A polymer is a molecule with high molecular weight constituted by identical or different repeating units (monomers) hold together as a chain by covalent bonds. Nucleic acids, polysaccharides and proteins are biological macromolecules essential for living organism with inner biocompatibility and essential biological activity for health. The biomedical and pharmaceutical applications of natural polymers face some limitations associated to difficult isolation and purification processes connected to the risk of immunological reactions once applied in the human body. The design of synthetic polymers takes inspiration from the main features of natural polymers, with the final goal to overcome their limitations and meet the therapeutic medical needs of patients. The combination of natural and synthetic polymers results in biomaterials with unique and complementary characteristics. The main goal of the present thesis is the development of new biomaterials composed of hybrid and synthetic polymers formulated as hydrogels and nanoparticles for application in drug delivery, tissue engineering, and brain targeting. The central theme of this thesis is presented in Chapter 2. Temperature responsive polymers belong to the class of smart polymers, that, in aqueous solutions, lead to the formation of stimuli responsive hydrogels. For biomedical and pharmaceutical applications, hydrogels must satisfy precise properties that are explained in this section. The chapter mainly focuses on the use of hydrogels for protein release, the ways proteins can be loaded and subsequently delivered in a controlled way from the gel network are explained. The application of hydrogels in tissue engineering is presented and deeply investigated in the following Chapter 3, with particular emphasis on the most recent applications in cartilage repair. After a brief presentation of natural and synthetic hydrogels, the discussion converges on the use of gel networks for the delivery of bioactive molecules such as growth factors mostly involved as signaling and stimulating molecules for cells proliferation, growth and differentiation. Growth factors are rapidly degraded in physiological conditions, being therefore unable to reach the injury site and to unroll their work. The encapsulation into polymeric scaffolds is an efficacious approach to overcome these disadvantages and provide an in-situ release of bioactive proteins. Other strategies for cartilage restoration regards the encapsulation of genetic material encoding for growth factors and the enclosure of platelet rich plasma (PRP) into the hydrogel scaffold. Chapter 4 introduces the principal biomaterial of this thesis. The aim of the work is to develop a fast-gelling hydrogels composed of a central hydrophilic block of poly ethylene glycol (PEG) flanked by two thermosensitive chains of poly(hydroxypropyl methacrylamide) (p(HPMAm-lac)) derivatized with vinyl groups and cross-linked, by Michael addition with thiolated hyaluronic acid (HA-SH) in a vinyl sulfone- thiol groups ratio of 1:1. Vinyl sulfonated triblock copolymers were highly reactive towards thiol groups giving rise to hydrogels with rapid gelation time and with complete conversion of vinyl sulfone groups. The hydrogels showed increasing G' values, decreasing gelation temperature (considered as temperature at which G'=G''), greater swelling and faster degradation kinetics at decreasing thiolation degree of hyaluronic acid. Cells viability, evaluated by MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay for 21 days on mouse bone marrow stromal cell (BMSCs) and NIH 3T3 mouse embryonic fibroblasts, showed that cells viability was preserved, and cells maintained their shape for the considered timescale. Chapter 5 reports on the in vivo biocompatibility study of the tandem cross-linked vinyl sulfone bearing p(HPMA-lac)-PEG-p(HPMA-lac) based hydrogel when injected into Balb/c mice. The hydrogel was easily injected both intramuscularly and subcutaneously and immediately cross-linked in a stable network at the site of administration, as confirmed by ultranosography monitoring. Bone marrow cells of mice treated with hydrogels, after been cultured in medium, showed an important decrease of pro-inflammatory cytokines and chemokines. The decrease is timedependent, possibly due to the anti-inflammatory effect of hyaluronic acid released during the degradation of the hydrogel. It was concluded that the presented cross-linked hydrogel was a good candidate for tissue engineering applications and for drug delivery. The feasibility of the application of the vinyl sulfone bearing p(HPMA-lac)-PEG-p(HPMA-lac) based hydrogels in articular cartilage defects is the topic of Chapter 6, where the hydrogel was loaded with platelet rich plasma extracted from equine blood samples. In vitro studies and rheological analysis showed that the hydrogel provided a protective and strong network and increased the mechanical strength of platelet derived fibrin gel. The release studies focused on PDGF-BB and TGF-Î²1 and is an ongoing work, as well as the viability and