Introduction: Estradiol(E2), a female hormone produced by ovary, plays a vital role in the reproductive cycle and metabolism[1]. In recent epidemiology studies, cardiovascular disease rates are significantly different between premenopausal and postmenopausal women, suggesting the cardio-protective effects of E2[2]-[4]. However, estrogen replacement therapies in cardiovascular diseases were in dispute because: 1) poorly water-solubility of E2 limits its bioavailability; 2) long-term use of E2 will trigger the genomic action and hence induce a higher risk of cancer. Chitosan is a natural polysaccharide composed of β (1-4) linked N-acetyl-D-glucosamine residues[5],[6]. In our previous study, we found that using membrane-mimicking moiety phosphorylcholine (PC) to substitute certain amounts of primary amine in the chitosan backbone demonstrate prominent biocompatibility nature[7]. In present study, the objective is to covalently conjugate non-soluble E2 to the CH-PC polymer backbone using a prodrug strategy for the development of a novel extracellular matrix (ECM).
Materials and Methods: 17α-ethinylestradiol-benzoic acid was prepared as described in a following a known procedure[8]. Chitosan (CH) (molecular weight ranges from 50~100K) was purchased from Fisher Scientific Inc. A further deacetylation reaction led to a higher DDA (~98%). Phosphorylcholine-glyceraldehyde (PC-CHO) was prepared as described previously[9]. CH-PC-E2 samples were characterized by 1H NMR, UV/vis and FTIR/ATR spectra and were prepared in PBS. CH-PC-E2 hydrogel films were prepared using drop-casting approach and corresponding mechanical properties were analyzed by QCM-D. Cell culture was performed on EA.hy926 cell line.
Results and Discussion: Conjugation of E2 to CH-PC backbone was validated by 1H NMR spectrum (Figure 1A). Particularly, the aromatic protons of idobenzoic acid linker were shown at 8.23 and 7.92 ppm. In aqueous solution phase, CH-PC-E2 tends to form self-aggregates mainly due to hydrophobic E2 residues and hydrophilic CH-PC main chains (Rh value ~63nm, Figure 1B). By QCM-D analysis, the CH-PC-E2 demonstrated hydrogel nature after deposition to the interface due to the crosslinking via ionic pairing within PC groups and E2 microdomains (Figure 1C). The elastic modulus of CH-PC-E2 hydrogel films are ~0.7 MPa, in the similar magnitude of cardiac regeneration materials, such as Dacron (~0.6MPa)[10].
Figure 2 shows microscopic images (A-C) and corresponding fluorescence images (D-F) obtained from culturing EA.hy926 cells on the CH-PC-E2 films. The green regions reflect accumulated intercellular distribution of NO. By analyzing the fluorescence intensities, we found that CH-PC-E2 film significantly stimulated higher NO release (1.65 times) compared to the CH-PC films. Moreover, the increment was diminished in the cells pre-treated with inhibitor ICI 182,780.
Conclusions: The present research aims to covalently graft non-soluble E2 to CH-PC polymer backbone and develop formulation methods in depth. It also successfully demonstrates that the protective effect of CH-PC-E2 hydrogel films on cardiovascular system through investigation of NO stimulation. Biomedical applications of CH-PC-E2 as a novel platform for the cardiovascular regenerative medicine, e.g. to enhance the reendothelialization process or promotes the recovery of the endothelium, will be carried out in future.
This work was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC) and the World Premier International Research Center Initiative (WPI) in Japan.; Baowen Qi acknowledges the financial support by the NIMS Internship Program 2011 and 2013.
References:
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