Background/Objectives: RNA interference (RNAi) is a promising strategy for mitigating diseases at the molecular level. However, RNAi is limited by its instability in biological fluids and impermeability to cellular membranes. In response, our lab has previously patented a non-ionizable lipid nanoparticle (LNP) platform (R8-PLP) for RNAi therapeutic delivery. This formulation incorporates 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG) to improve particle stability and drug retention. However, long-anchored PEGylated lipids like DSPE-PEG may impair internalization and stimulate immune responses. The literature suggests substituting short-anchored PEGylated-lipids like 1,2-dimyristoyl-rac-glycero-3-[methoxy(polyethylene glycol)-2000] (DMG-PEG) to attenuate these effects. Here, we evaluated whether substituting DMG-PEG for DSPE-PEG in our R8-PLP would improve in vitro cellular delivery and gene transfection without compromising in vitro critical quality attributes (CQAs) or increasing cytotoxicity. Methods: CQAs [encapsulation efficiency (EE%), particle size (nm), homogeneity (polydispersity index; PDI), and membrane zeta-potential] were assessed at assembly and after storage for up to 28 days at 4 °C. Additionally, in-serum stability at 4 °C and serum release kinetics at 37 °C were assessed. Human aortic smooth muscle cells (HASMCs) were treated with R8-PLPs and analyzed for cellular uptake (fluorometry), cytotoxicity (LIVE/DEAD stain), and gene modulation (qPCR). Results: DMG-PEG incorporation at variable mol% did not alter R8-PLP size, homogeneity, or siRNA EE% at assembly or after long-term storage, but did accelerate siRNA release kinetic profiles compared to DSPE-PEG controls. DMG-PEG substitution enhanced cellular uptake compared to DSPE-PEG R8-PLPs without increasing cytotoxicity. DMG-PEG incorporation also achieved significant silencing versus non-treated controls but did not improve gene silencing compared to DSPE-PEG R8-PLPs. Conclusions: Thus, DMG-PEG substitution did not enhance R8-PLP in vitro gene modulation efficacy despite improving cellular uptake and maintaining CQAs.

This article presents the rationale, challenges, and adaptive strategies employed during the initiation and execution of the arteriovenous (AV) access trialda multicenter randomized controlled trial (RCT) comparing AV fistulas and AV grafts for hemodialysis in older adults with major comorbidities. Motivated by shifts in epidemiologic landscapes and evolving guidelines moving away from a fistula-first approach and to more patient-centric approaches, the objective of this randomized controlled trial was to fill critical knowledge gaps in determining the optimal vascular access for this complex patient population. We outline the challenges encountered in patient recruitment along with measures employed to overcome these obstacles in recruitment. We emphasize the pivotal role of continuous research in overcoming these challenges, underscoring its necessity to achieve a thorough comprehension of optimal vascular access strategies for this complex patient population. (JVS-Vascular Insights 2024;2:100108.)

This article presents the rationale, challenges, and adaptive strategies employed during the initiation and execution of the AV Access trial—a multicenter randomized controlled trial (RCT) comparing arteriovenous fistulas (AVFs) and arteriovenous grafts (AVGs) for hemodialysis in older adults with major comorbidities. Motivated by shifts in epidemiologic landscapes and evolving guidelines moving away from a Fistula First approach and to more patient-centric approaches, the objective of this RCT is to fill critical knowledge gaps in determining the optimal vascular access for this complex patient population. We outline the challenges encountered in patient recruitment along with measures employed to overcome these obstacles in recruitment. We emphasize the pivotal role of continuous research in overcoming these challenges, underscoring its necessity to achieve a thorough comprehension of optimal vascular access strategies for this complex patient population.

