by coupling intrinsic dissolution test with chemometric analysis as well as by dynamic vapour sorption measurements. The dissolution behaviour of the &alpha; form stems mainly from the transition to &beta; form that occurs upon exposition to relative humidity higher than 40%. The &alpha; form converted more rapidly than the amorphous form due to the smaller supersaturation ratio. It can be concluded that, due to its marked tendency to transform into &beta; form, the dissolution test for the &alpha; form, even if conducted according to compendial procedures, needs to be accompanied by a panel of further tests that allow to uniquely identify the solid phase under investigation