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Multifunctional nanoparticles for doxycycline delivery towards localized elastic matrix stabilization and regenerative repair.

ABSTRACT: Abdominal aortic aneurysms (AAAs) are abnormal expansions of the aortic wall, typically characterized by chronic up-regulation of matrix metalloproteases (MMPs)-2 and -9. These MMPs degrade elastin and elastic matrix within the aortic wall, leading to a progressive loss of elasticity of the abdominal aorta as the condition progresses. Doxycycline (DOX) is a tetracycline-based antibiotic which has shown significant promise in delaying and slowing the growth of AAAs in both clinical studies and animal models. However, it has been found to inhibit elastic matrix deposition by vascular cells at dosages in the ?g ml(-1) range, which is typically observed in the circulation, in addition to systemic side effects, following oral dosage. In this paper, we describe the development of DOX-loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles for localized, controlled and sustained DOX delivery towards AAA therapy. Further, we demonstrate that surface functionalization of these nanoparticles with cationic amphiphiles not only imparts them with a positive charge for potentially enhanced aortic uptake, but also enables enhanced elastin binding via hydrophobic interactions, as well as up-regulating activity of the elastin crosslinking enzyme lysyl oxidase. In addition to the DOX released from the nanoparticles being effective in inhibiting MMP-2 production and activity, we also demonstrate that surface functionalization of the nanoparticles cationic amphiphiles may also play a role in MMP-2 inhibition via (i) electrostatic interactions with negatively charged residues in the active-site of MMP-2 or (ii) steric blockade of the active site on account of the presence of two dodecyl chains in the DMAB molecule. Thus, in addition to enhanced aortic uptake and retention illustrated in studies by other groups, we have demonstrated that cationic functionalization of PLGA nanoparticles enhances elastogenic outcomes by targeted binding to elastin, as well as their potential to inhibit elastolysis. These results establish their multifunctionality as a localized delivery system for AAA therapy. Overall, this delivery system has the potential to enhance regenerative outcomes at sites of proteolytic matrix disruption/degradation by enabling targeted, controlled and long-term release of therapeutic agents.

SUBMITTER: Sivaraman B

PROVIDER: S-EPMC3622827 | biostudies-literature | 2013 May

REPOSITORIES: biostudies-literature

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Multifunctional nanoparticles for doxycycline delivery towards localized elastic matrix stabilization and regenerative repair.

Sivaraman Balakrishnan B Ramamurthi Anand A

Acta biomaterialia 20130129 5

Abdominal aortic aneurysms (AAAs) are abnormal expansions of the aortic wall, typically characterized by chronic up-regulation of matrix metalloproteases (MMPs)-2 and -9. These MMPs degrade elastin and elastic matrix within the aortic wall, leading to a progressive loss of elasticity of the abdominal aorta as the condition progresses. Doxycycline (DOX) is a tetracycline-based antibiotic which has shown significant promise in delaying and slowing the growth of AAAs in both clinical studies and an ...[more]

PMID: 23376127

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Project description:Elastic fibers do not naturally regenerate in many proteolytic disorders, such as in abdominal aortic aneurysms, and prevent restoration of tissue homeostasis. We have shown drug-based attenuation of the stress-activated protein kinase, JNK-2 to stimulate elastic matrix neoassembly and to attenuate cellular proteolytic activity. We now investigate if JNK2 gene knockdown with small interfering RNA (siRNA) provides greater specificity of action and improved regenerative/antiproteolytic outcomes in a proteolytic injury culture model of rat aneurysmal smooth muscle cells (EaRASMCs). A siRNA dose of 12.5 nM delivered with a transfection reagent significantly enhanced downstream elastic fiber assembly and maturation versus untreated EaRASMC cultures. The optimal siRNA dose was also delivered as a complex with a polymeric transfection vector, polyethyleneimine (PEI) in preparation for future in vivo delivery. Linear 25 kDa PEI-siRNA (5:1 molar ratio of amine to phosphate) and linear 40 kDa PEI-siRNA (2.5:1 ratio) were effective in downregulating the JNK2 gene, and significantly increasing expression of elastic fiber assembly proteins, and decreases in elastolytic matrix metalloprotease-2 versus treatment controls to significantly increase mature elastic fiber assembly. The current work has identified siRNA dosing and siRNA-PEI complexing conditions that are safe and efficient in stimulating processes contributing to improved elastic matrix neoassembly via JNK2 gene knockdown. The results represent a mechanistic basis of a broader therapeutic approach to reverse elastic matrix pathophysiology in tissue disorders involving aberrations of elastic matrix homeostasis, such as in aortic aneurysms. Impact statement The elastic matrix and elastic fibers are key components of the structural extracellular matrix of elastic tissues and are essential to their stretch and recoil and to maintain healthy cell phenotype. Regeneration and repair of elastic matrix is naturally poor and impaired and is an unresolved challenge in tissue engineering. In this work, we investigate a novel gene silencing approach based on inhibiting the JNK2 gene, which provides significant downstream benefits to elastic fiber assembly and maturation. Combined with novel delivery strategies such as nanoparticles, we expect our approach to effect in situ elastic matrix repair in the future.

Project description:Abdominal aortic aneurysms (AAA) are localized expansions of the abdominal aorta that develop due to chronic proteolytic disruption of the structural extracellular matrix (ECM) components (elastin and collagen) within the aorta wall. Major limitations in arresting or reversing AAAs lie in naturally poor and aberrant regeneration and repair of elastic matrix structures in the aorta wall. Bone marrow derived mesenchymal stem cells (BM-MSCs) have emerged as a promising regenerative tool and their therapeutic effects are also known to be effected through their paracrine secretions. Extracellular vesicles (EVs) present in these secretions have emerged as critical cellular component in facilitating many therapeutic benefits of MSCs. EV treatment is thus potentially appealing as a stem cell-inspired cell-free approach to avoid possible phenotypic plasticity of MSCs in vivo. In this study, we investigated the thus far unknown effects of BM-MSC derived EVs on vascular elastic matrix repair in the context of AAA treatment. EVs isolated from BM-MSC source were characterized and their pro-regenerative and their anti-proteolytic effects were evaluated on our established in vitro experimental conditions derived from AAA rat model. Our studies revealed the efficacy of BM-MSC derived EVs in attenuating the proteolytic activity and also in imparting elastic matrix regenerative benefits under aneurysmal environment. Interestingly, compared to cell culture conditioned media (CCM), EVs demonstrated superior regenerative and anti-proteolytic benefits in a proteolytic injury culture model of AAA. From these studies, it appears that EVs derived from BM-MSCs could be beneficial in undertaking a reparative effort in AAA induced degeneration of vascular tissue. Statement of Significance Abdominal aortic aneurysms (AAAs) are localized, rupture-prone expansions of the aorta which result from loss of wall flexibility due to enzymatic breakdown of elastic fibers. There are no established alternatives to surgery, which possess high risk for the mostly elderly patients. Our previous studies have established the elastic regenerative and reparative effect of cell culture secretions derived from adult stem cell source. In this study, we propose to isolate extracellular vesicles (exosomes) from these secretions and evaluate their regenerative benefits in AAA smooth muscle cell culture model. This simple and innovative treatment approach has the potential to arrest or reverse AAA growth to rupture, not possible so far.

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Multifunctional nanoparticles for doxycycline delivery towards localized elastic matrix stabilization and regenerative repair.

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Multifunctional nanoparticles for doxycycline delivery towards localized elastic matrix stabilization and regenerative repair.

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