Objectives Dialysis access creation is a common outpatient procedure that can be completed using general, regional, or local anesthetic techniques. There are few endorsed guidelines regarding opioid-based pain control following fistula creation. The purpose of this study was to determine whether utilization of regional anesthesia (RA) is associated with the decreased use of narcotics postoperatively. Methods We performed a prospective cohort study including all patients undergoing arteriovenous fistula creation with one vascular surgeon from August 2019 to February 2020. Patients were selected for regional versus general anesthesia. Selection for anesthesia type was determined by the primary anesthesiologist. Patients selected for RA underwent supraclavicular brachial plexus block with 30 cm(3) of 0.5% ropivacaine. Patients were seen in clinic follow-up and completed a questionnaire regarding their postoperative opiate use and pain control. Results In the study period, 52 patients underwent arteriovenous fistula creation and completed the follow-up questionnaire. Forty patients received RA. Seventy-five percent of patients sent home with a narcotic prescription filled the prescription. There was a significant difference in postoperative opioid use between the two study groups. Patients who received regional block took on average 3.3 pills totaling 16.5 morphine milligram equivalents, whereas patients who received general anesthesia took on average 6.64 pills totaling 33.2 morphine milligram equivalents (P = 0.04). Conclusions Morbidity and mortality related to opiate use continues to be a public health issue in the United States. This study demonstrates that regional anesthetic techniques in comparison to general anesthesia can result in a significant decrease in postoperative opiate consumption.

Vascular interventions result in the disruption of the tunica intima and the exposure of sub-endothelial matrix proteins. Nanoparticles designed to bind to these exposed matrices could provide targeted drug delivery systems aimed at inhibiting dysfunctional vascular remodeling and improving intervention outcomes. Here, we present the progress in the development of targeted liposomal nanocarriers designed for preferential collagen IV binding under simulated static vascular flow conditions. PEGylated liposomes (PLPs), previously established as effective delivery systems in vascular cells types, served as non-targeting controls. Collagen-targeting liposomes (CT-PLPs) were formed by conjugating established collagen-binding peptides to modified lipid heads via click chemistry (CTL), and inserting them at varying mol% either at the time of PLP assembly or via micellar transfer. All groups included fluorescently labeled lipid species for imaging and quantification. Liposomes were exposed to collagen IV matrices statically or via hemodynamic flow, and binding was measured via fluorometric analyses. CT-PLPs formed with 5 mol% CTL at the time of assembly demonstrated the highest binding affinity to collagen IV under static conditions, while maintaining a nanoparticle characterization profile of ~50 nm size and a homogeneity polydispersity index (PDI) of ~0.2 favorable for clinical translation. When liposomes were exposed to collagen matrices within a pressurized flow system, empirically defined CT-PLPs demonstrated significant binding at shear stresses mimetic of physiological through pathological conditions in both the venous and arterial architectures. Furthermore, when human saphenous vein explants were perfused with liposomes within a closed bioreactor system, CT-PLPs demonstrated significant ex vivo binding to diseased vascular tissue. Ongoing studies aim to further develop CT-PLPs for controlled targeting in a rodent model of vascular injury. The CT-PLP nanocarriers established here show promise as the framework for a spatially controlled delivery platform for future application in targeted vascular therapeutics.

Lipid nanoparticles have become increasingly popular delivery platforms in the field of gene therapy, but bench-to-bedside success has been limited. Many liposomal gene vectors are comprised of synthetic cationic lipids, which are associated with lipid-induced cytotoxicity and immunogenicity. Natural, non-cationic PEGylated liposomes (PLPs) demonstrate favorable biocompatibility profiles but are not considered viable gene delivery vehicles due to inefficient nucleic acid loading and reduced cellular uptake. PLPs can be modified with cell-penetrating peptides (CPPs) to enhance the intracellular delivery of liposomal cargo but encapsulate leakage upon CPP-PLP assembly is problematic. Here, we aimed to identify parameters that overcome these performance barriers by incorporating nucleic acid condensers during CPP-PLP assembly and screening variable ethanol injection parameters for optimization. CPP-PLPs were formed with R8-amphiphiles via pre-insertion, post-insertion and post-conjugation techniques and liposomes were characterized for size, surface charge, homogeneity, siRNA encapsulation efficiency and retention and cell associative properties. Herein we demonstrate that pre-insertion of stearylated R8 into PLPs is an efficient method to produce non-cationic CPP-PLPs and we provide additional assembly parameter specifications for a modified ethanol injection technique that is optimized for siRNA encapsulation/retention and enhanced cell association. This assembly technique could provide improved clinical translation of liposomal based gene therapy applications.

