Polylactic acid based materials and nanostructured multilayers for cardiovascular devices and wound healing

i. Poly(L-lactic acid), which is the current gold standard to fabricate bioresorbable stents, was modified by blending, with the aim to prepare a material with lower degradation time and improved mechanical properties. Binary blends of poly(L-lactic) acid (PLLA1: 80428 Da and PLLA2: 201790 Da) and poly(lactide-co-glycolide) (PLGA1) (LA:GA = 50:50 mol:mol; 32030 Da) with various compositions (100/0; 75/25: 50/50; 25/75; 0/100 wt./wt.) were prepared by solution casting. SEM analysis showed a biphasic morphology. Some degree of blend compatibility was suggested by the measured glass transition temperatures of blends with respect to pure components. Tensile mechanical properties evidenced higher compatibility for blends based on PLLA1 and for PLLA2/PLGA1 75/25 composition. Blends containing 75 wt.% PLGA were selected for further characterizations due to their superior mechanical properties. In vitro degradation tests showed PLGA rapid degradation within 2-4 weeks, leading to porous samples. A limited inflammatory reaction resulted from subcutaneous implantation of blend samples in Balb-c mice, suggesting their suitability for biomedical applications. Effect of macrophages adhesion and related cytokines release on endothelial cells (PAOEC) proliferation and migration was evaluated on PLLA2/PLGA1 75/25 blend compared to pure polymers. Slight differences were shown by the macrophages reaction when in contact with PLLA2, PLGA1 or PLLA2/PLGA1 75/25 blend. However, these differences showed to differently enhance endothelial cells behaviour in terms of healing from a wound scratch, in particular PLLA2/PLGA1 75/25 promoted a fast healing. To reduce the blend fast degradation rate, PLGA2 (LA:GA = 50:50 mol:mol, Mw = 77000 – 106000 Da) was used instead of PLGA1, in blends with PLLA1 and PLLA2, as it possesses a lower degradation rate than PLGA1. Binary blends were prepared with different compositions (PLLA1 (or PLLA2)/PLGA2 25/75; 50/50; 75/25). The fractured sections of blend film samples showed the presence of two phases, suggesting immiscibility. However, dispersed domains were of lower size as compared to the corresponding blends containing PLGA1, suggesting a superior compatibility degree with increasing the LA content of poly(lactic acid-co-glycolic acid) phase. The glass transition behaviour of PLLA2/PLGA2 compositions indicated some compatibility between the polymers. Tensile mechanical analysis showed that the maximum tensile strength (max) decreased significantly with increasing the amount of PLGA2 into the blends. The max of blend samples was higher than that predicted by the rule of mixtures for PLLA1/PLGA2 75/25 (max = 26 MPa) and PLLA2/PLGA2 75/25 blend (max = 39 MPa). Young’s modulus (E) behavior as function of amount of PLGA2 into the blends was similar to that of maximum tensile strength. The E of blend samples was higher than that predicted by the rule of mixtures only for PLLA1/PLGA2 75/25 composition (E = 435 MPa). The obtained results led to the selection of PLLA2/PLGA2 75/25 and PLLA1/PLGA2 75/25 blends for further characterisation, due to their superior compatibility and mechanical properties compared to the other blend compositions. To further improve the compatibility between the components in PLLA1/PLGA2 and PLLA2/PLGA2 blends (and consequently to increase the blend mechanical properties), the effect of three different compatibilizers - polycaprolactone (PCL), poly(D,L-lactide-co-caprolactone) (PCL-co-PLA) and poly(D,L-lactide-co-caprolactone) (PLA-co-PCL -co-PGA) – was compared. In compatibilized blends, a reduction of the size of the dispersed phase inclusions (< 1μm) and a less distinct interface between the phases was detected, as compared to uncompatibilised blends with the same weight ratio between the components. The Tg behaviour confirmed the increase of the compatibility between the phases when a compatibilizer was used, in particular the lower ΔTgs between blend components was obtained when 1% PCL was added to PLLA1/PLGA2 75/25 blend (ΔTg = 5.8 °C) and to PLLA2/PLGA2 75/25 (ΔTg = 6.2 °C) . Compatibilized PLLA2/PLGA2 75/25 blends showed better mechanical behavior with respect to compatibilized PLLA1/PLGA2 75/25 blends. Among, compatibilised PLLA2/PLGA2 75/25 blends, some improvements of the mechanical performance over uncompatibilised PLLA2/PLGA2 75/25 (E = 383 MPa; σmax = 40 MPa) blends were obtained for PLLA2/PLGA2 75/25 + 2% PCL (σmax =52.1 MPa, E= 382.5 MPa, ε=31.9%). Due to the small differences in mechanical performance of compatibilised and uncompatibilised PLLA2/PLGA2 75/25 blends, the uncompatibilised PLLA2/PLGA2 75/25 blend was selected as optimal