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Biomaterials

Biomaterials - Poster Session E

Poster O7 - Local Delivery of Sodium Butyrate to Attenuate Neointimal Hyperplasia Formation After Arterial Injury

Saturday, October 26, 2024
10:00 AM – 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  Peripheral vascular disease occurs as a result of atherosclerosis, where fatty plaques narrow the arterial lumen, impairing blood flow. In order to restore blood flow, a variety of non-invasive techniques, such as balloon angioplasty and stenting, or open surgery techniques, such as endarterectomy or graft implantation, are performed [1]. All of these techniques involve significant vascular manipulation, which can injure the vascular smooth muscle cells (VSMCs) in the medial layer of the artery. This leads the VSMCs to switch to a more synthetic, proliferative phenotype, and promotes VSMC migration into the intima, VSMC proliferation, and irregular deposition of ECM, leading to the re-narrowing of the new or re-opened lumen. This remodeling process, called neointimal hyperplasia, negatively impacts the long-term patency of vascular grafts and stented arteries [2] Butyrate, a short chain fatty acid derivative from dietary fiber digestion, has been a subject of interest in its role in cardiovascular disease. Butyrate in particular has been studied for its antiproliferative effects [3]. In vivo studies discovered that butyrate diet supplementation in vancomycin-treated mice decreased restenosis after femoral wire injury [4]. In vitro studies also established that butyrate prevented proliferation of synthetic VSMCs [5]. The hypothesis of this study is that sustained butyrate delivered locally via poly(lactic-co-glycolic) (PLGA) microspheres to injured arterial tissue will prevent the over-proliferation of VSMCs, reducing neointimal thickness and improving patency of arteries after surgical manipulation.

Materials and

Methods:  Microspheres were fabricated using a water-in-oil-in-water double emulsion process (Figure 1A). Briefly, PLGA dissolved in dichloromethane (DCM) was vortexed with sodium butyrate in distilled water to form an emulsion, then combined with 2% poly vinyl alcohol. This emulsion was added to water, stirred to allow sufficient microsphere assembly, and lyophilized after washing. Scanning electron microscopy (SEM) imaging and size distribution via light microscopy image analysis were used to characterize the microspheres. Butyrate encapsulation efficiency was determined by dissolving the microspheres in DCM, eluting the butyrate with distilled water, and reading the absorbance of the water phase at 230 nm. Release studies were also conducted by incubating the microspheres in PBS at 37℃, collecting the supernatant, and reading the absorbance at 230 nm. After microsphere characterization, the microspheres were piloted in a rat carotid artery balloon angioplasty model. Briefly, carotid balloon angioplasty was performed on 5-month-old Sprague Dawley rats to induce arterial injury and subsequent neointimal hyperplasia formation. 1 mg of microspheres was suspended in 50 µL of fibrinogen, and after arterial injury, an equal volume of thrombin was added. The whole solution was dropped onto the perivascular area of the injured artery, and allowed to gel for 5 minutes before closing the wound. Rats were monitored and subsequently euthanized at 2 weeks, and both the injured and contralateral carotids were collected. Carotids were fixed, processed, and embedded in paraffin. Carotids underwent H&E staining to assess neointimal morphology and thickness.

Results, Conclusions, and Discussions: Results and

Discussion: Light microscopy analysis of microsphere size showed that the fabrication process produced a wide range of microsphere sizes, with an average diameter of 50.69±42.66 µm in butyrate-loaded microspheres, and 79.27±56.75 µm in empty microspheres, and an encapsulation efficiency of 54% (Figure 1B). SEM imaging indicated that butyrate incorporation did not affect the outer surface of the microspheres (Figure 1C). Finally, initial findings of the drug release study showed a burst release of butyrate within the first few hours, and a total of 70% of loaded drug released over 2 weeks, and 75% of the loaded butyrate released over 4 weeks (Figure 1D). In situ images of the microsphere-loaded gel administered peri-vascularly are provided in Figure 2A. Morphometric analysis of H&E-stained carotid artery sections showed that the balloon injury successfully induced neointimal remodeling and thickening 2 weeks post angioplasty, indicated by the narrowed lumen of the left carotid compared to the right carotid. Neointimal remodeling was present in both control groups (injury alone and injury + vehicle control). In contrast, minimal neointimal hyperplasia was noted post localized butyrate treatment with the microsphere-loaded gels. Interestingly, profound periadventitial remodeling, indicated by increased adventitial thickening and abundant immune cell infiltration could be observed in the butyrate-treated carotid (Figure 2B). This can be attributed to the use of the fibrin gel as opposed to a synthetic gel, however additional animal models would be necessary in order to adequately characterize the effects of butyrate on neointimal hyperplasia.

Conclusions: Preliminary characterization studies indicate that encapsulation of butyrate within the microspheres was successful, and does not affect the formulation or surface of the microspheres. Additionally, butyrate was successfully released from the microspheres, with 70% of the encapsulated butyrate released after two weeks. Pilot in vivo data suggests potential neointima-mitigating efficacy of localized butyrate delivery, although future studies with a larger animal cohort are necessary to fully characterize the therapeutic efficacy and safety profiles of the proposed butyrate releasing microsphere-loaded gels.

Acknowledgements (Optional):  Citations:
1. Lee, T. & Haq, N. U. New Developments in Our Understanding of Neointimal Hyperplasia. Adv. Chronic Kidney Dis. 22, 431–437 (2015).
2. Melnik, T., Jordan, O., Corpataux, J.-M., Delie, F. & Saucy, F. Pharmacological prevention of intimal hyperplasia: A state-of-the-art review. Pharmacol. Ther. 235, 108157 (2022).
3. Mathew, O. P., Ranganna, K. & Milton, S. G. Involvement of the Antioxidant Effect and Anti-inflammatory Response in Butyrate-Inhibited Vascular Smooth Muscle Cell Proliferation. Pharmaceuticals 7, 1008–1027 (2014).
4. Ho, K. J. et al. Vancomycin treatment and butyrate supplementation modulate gut microbe composition and severity of neointimal hyperplasia after arterial injury. Physiol. Rep. 3, e12627 (2015).
5. Ranganna, K., Yatsu, F. M., Hayes, B. E. & Jayakumar, A. Butyrate inhibits proliferation-induced Proliferating Cell Nuclear Antigen expression (PCNA) in rat vascular smooth muscle cells.