Distal exposure of the internal carotid artery is technically challenging and often inaccessible from a standard lateral neck incision. The double mandibular osteotomy (DMO) technique is described as an adjunct for vascular exposure. To date, no case series within the literature explores the technical and functional outcomes. The purpose of this study is to report our institutional results for exposure of skull base carotid artery pathology.

Background: Anecdotal experience demonstrates the existence of patients with superiorly located carotid stenosis, neoplasms, or aneurysms where the mandible obstructs effective surgical access using standard techniques. As carotid pathology extends anatomically beyond the limits of standard operative technique, additional exposure becomes paramount to safely and effectively address the lesion. Double mandibular osteotomy (DMO) is one of several techniques to obtain additional exposure to high-carotid pathology; however, there is no large series to address the outcomes of patients undergoing this procedure. Methods: A retrospective case series was performed for all patients undergoing surgery for carotid pathology from 2011-2019 that could not be approached with standard cervical incision. The primary predictor variable was higheanatomic carotid pathology necessitating DMO. The primary outcome variable was early and late complications sustained by patients. Results: Fifteen patients met study criteria and underwent 16 DMOs to access high-carotid pathology including carotid stenosis (n = 8 patients), carotid aneurysm (n = 2 patients), and carotid body tumor (n = 8 patients). Two patients had dual ipsilateral pathology with one patient having both carotid artery stenosis and aneurysm, and the other patient diagnosed with carotid artery stenosis and carotid body tumor. One patient had bilateral carotid artery stenosis, each requiring high anatomic exposure for treatment. Early complications occurred in 8 patients. Five patients experienced significant dysphagia requiring enteral feeding, and 2 patients developed malocclusion directly related to the double mandibular osteotomy. One patient experienced contralateral cortical watershed infarcts. Late complications included one patient developing osteomyelitis of the mandible, and this patient also developed distal mandibular segment screw exposure. The comparison of the outcome groups for categorical predictor variables using Fisher's exact test detected no statistically significant differences for gender, hypertension, hyperlipidemia, type 2 diabetes, chronic obstructive pulmonary disease, tobacco use, chronic kidney disease, or cerebrovascular disease. For the continuous variable comparisons, independent-samples t-tests detected no difference between the complication groups for age, operative time, or years of follow-up. No significant differences were found between the groups for body mass index or intraoperative blood loss. Conclusions: The double mandibular osteotomy provides excellent exposure and surgical access to the distal internal carotid artery for repair of vascular pathology with acceptable outcomes and long-term complications compared with previously reported techniques. Because of the early complications realized with the DMO, we recommend the procedure for symptomatic patients with a high risk of failing medical therapy alone and not appropriate for endovascular treatment as well as those patients with tumors requiring surgical intervention.

Background: Intimal hyperplasia (IH) is the most common indicator for secondary intervention in peripheral vascular disease. Matrix metalloproteinases (MMPs) play a role in IH development due to their degradation of the extracellular matrix. Doxycycline (Doxy), a member of the tetracycline family of antibiotics, is a potent MMP inhibitor. We have previously shown that Doxy inhibits MMP activity and vascular smooth muscle cell migration in vitro. We hypothesized that Doxy would decrease MMP activity in vivo and inhibit the development of IH in a rodent model of vascular injury. Methods and Results: Doxy (400 mg/pellet) was delivered by a slow-release pellet implanted 3 days prior to or at the time of balloon angioplasty (BA) of the common carotid artery in female rats. At 14 days post-BA, intima-to-media (I:M) ratios were 0.77 +/- 0.21 and 1.04 +/- 0.32 in the Doxy treated groups, respectively, compared to 1.25 +/- 0.26 in the control group (P = not significant; n = 3). Additionally, the tested dose of Doxy in either group had no inhibitory effect on membrane type 1-MMP or MMP-2 tissue levels, as measured by immunohistochemistry, or on systemic levels of MMP, as measured by total MMP serum levels using enzyme-linked immunosorbent assay. At 14 days post-BA, VSMC proliferation in the injured artery was increased to Doxy treatment prior to and at the time of surgery (23.5 +/- 3.4 and 27.2 +/- 3.9%, respectively), compared to control (11.4 +/- 0.4%; n = 3), as measured by proliferating cellular nuclear antigen immunostaining. Conclusions: In our in vivo model of vascular injury, systemic Doxy administration prior to or at the time of vascular injury does not significantly hinder the progression of IH development. Additional doses and routes of administration could be examined in order to correlate therapeutic serum levels of Doxy with effective MMP inhibition in serum and arterial tissue. However, alternative drug delivery systems are needed in order to optimize therapeutic administration of targeted MMP inhibitors for the prevention of IH development.