blend composition for the development of bioresorbable stents or coatings for metal stents. In vitro degradation tests showed that the weight of PLLA2/PLGA2 75/25 samples did not vary significantly after 2-8 weeks incubation time in PBS. In vitro cells tests performed on PLLA2/PLGA2 75/25 blend using endothelial cells (PAOEC) for 72h days showed that cells adhered and proliferated well on them as compared to the control. Some preliminary tests were performed for the incorporation of Tacrolimus within PLLA2/PLGA2 75/25 blend, however the drug was not released after 30 days incubation in PBS, suggesting that the method for the incorporation of the drug into the blend must be optimised. Moreover, preliminary experiments were carried out for the fabrication of tubular conduits based on PLLA2/PLGA2 75/25 blend by a solution dipping – rotating mandrel technique, from which it is possible to fabricate stents by laser ablation. As a conclusion, in this thesis work, a suitable polymeric material for stent preparation was selected (PLLA2/PLGA2 75/25 blend). In the future this material will be used to prepare drug eluting stents by laser ablation of tubular constructs or drug eluting coating for stainless steel stents. Additional mechanical tests on model polymer stents will be necessary to assess PLLA2/PLGA2 75/25 blend suitability for stent applications. ii. To achieve the second aim of this thesis work, proper surface modification techniques were developed for both the polymer material and stainless steel substrates. Nanocoatings were obtained by applying the layer-by-layer (LbL) technique to coat PLLA2 and PLLA2/PLGA2 75/25 films and stainless steel plates (kindly purchased from C.I.D. s.r.l.) with the purpose to confer antithrombogenic properties to the selected material for the fabrication of bioabsorbable stents. Two different LbL deposition methods were developed and characterized: (i) method 1 used heparin (HE) and poly(diallyldimethylammonium chloride) (PDDA) as polyanion and polycation, respectively; (ii) method 2 used poly(styrene sulfonate) (PSS) as polyanion, PDDA as polycation and HE as the last deposited polyanion. A surface priming treatment was applied before depositing LbL coating. Heparin was selected because it has the highest negative charge density of any known biological macromolecule and anticoagulant properties; whereas PDDA and PSS were selected because are biocompatible and FDA approved. PLLA2 model films were pre-functionalized through aminolysis by using 1,6-hexamethylenediamine reagent with different parameter sets: reagent concentration (C), aminolysis treatment times (t) and temperatures (T). The optimal treatment parameters for PLLA2 cast films were selected on the basis of contact angle values and surface morphology and were found to be: C = 0.08 g/ml, t = 12 min and T = 37 °C. In details, PLLA2 contact angle decreased from 74 °C to about 65°C after aminolysis under the above parameters, while surface morphology was not altered. The effectiveness of the selected aminolysis treatment was confirmed both by XPS analysis, which showed the appearance of the N1s peak due to the formation of amino groups, and by a colorimetric method for amino groups quantification. The same aminolysis treatment was successfully performed on PLLA2/PLGA 75/25 films, as demonstrated by the obtained wettability (contact angle decreased from 81 °C to 70 °C ) and by amino groups quantification by a colorimetric method (NH2 concentration increased from 3 to 26 ng/mm2). LbL coatings were then deposited on aminolysed PLLA2/PLGA 75/25 film. The surface wettability of blend samples coated according to method 2 was higher compared to that of blend samples coated according to method 1: after the deposition of the 14th layer, static contact angle was around 50° and 67°, for samples modified according to method 2 and method 1,respectively. FTIR-ATR spectra of LbL samples coated according to method 2 demonstrated the presence of PSS/PDDA on the sample surface after the deposition of 14 layers . On the contrary, FTIR-ATR spectra did not vary after LbL deposition performed according to method 1. These results suggested that the treatment according to method 2 resulted in the formation of more homogeneous and thicker LbL coatings compared to method 1. XPS analysis and a colorimetric method employing toluidine blue (indicating the presence of HE and PSS) confirmed the successful deposition of the polyelectrolytes on the blend surface coated by method 2. The stainless steel (SS) plate surface was activated by incubation in an alkaline solution to expose hydroxyl groups and then priming treatment was performed using 3-aminopropyl triethoxysilane (APTES). APTES coating consisted of globular submicrometer domains