proliferation of equine mesenchymal stem cells. Chapter 7 presents a thermosensitive triblock copolymer of p(HPMA-lac)-PEG-p(HPMA-lac) partly functionalized with methacrylic, acrylic and vinyl sulfone groups and cross-linked with thiolated hyaluronic acid (HA-SH) to form in situ jellying hydrogel. The controlled release of the glycopeptide vancomycin in phosphate buffer (PBS buffer, 150 mM, pH 7.4) at 37 Â°C was achieved for at least 5 days. Vancomycin, released from hydrogels, was tested for its antimicrobial activity on a gram positive bacterium, Staphylococcus Aureus, whose growth was inhibited by released vancomycin to the same extent vancomycin positive control solutions. Hydrogels with higher amount of hyaluronic acid released vancomycin with slower rates compared to hydrogels with lower polysaccharide content. An ionic interaction between the positive charged vancomycin and the negative hyaluronic acid was postulated and demonstrated. This hypothesis was confirmed by release studies in borate buffered saline (BBS buffer, 10 mM, pH 8.5) at 37Â°C, where the vancomycin is negative charged and was diffusionally and more rapidly released as compared to hydrogels at pH 7.4. Moreover, it was found that the encapsulation into hydrogel networks prevented vancomycin deamidation. The developed hydrogel system proved efficacious as a potential antibacterial depot to prevent orthopaedic implant associated infections. The topic of stimuli sensitive polymers inspired the research described in Chapter 8, where a novel dual responsive triblock copolymer system, sensitive to themperature and controllably degradable at acidic pH, composed of a central chain of polyethylene glycol (PEG) flanked by two identical chains with pendent cyclic ortho ester groups, was developed. The side chains give the acid sensitive behavior to the polymer while, the copolymerization with PEG middle block resulted in thermosensitive gel forming polymers. Several triblock copolymers with different molecular weight were prepared and formulated as physical hydrogel networks held together by secondary forces (mainly hydrophobic interactions). The molecular weights of the polymers and the amount of polymers into the hydrogels directly influenced the gelation times of the hydrogels. The perspective is to evaluate the swelling behavior of the hydrogels at different pHs and to stabilize the polymer network by the use of chemical cross-links to ensure higher mechanical strength. One examples of chemical cross-link is the Michael addition reaction among acrylic, methacrylic or vinyl sulfone groups and thiolated ones. In-vitro viability on mesenchimal cells will be investigated to assure the lack of toxicity and the potential feasibility of the hydrogels for in-vivo applications. Recently, several studies reported on nanohydrogels systems for biomedical applications. Nanohydrogels are hydrogel based nanoparticles in the nanometer scale from tens to hundreds of nanometers that combine the advantages of hydrogels and nanoparticles for drug formulation and delivery, which include controllable drug release, high stability in physiological media, distinct responsiveness to environmental factors such as pH and temperature, high cellular uptake due to the endocytosis mechanisms, long half-life in circulation by appropriate surface modification and drug targeting by conjugation of ligand onto the surface of hydrogel nanoparticles. Future development of the poly ortho esters-PEG- poly ortho esters triblock copolymers as nanohydrogel systems is an attractive perspective. In Chapter 9 a new adenosine conjugated polylactic-co-glycolic acid-polyethylene glycol PLGA-PEG block copolymer formulated in the form of solid nanoparticle for brain targeting was investigated and developed. The nanoparticle core consisted of hydrophobic PLGA shielded by an external corona of PEG surface decorated with adenosine, chosen as model ligand to specifically target nucleoside transporters present on the blood brain barrier (BBB). Adenosine was linked to the PEG via its amino group in position 6' of the adenine. Homogenous polymer distribution, particle size of 200 nm and spherical shape were assessed. Rhodamine 6G, a highly fluorescent dye was encapsulated into the polymeric nanoparticle and used for in vivo studies to determine circulation time and brain uptake of the nanoparticles upon intravenous administration in mice. Release studies from nanoparticles are ongoing. In vitro stability, cell viability, haemolysis and platelet aggregation of nanoparticles will be tested. Further development of this research topic will be the development of PLGA-PEG-Adenosine copolymers conjugated via the hydroxyl group in position 5' of adenosine ribose, which, according to structure activity relationship studies (SAR) is not involved in the binding of adenosine with transporters.
|Titolo:||Novel polymeric delivery systems for pharmaceutical and biomedical applications: from synthesis to in vivo feasibility studies|
|Data di pubblicazione:||20-mar-2015|
|Appare nelle tipologie:||Tesi di dottorato (Pregresso)|