The polyherbal blend Zyflamend™ has been shown to have anti-inflammatory properties and attenuate inflammatory-modulated pathologies. Fish oils have also been shown to have cardioprotective properties. However, the beneficial effects of their combination have not been investigated. Intimal hyperplasia (IH), a pathological remodeling response of a vessel to injury, is heavily regulated by an immune-mediated reaction. The objective of this study was to determine if dietary supplementation with Zyflamend and/or Wholemega could affect inflammatory-dependent vascular remodeling mechanisms when provided at human equivalent doses. Based on their anti-inflammatory properties and protective benefits demonstrated in previous pre-clinical studies, we hypothesized administration of these supplements would prevent IH in an animal model of vascular injury. The diets of aged male rats were supplemented with human equivalent doses of Zyflamend (Zyf) and/or Wholemega (WMega) or placebo (Plac) for 1wk prior to balloon angioplasty (BA)-induced injury of the left carotid artery. At 28d post-injury morphometric analysis of carotid tissue revealed IH was decreased in Zyf + WMega animals compared to placebo, while Zyf or WMega independently had no significant effect. Serum cytokine screening indicated injury-induced interleukin family isoforms, interferon-γ, and macrophage inflammatory proteins were downregulated by Zyf + WMega. Immunohistochemical staining for monocyte/macrophage phenotypic markers revealed that while overall monocyte/macrophage vessel infiltration was not affected, Zyf + WMega limited the alternative differentiation of M2 macrophages and reduced the presence of myofibroblasts in the injured vessel wall. In summary, dietary supplementation with Zyf + WMega attenuated the acute inflammatory response following vascular injury and inhibited IH development in vivo.

Objectives: Nanoparticles designed to localize to areas of vascular perturbations could provide a targeted delivery system for gene therapeutics aimed at improving intervention outcomes. PEGylated liposomes (PLPs) are potential delivery vectors, and PEG residues provide a scaffold for multifunctional surface modifications. We have previously developed PLPs functionalized with collagen targeting peptides (CTP-PLPs) that preferentially bind collagen IV, an abundant sub-endothelial matrix protein exposed during vascular intervention. Here we present dual ligand-modified liposomes with multifunctional potential, building on our CTP-PLP platform by simultaneously incorporating cell-penetrating peptides (R8) to enhance liposomal cell association and nucleic acid delivery. Methods: PLPs were formed with DOPC-PEG + 30mol% cholesterol + 0.1mol% Rhodamine-DOPE, and siRNA loaded via EtOH injection. CTP-PLPs were formed as PLPs + 5mol% CTP-modified-DSPE, as previously reported. Dual-ligand modified liposomes (R8-CTP-PLPs) incorporated CTP concurrently with 5-10mol% sterylated-R8. Vascular smooth muscle cells (VSMC) were treated for 24hr at 100uM lipid for cellular association, quantified via fluorimetry, or at 400nM siRNA encapsulate for gene silencing, quantified via qPCR. Results: While CTP-PLPs increased VSMC association by 1.6-fold compared to PLP, R8-CTP-PLPs increased association by 3.6-fold (5mol%R8) and 10.4-fold (10mol%R8; Fig1A). Likewise, siRNA delivery via R8-CTP-PLPs resulted in enhanced gene silencing compared to CTP-PLPs and non-treated controls (Fig1B). Conclusions: R8-CTP-PLP nanocarriers established here show promise as the framework for a spatially controlled drug delivery platform for targeted vascular therapeutics. Ongoing studies aim to elucidate R8-CTL-PLP vessel wall binding and targeted gene silencing in a dynamic living environment via ex vivo vessel perfusion and in vivo rodent models of vascular injury.