and autofluorescence of APTES coating was indicative of the multilayered structure of APTES coatings on stainless steel substrates. XPS analysis demonstrated the presence of amino groups on the APTES-functionalised surface. LbL was performed on APTES modified stainless steel samples using both method 1 and method 2. FTIR-ATR spectra of coated samples evidenced the presence of typical absorption peaks of polyelectrolytes, confirming the successful LbL deposition for both methods. Static contact angle measurements showed that method 2 allowed the obtainment of surfaces with increasing hydrophilicity with increasing the layer number: after the deposition of 10 layers, static contact angle was lower than the value of pure polyelectrolytes. On the contrary, surface contact angle of samples modified using method 1 approached the contact angle values of polyelectrolytes after the deposition of 13-14 layers. This result suggested that method 2 allowed the obtainment of a more homogeneous LbL coating. Method 2 was thus selected also for stainless steel functionalisation. XPS analysis confirmed the successful deposition of the polyelectrolytes on the LbL coated sample surface. A colorimetric method employing toluidine blue was also used to detect the polyanion deposition. As a conclusion, in this work specific surface priming methods were developed for prefunctionalisation of stainless steel substrates and PLLA2/PLGA 75/25 samples. Moreover, a LbL method was optimized for the coating of pre-functionalised stainless steel and PLLA2/PLGA 75/25 samples. The aim of developed LbL coatings was that to expose HE to allow the obtainment of samples with anti-thrombogenic properties; moreover, HE could be exploited for the surface functionalisation with bioactive peptides, able to electrostatically interact with this glycosaminoglycan and exposing selective bioactive recognition sequences for interaction with receptors on endothelial cells. As an example, the KKKKKKSGSSGKCRRETAWAC peptide could be employed: CRETTAWAC sequence is able to favor EC adhesion while it hinders platelet attachment, KKKKKK is necessary for electrostatic interaction with HE and SGSSGK is a spacer sequence. This thesis work was focused on materials for cardiovascular applications, with reference to stenting. However, the possible transfer of the materials prepared and the experimental approach developed in vascular tissue engineering and wound healing was investigated. Therefore, model aminolysed PLLA2 films were prepared and coated by the LbL technique with suitable polyelectrolytes. The aim was that to prepare LbL coated PLLA2 substrates to be used in the form of porous tubular conduits for vascular tissue engineering or fibrous substrates for wound healing. For the preparation of fibrous substrates for wound healing, two different techniques were applied: wet-spinning and deposition by Pressure Assisted Microsyringe (PAM). The polyelectrolytes selected for the multilayered nanocoating were: heparin (HE), as polyanion, due to its high negative charge and ability to bind growth factors for wound healing or bioactive peptides favoring endothelisation (depending on the final application), and chitosan (CH), as polycation, due to its antimicrobial properties and biocompatibility. PLLA2 microfibers (50-90 μm) were prepared by the wet-spinning technique, using 7% (wt/v) PLLA2 solution concentration and 6mL/h spin rate. Microstructures were also prepared by PAM technique, extruding a polymeric solution (5% wt/v concentration) through a needle onto a glass slide, fabricating grids with regular geometry and hexagonal pores (diameter = 500 µm). Aminolysis treatment (0.08 g/mL, for 12 min at 37°C) was successfully performed on PLLA2 films and PLLA2 microfibers; however PAM microstructures underwent degradation and fragmentation after aminolysis due to their limited thickness (diameter of 5 μm). For this reason, they could not be used further for LbL coating. After aminolysis, film and microfiber samples were coated with 20 alternate layers of HE and CH. Surface wettability analysis showed alternate values of the contact angles as a function of the layer number, varying between the characteristic HE (40° C) and CH contact angles (60 °C). The presence of the two polyelectrolytes in the multilayer coating was also confirmed by XPS spectra, which showed the presence of the N1s peak, associated with both HE and CH deposition, and S2p peak, due to HE deposition. As a conclusion, HE/CH-LbL coated PLLA2 was found to be suitable for the fabrication of micrometric fibers by wet spinning, which in the future could be used to prepare porous scaffolds of desired shape (flat membranes or tubular constructs) by fiber compression molding in a suitable mold.