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Drug Delivery

Drug Delivery - Poster Session E

Poster W16 - Doxycycline-loaded Extracellular Vesicles for Elastic Matrix Regenerative Repair

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

Introduction:  Abdominal aortic aneurysms (AAAs) are localized dilatations of the abdominal aorta that result from chronic overexpression of matrix metalloproteinases (MMPs) that break down the elastic matrix in the vessel wall. AAAs grow to ultimate rupture which is mostly fatal1. Current management of AAAs is primarily surgical or endovascular which tend to be risky for the mostly older AAA patients2. There is no established therapy to reverse matrix pathophysiology to arrest or slow AAA growth3. A critical limitation lies in the inherently poor ability of vascular smooth muscle cells (SMCs) to repair or regenerate elastic fibers when degraded. We have shown stem cell derived extracellular vesicles (EVs) to attenuate MMPs and stimulate elastic matrix assembly by aneurysmal SMCs4,5. We have also separately determined that the antibiotic drug, Doxycycline (Dox) at subclinical doses, not only attenuates MMPs but also stimulates new elastic fiber assembly6. Both effects of EVs and Dox are mediated by their attenuation of mitogen activated protein kinases (MAPKs) such as JNK and ERK1/2. To provide synergy of effect, in this work, we have sought to utilize EVs as nanovehicles for controlled release of loaded Dox for the purpose of elastic matrix regenerative repair for alleviation of AAAs.

Materials and

Methods:  EVs from bovine milk were extracted and isolated using differential ultracentrifugation method7. We standardized a technique to incorporate Dox into EVs using incubation and electroporation (GenePulser XcellTM) method and characterized Dox release kinetics over time. Atomic force microscopy (AFM) and differential light scattering (DLS) techniques were used to study pre- and post-loading characteristics (size, shape, loading efficacy) of EVs. Dox-EV cytotoxicity was assessed in vitro cultures of aneurysmal SMCs from an elastase injury rat AAA model using a Live/Dead assay® and MTT assay. Western blot for MMP2, JNK and ERK1/2 were carried out to assess protein expression levels post Dox/Dox-EV treatment.

Results, Conclusions, and Discussions: Results and

Discussion: We observed controlled and sustained release of Dox from EVs when incubated in PBS (pH 7.4); this involved an initial burst release followed by a prolonged and steady release over 72 h. AFM micrographs showed a minimal change in EV morphology post- loading with Dox, suggesting that their structural integrity was maintained. DLS measurements indicated a slight decrease in average particle size. The LIVE/DEAD and MTT assays indicated that Dox-EVs elicited lower cytotoxicity and promoted higher viability of aneurysmal SMCs vs. cultures treated with Dox alone. Western blot studies revealed Dox-EV treatment was more effective in downregulating MMP2, JNK and ERK1/2 expression, compared to free Dox, at equivalent Dox delivery doses. Overall, our ongoing study has shown that Dox-loaded EVs enable sustained and predictable drug release with maintained structural integrity, less cytotoxicity, with enhanced efficacy in downregulating MAPKs and downstream elastolytic MMPs compared to free Dox.

Conclusion: By leveraging EVs as functional nanocarriers and incorporating Dox with well-established anti-MMP effects, this study presents a promising approach for matrix remodeling in AAA. The positive outcomes observed in this study thus warrant additional investigations in in-vivo models, which we look forward to exploring in our future research.

Acknowledgements (Optional):