Vascular interventions inherently result in disruption of the tunica intima and exposure of subendothelial matrix proteins. Spatially controlled nanoparticles designed to colocalize to these exposed matrices could provide a targeted drug delivery system aimed at inhibiting dysfunctional vascular remodeling and improving intervention outcomes. We have previously reported the development of a surface-modified liposomal platform designed to preferentially bind collagen type IV, an abundant subendothelial matrix protein, in a static in vitro environment. Here we present our progress in this discovery-driven liposomal nanocarrier system by validating its collagen IV binding affinity under simulated physiological and pathologic vascular flow conditions. Nontargeting PEGylated liposomes (PLP) were formed with bulk DOPC-PEG + 30 mol% cholesterol + 0.1 mol% Rhodamine-DOPE. Collagen-targeting liposomes (CT-PLP) were formed by reacting DSPE-PEG-DBCO to previously established collagen binding peptides (CBP), via copper-free click chemistry, and inserting 5 mol% CBP-modified lipids to base PLPs at lipid hydration. Collagen IV matrices were dried at 3 μg/cm2, and hemodynamic liposome binding was assayed by live fluorescence microscopy over 60 minutes simulated flow at 5 to 115 dynes-s/cm2 using a closed parallel-plate flow chamber. Liposome binding was quantified by mean fluorescent intensity of bound Rhodamine-labeled lipid normalized to background. Under continuous flow, CT-PLPs demonstrated increased binding to collagen matrices over time at all tested sheer stress conditions (Fig, A). After 60 minutes of flow, CT-PLPs demonstrated remarkable binding under all conditions while nontargeted PLP control binding was negligible (Fig, B). CT-PLPs demonstrated an affinity for collagen IV binding under simulated hemodynamic flow at sheer stress conditions ranging from venous physiological to pathologic flow and arterial physiological to pathologic flow. CT-PLP nanocarriers established here show promise as the framework for a spatially controlled drug delivery platform for future application in targeted vascular therapeutics. Ongoing studies aim to demonstrate CT-PLP binding capacity in situ via ex vivo human saphenous vein and femoral artery perfusion under normal and elevated sheer stress.

