Development and characterization of angiogenic microneedles

Angiogenesis plays a crucial role in tissue repair processes, ensuring the continuation of important cellular processes such as stem cell differentiation, cell proliferation, and migration during tissue repair. It also plays a significant role in reducing the impact of hypoxia conditions caused by tissue damage by facilitating oxygen transport to the tissue. Estrogen hormones are known to be particularly effective in angiogenesis during mammalian ovulation. In recent years, microneedle (MN) patches, due to their penetration-enhancing properties, are considered ideal platforms for transdermal application of various therapeutics. This thesis aims to develop angiogenic microneedles containing estrogen (estrone (E1), ß-estradiol (E2), estriol (E3)). For this purpose, microneedles were prepared by blending chitosan (CH) and poly(vinyl alcohol) (PVA) polymers with different crosslinkers (glutaraldehyde, Glu, tetraethyl orthosilicate (TEOS)), and their mechanical properties were compared. Additionally, the angiogenic effects and in vivo responses of microneedles containing different estrogen derivatives were investigated. The morphologies of the produced microneedles were examined using light and scanning electron microscopy (SEM). Microneedles containing E1 and E2 exhibited square-based pyramidal structures with approximately 650 μm needle height and a smooth needle structure, while the morphology of E3-containing microneedles was disrupted, and the number of smooth needles decreased. On the other hand, microneedles crosslinked with TEOS and Glu containing E1 and E2 showed similar morphological appearances, with smooth and regularly arranged needles. The mechanical characterization of microneedles crosslinked with TEOS was examined using a texture analyzer, revealing that the TEOS concentration and exposure time to glutaraldehyde vapor significantly affect the mechanical properties of the microneedles. TEOS-containing MN-T groups exhibited more flexibility and higher fracture strength compared to MN groups without TEOS, while E1 or E2 did not cause significant changes in mechanical properties. Furthermore, microneedles exposed to Glu vapor containing E1 or E2 became more fragile as the duration of Glu vapor application increased. When the water retention capacities of microneedles were examined, it was observed that TEOS-crosslinked microneedles did not have sufficient crosslinking, dissolved in water, and did not exhibit a stable structure. Microneedles exposed to Glu vapor were stable on the 8th day with a maximum mass loss of 1%. The water retention capacity of Glu-crosslinked microneedles decreased with increasing glutaraldehyde vapor exposure time. The water retention capacity of microneedles containing E2 and exposed to Glu vapor for 15, 30, and 60 minutes was measured as 88%, 49%, and 31%, respectively. The effects of estrogen-loaded MNs on angiogenesis were examined using in ovo CAM experiments. In all estrogen-loaded microneedles, statistically significantly higher vascularization was observed compared to non-loaded groups (p<0.05). When compared among themselves, the highest vascularization was observed in the E3-loaded microneedle group, followed by the E2 and E1 groups, respectively. However, no statistically significant difference in vascularization levels was observed among the E3, E2, and E1 groups (p>0.05). This result indicates that different estrogen derivatives have similar effects on angiogenesis. Ultimately, it was observed that microneedles had a safe dose of angiogenic effect. For the evaluation of the in vivo response of the developed microneedles, microneedles containing E2 and exposed to glutaraldehyde vapor were selected and implanted subcutaneously into rats. Macroscopic examination of the samples taken from the abdominal region 14 days after the in vivo implantation of MN patches revealed that the MN patches integrated with the tissue by being incorporated into the connective tissue. It was observed that both the control group and estrogen-loaded microneedles showed foreign body granulomatous reaction after implantation, which was considered an expected response. On the other hand, increased presence of inflammatory cells and vascularization were observed in estrogen-loaded microneedles compared to the control group. This finding indicates that estrogen can trigger angiogenic and inflammatory responses. Additionally, it was observed that estrogen-containing microneedles stimulated fibroblast activity and increased collagen fibril accumulation.