Development of new in vitro models based on the use of electrospun scaffolds and their imaging by multiphoton microscopy coupled with fluorescence lifetime imaging microscopy

2016 - 2017In this thesis, new possible applications of electrospun scaffolds are presented. Besides, the interaction of human dermal fibroblasts (HDFs) with the materials has been investigated using multiphoton microscopy (MPM) coupled with fluorescence lifetime imaging microscopy (FLIM), using a non-invasive, marker-free approach. In the first part of the thesis, pure poly-L-lactide (PLA) scaffolds were obtained and characterized as delivery systems for Diclofenac sodium salt (DCF) and a synthetically obtained prodrug of it for the treatment of actinic keratosis. The Diclofenac prodrug was obtained via solid phase peptide synthesis using a versatile, clean, high yielding procedure. Besides, the drug encapsulation and its release from the scaffold could be imaged using MPM. Moreover, when working with the unmodified DCF we were able to control the release profile by adding small amounts of dimethyl sulfoxide. The DCF-loaded scaffold was used as a delivery system to induce in vitro cell death in HDFs. The target cells were imaged using MPM coupled with FLIM, using a non-invasive, marker-free in vitro model to investigate drug effects. In the last part of the thesis, we produced and characterized different hybrid gelatin/PLA scaffolds. In this case, the goal was to obtain well-blended scaffolds with tunable properties, such as porosity, hydrophobicity and wettability... [edited by Author]XXX cicl

Electrospun poly-L-lactide scaffold for the controlled and targeted delivery of a synthetically obtained Diclofenac prodrug to treat actinic keratosis

Abstract Actinic Keratosis' (AKs) are small skin lesions that are related to a prolonged sun-damage, which can develop into invasive squamous cell carcinoma (SCC) when left untreated. Effective, specific and well tolerable therapies to cure AKs are still of great interest. Diclofenac (DCF) is the current gold standard for the local treatment of AKs in terms of costs, effectiveness, side effects and tolerability. In this work, an electrospun polylactic acid (PLA) scaffold loaded with a synthetic DCF prodrug was developed and characterized. Specifically, the prodrug was successfully synthetized by binding DCF to a glycine residue via solid phase peptide synthesis (SPPS) and then incorporated in an electrospun PLA scaffold. The drug encapsulation was verified using multiphoton microscopy (MPM) and its scaffold release was spectrophotometrically monitored and confirmed with MPM. The scaffold was further characterized with scanning electron microscopy (SEM), tensile testing and contact angle measurements. Its biocompatibility was verified by performing a cell proliferation assay and compared to PLA scaffolds containing the same amount of DCF sodium salt (DCFONa). Finally, the effect of the electrospun scaffolds on human dermal fibroblasts (HDFs) morphology and metabolism was investigated by combining MPM with fluorescence lifetime imaging microscopy (FLIM). The obtained results suggest that the obtained scaffold could be suitable for the controlled and targeted delivery of the synthesized prodrug for the treatment of AKs. Statement of Significance Electrospun scaffolds are of growing interest as materials for a controlled drug delivery. In this work, an electrospun polylactic acid scaffold containing a synthetically obtained Diclofenac prodrug is proposed as a novel substrate for the topical treatment of actinic keratosis. A controlled drug delivery targeted to the area of interest could enhance the efficacy of the therapy and favor the healing process. The prodrug was synthesized via solid phase, employing a clean and versatile approach to obtain Diclofenac derivatives. Here, we used multiphoton microscopy to image drug encapsulation within the fibrous scaffold and fluorescence lifetime imaging microscopy to investigate Diclofenac effects and potential mechanisms of action

Non-invasive characterization of hybrid gelatin:poly-L-lactide electrospun scaffolds using second harmonic generation and multiphoton imaging

In this work we generated hybrid gelatin:poly-l-lactide electrospun scaffolds and implemented non-invasive methods to characterize them

Photoinduced Thiol-ene Chemistry Applied to the Synthesis of Self-Assembling Elastin-Inspired Glycopeptides

Abstract: Synthetic (glyco)peptides inspired by proteins able to self-assemble are appealing biomaterials in the field of tissue engineering and regenerative medicine. Herein, for the first time, taking advantage of thiol-ene chemistry coupled to solid-phase peptide synthesis, a self-assembling peptide inspired by elastin protein was bioconjugated to three carbohydrates in order to obtain the corresponding glycopeptides. They were studied at the molecular and supramolecular level. The results show that the carbohydrate influences the molecular conformation of the glycopeptide and its self-aggregation properties as well. As future perspective, the results could enable us to tune the final self-aggregation properties of the glycopeptide by changing the sugar moiety

Nanocellulose and elastin act as plasticizers of Electrospun bio-inspired scaffolds.

In this work, we produced cross-linked electrospun hybrid scaffolds composed of gelatin/poly-D,L-lactide, gelatin/poly-D,L-lactide/nanocellulose and gelatin/poly-D,L-lactide/cellulose nanocrystals/elastin. Fourier-Transform Infrared Spectroscopy, X-Ray Diffraction, and High Performance Liquid Chromatography demonstrated the complete embedding of each com-ponent in the hybrid scaffolds. Degree of cross-linking was quantified by using a 2,4,6-trinitrobenzenesulfonic acid assay and Attenuated Total Reflectance Spectroscopy revealed the effectiveness of cross-linking reaction. Noteworthy, the interconnected porous structure revelead in un-cross-linked scaffolds endured even after cross-linking. Scaffolds were characterized in water through contact angle showing total wettability. We throughout investigated mechanical properties by Uniaxial Tensile Testing showing that even in the dry-state nanocellulose and elastin containing scaffolds exhibit higher elongation at rupture compared to pure gelatin/poly-D,L-lactide. Therefore, we succeed in tuning the toughness of the scaffolds by modulating composition. In order to use scaffolds as medical devices, we assayed fibroblasts on scaffolds extraction media establishing that they were non-cytotoxic. Finally the attachment and proliferation of fibroblasts on the surface of different scaffolds was evaluate