Systematic literature reviews remain inconclusive on defining optimal thrombectomy methods to treat occluded hemodialysis access. Recent changes in Centers for Medicare and Medicaid Services bundling/reimbursement for endovascular thrombectomy procedures have led to renewed interest in delivering cost-effective care. Percutaneous mechanical rotational thrombectomy (RT) devices (Cleaner XT; Argon Medical Devices, Plano, Tex) have been developed to compete with traditional pharmacomechanical thrombectomy (PMT) devices (AngioJet with tissue plasminogen activator; Boston Scientific, Marlborough, Mass) for percutaneous thrombectomy procedures. The objective of this study was to evaluate clinical and economic factors of open surgical thrombectomy (OST) compared with contemporary percutaneous mechanical thrombectomy (PERC) devices for thrombosed hemodialysis vascular access. Data analysis was performed from a tertiary care hospital prospectively maintained hemodialysis database between 2012 and 2016. All patients undergoing outpatient thrombectomy for occluded hemodialysis access were captured. Clinical end points included arteriovenous access type, thrombectomy method, operative time, estimated blood loss, and intervention success rates. Financial end points included direct cost, total cost, operating margin, and total margin. Additional subgroup analysis of the percutaneous thrombectomy group was performed to compare pharmacomechanical with rotational techniques. Analysis using univariate and multivariate measures was performed with significance assigned as P < .05. Between 2012 and 2016, there were 1072 hemodialysis arteriovenous accesses (88.3% arteriovenous fistula [n = 947]; 11.7% arteriovenous graft [n = 125]) created; 391 patients required thrombectomy (OST, n = 64; PERC, n = 327), and 670 thrombectomy procedures were performed (OST, n = 71; PERC, n = 599). Arteriovenous graft was the predominant type of access requiring thrombectomy (77% arteriovenous graft [n = 506] vs 23% arteriovenous fistula [n = 151]; OST, 97.2%; PERC, 74.5%; P = NS). Most accesses were in the upper extremity (upper extremity, 90.7% [n = 592]; lower extremity, 9.3% [n = 60]; OST, 87.5%; PERC, 91.2%; P = NS). Despite that more diabetic patients were treated with PERC, no other statistical difference between groups was observed in gender or comorbidities, including hypertension, coronary artery disease, congestive heart failure, tobacco abuse, and hypercoagulable disorder. Concurrent intervention with outflow venous angioplasty was the most common single intervention (52.7%). Each thrombectomy method had high technical success rates (OST, 95.8%; PERC, 95.7%; P = NS). Subgroup analysis between percutaneous methods (42.7% RT [n = 256], 53.1% PMT [n = 318], 4.2% RT + PMT [n = 25]) showed that RT therapy had lower estimated blood loss (P < .001) and shorter operative time (RT, 48 minutes; PMT, 52 minutes; P < .03). The highest successful thrombectomy rate was RT (97.7% RT vs 94.7%; P < .07). Financially, all economic end points, specifically direct supply cost (OST, $3334; RT, $3241; PMT, $3529; RT + PMT, $4414; P < .001), total cost (OST, $7220; RT, $6291; PMT, $7317; RT + PMT, $9735; P = .001), operating margin (OST, $1331; RT, $−360; PMT, $−311; RT + PMT, $−1154; P < .001), and total margin (OST, $−894; RT, $−2200; PMT, $−2902; RT + PMT, $−4596; P < .001), favored OST therapy. With equal success rates between hemodialysis access thrombectomy methods, RT has the lowest operating and total cost, whereas OST has significantly better profit margins in a hospital-based setting. All thrombectomy procedure types had a negative profit margin in an outpatient hospital-based setting. PERC using RT decreases operative time, blood loss, and cost compared with PMT. Combination percutaneous thrombectomy leads to the greatest financial loss compared with any single therapy. With recent reimbursement/bundling changes for percutaneous hemodialysis thrombectomy, OST should be considered first-line treatment for occluded hemodialysis access in a hospital-based setting, whereas RT may be more cost-efficient in office-based settings as the cost profile is more favorable.

Liposomal delivery systems (LDSs) have been at the forefront of medicinal nanotechnology for over three decades. Increasing LDS association to target cells and cargo delivery is crucial to bolstering overall nanodrug efficacy. Our laboratory aims to develop LDSs for molecular therapeutics aimed at vascular pathology. We have previously established a liposome platform that is an effective delivery system for RNA interference in vascular cell types by using polyethylene glycol (PEG) decorated liposomes bearing an octa-arginine (R8) cell penetrating peptide (CPP). Further tailoring liposome membranes to mimic vascular cell membrane lipid constituents may be a promising strategy for increasing cargo delivery. Here we aimed to develop liposomal formulations that could make use of diacylglycerol (DAG) and phosphatidylserine (PS), naturally occurring lipid species that are known to influence vascular cell function, as a facile and efficient means to increase nanodrug efficacy without compromising clinical viability. We investigated the ability of DAG and PS to amplify the cellular uptake of our previously established LDS platform loaded with small interfering ribonucleic acid (siRNA) cargo. Cellular fluorescence microscopy experiments were performed in conjunction with quantitative cell association assays and cytotoxicity assays to analyze the effect of DAG/PS on the differential delivery of fluorescently-tagged liposomes to vascular smooth muscle cells (VSMCs) and vascular endothelial cells (VECs) and on liposomal-mediated toxicity. In these studies, significant, dose-dependent increases in association to target cells were observed, as well as cell-type specific effects on cell viability. The stability and encapsulation-efficiency of the DAG/PS-modified LDSs were analyzed by standard nanoparticle characterization methods, and siRNA transfection efficacy was quantified to gauge delivery potential as a function of DAG/PS modification. Our results suggest that the signaling lipids tested here imbue our LDS architectures with increased therapeutic potential, without compromising stability, encapsulation efficiency, or biocompatibility, thus presenting a natural strategy to increase nanodrug efficacy and specificity.

