Clinical-grade Doxorubicin encapsulated low temperature sensitive liposomes (LTSLs) were combined with a clinical magnetic resonance-guided high intensity focused ultrasound (MR-HIFU) platform to investigate in-vivo image-guided drug delivery. Plasma pharmacokinetics were determined in 3 rabbits. Fifteen rabbits with Vx2 tumors within superficial thigh muscle were randomly assigned into three treatment groups: 1) free doxorubicin, 2) LTSL and 3) LTSL+MR-HIFU. For the LTSL+MR-HIFU group, mild hyperthermia (40–41°C) was applied to the tumors using an MR-HIFU system. Image-guided non-invasive hyperthermia was applied for a total of 30 min, completed within 1 hour after LTSL infusion. High-pressure liquid chromatography (HPLC) analysis of the harvested tumor and organ/tissue homogenates was performed to determine doxorubicin concentration. Fluorescence microscopy was performed to determine doxorubicin spatial distribution in the tumors. Sonication of Vx2 tumors resulted in accurate (mean=40.5±0.1°C) and spatially homogenous (SD=1.0°C) temperature control in the target region. LTSL+MR-HIFU resulted in significantly higher tumor doxorubicin concentrations (7.6- and 3.4-fold greater compared to free doxorubicin and LTSL respectively, p<0.05, Newman-Keuls). This improved tumor concentration was achieved despite heating <25% of the tumor volume. Free doxorubicin and LTSL treatments appeared to deliver more drug in the tumor periphery as compared to the tumor core. In contrast, LTSL+MR-HIFU treatment suggested an improved distribution with doxorubicin found in both the tumor periphery and core. Doxorubicin bio-distribution in non-tumor organs/tissues was fairly similar between treatment groups. This technique has potential for clinical translation as an image-guided method to deliver drug to a solid tumor.

Although abdominal aortic aneurysms (AAA) can be potentially stabilized by inhibiting inflammatory cell recruitment and their release of proteolytic enzymes, active AAA regression is not possible without regeneration of new elastic matrix structures. Unfortunately, postneonatal vascular smooth muscle cells (SMCs), healthy, and likely more so, diseased cells, poorly synthesize or remodel elastic fibers, impeding any effort directed at regenerative AAA treatment. Previously, we determined the eleastogenic benefits of oligomers (HA-o; 4-6 mers) of the glycosaminoglycan, hyaluronan (HA) and transforming growth factor-b1 (TGF-b1) to healthy SMCs. Since AAAs are often diagnosed only late in development when matrix disruption is severe, we now determine if elastogenic upregulation of SMCs from late-stage AAAs ( > 100% diameter increase) is possible. AAAs were induced by perfusion of rat infrarenal aortae with porcine pancreatic elastase. Elastic matrix degradation, vessel expansion (*120%), inflammatory cell infiltration, and enhanced activity of matrix-metalloproteases (MMPs) 2 and 9 resulted, paralleling human AAAs. Aneurysmal SMCs (EaRASMCs) maintained a diseased phenotype in 2D cell culture and exhibited patterns of gene expression different from healthy rat aortic SMCs (RASMCs). Relative to passage-matched healthy RASMCs, unstimulated EaRASMCs produced far less tropoelastin and matrix elastin. Exogenous TGF-b and HA-o (termed ''factors'') significantly decreased EaRASMC proliferation and enhanced tropoelastin synthesis, though only at the highest provided dose combination (20 mg/mL of HA-o, 10 ng/mL of TGF-b); despite such enhancement, tropoelastin amounts were only *40% of amounts synthesized by healthy RASMC cultures. Differently, elastic matrix synthesis was enhanced beyond amounts synthesized by healthy RASMCs (112%), even at lower doses of factors (2 mg/mL of HA-o and 5 ng/mL of TGF-b). The factors also enhanced elastic fiber deposition over untreated EaRASMC cultures and restored several genes whose expression was altered in EaRASMC cultures back to levels expressed by healthy RASMCs. However, the activity of MMPs 2 and 9 generated by EaRASMC cultures was unaffected by the factors/factor dose. The study confirms that SMCs from advanced AAAs can be elastogenically induced, although much higher doses of elastogenic factors are required for induction relative to healthy SMCs. Also, the factors do not appear to inhibit MMP activity, vital to preserve existing elastic matrix structures that serve as nucleation sites for new elastic fiber deposition. Thus, to enhance net accumulation of newly regenerated elastic matrix, toward possibly regressing AAAs, codelivery of MMP inhibitors may be necessitated.

Regression of abdominal aortic aneurysms (AAAs) via regeneration of new elastic matrix is constrained by poor elastin synthesis by adult vascular cells and absence of methods to stimulate the same. We recently showed hyaluronan oligomers (HA-o) and TGF-b1 (termed elastogenic factors) to enhance elastin synthesis and matrix formation by healthy rat aortic smooth muscle cells (RASMCs). We also determined that these factors could likewise elastogenically induce aneurysmal RASMCs isolated from periadventitial CaCl 2 -injury induced rat AAAs (aRASMCs). However, the factor doses should be increased for these diseased cell types, as even when induced, elastic matrix amounts are roughly one order of magnitude lower than those produced by healthy RASMCs. We presently investigate the dose-specific elastogenic effects of HA-o (0-20 mg/mL) and TGF-b1 (0-10 ng/mL) factors on aRASMCs and compare their phenotype and elastogenic responses to those of human AAA-derived SMCs (aHASMCs); we seek to determine whether aRASMCs are appropriate surrogate cell types to study in the context of inducing elastic matrix regeneration within human AAAs. The periadventitial CaCl 2 -injury model of AAAs exhibits many of the pathological characteristics of human AAAs, including similarities in terms of decreased SMC contractile activity, enhanced proliferation, and reduced elastogenic capacity of aneurysmal SMCs (relative to healthy SMCs) when isolated and expanded in culture. Both aRASMCs and aHASMCs can be elastogenically stimulated by HA-o and TGF-b1 and show broadly similar trends in their dosespecific responses to these factors. However, compared with aHASMCs, aRASMCs appear to be far less elastogenically inducible. This may be due to differences in maturity of the AAAs studied, with the CaCl 2 -injury induced aortal expansion barely qualifying as an aneurysm and the human AAA representing a more welldeveloped condition. Further study of SMCs from stage-matched CaCl 2 -injury induced rat aortal expansions and human AAAs will be necessary to more rigorously evaluate their basal and induced elastogenic responses.