Grimsley, Lauren B. MD, MBA; McNally, Michael M. MD; Buckley, Michael R. MD; Arnold, Joshua D. MD, FACS; Stevens, Scott MD; Freeman, Michael B. MD; Cantafio, Alex MD; Gregory, Lynellen B. MD; Grandas, Oscar H. MD, MBA, FACS Author Information

Introduction: Our laboratory aims to develop biocompatible nanocarriers for molecular therapeutics aimed at vascular pathology. We have previously established a liposome platform that is an effective delivery system for RNAi in vascular smooth muscle cells (VSMC). Tailoring liposome membranes to mimic vascular cell membrane lipid constituents may be a promising strategy for increased delivery to target cells. Here we test our previously established liposome platform with the incorporation of naturally occurring signaling lipids known to influence vascular cell function as a method to increase VSMC association. Methods: Established cell-penetrating neutral liposomes (R8-PLPs) were assembled and fluorescently tagged as previously described. The propensity of diacylglycerol (DAG) and/or phosphatidylserine (PS) to increase the association of R8-PLP to VSMCs was tested by the incorporation of gradient percentages DAG/PS alone and in combination at 5-20% membrane occupancy. Liposome stability and siRNA encapsulate retention was analyzed via dynamic light scattering and Ribo-green, respectively. Results: DAG and PS incorporation increased VSMC association of R8-PLP, with 10% PS increased over all other groups (P10; Fig1A). Combinatorial formulations were screened for optimal DAG content with PS fixed at 10%. DAG20%+PS10% (D20P10) performed best, with increased VSMC association over all other combinatorial groups or independent P10 modification (Fig1B). Stability profiles were consistent (~50nm size and ~80% drug retention) and not significantly different among groups. Conclusion: Signaling lipid incorporation into the nanocarrier architecture potentiates VSMC association of established R8-PLP liposomes, without sacrificing stability or drug retention. These results suggest cell mimetic tuning of liposomes to generate specificity and increase delivery efficacy is a viable strategy for advancing targeted liposomal drug delivery.

Vascular intervention results in intimal denudation, exposure of the subendothelial matrix, and subsequent intimal hyperplasia (IH), under the control of numerous remodeling mechanisms. Reduction of IH-induced restenosis may be achieved by manipulation of these remodeling pathways through targeted molecular inhibition. Spatially controlled nanoparticles designed to colocalize to exposed subendothelial matrices could provide an optimal delivery system for targeted vascular therapeutics. To his end, we aimed to develop the framework for a surface-modified liposomal drug delivery platform designed to preferentially bind collagen type IV. Nontargeted control liposomes (NTL) were formed with bulk DOPC-PEG, 30% cholesterol, and 0.1 mol% Rhodamine-DOPE. DSPE-PEG-DBCO lipids were conjugated to peptides previously shown to bind collagen IV via copper-free click chemistry and inserted at 5 mol% to form collagen-targeting liposomes (CTL), either at hydration (PreCTL) or by postinsertion via micellar transfer (PostCTL). Peptide conjugation was confirmed by matrix-assisted laser desorption/ionization time of flight, and liposomes were characterized by dynamic light scattering and electrophoretic mobility. Liposome binding was assayed on collagen IV matrices dried at 3 μg/cm2 and quantified by fluorescence at 0- to 24-hour static 37°C incubation. All liposome formulations exhibited a narrow size distribution (∼100 nm) and neutral-low positive charge. CTLs demonstrated a significant increase in binding vs NTLs (Fig). CTLs demonstrated significant affinity for collagen IV binding in a static environment compared with NTLs. Future studies aim to optimize the binding capacity of CTLs via further lipid modifications and under flow conditions mimicking vessel wall hemodynamics. Considering the efficacy demonstrated here, CTLs show promise as the framework for a spatially-controlled drug delivery platform for future application in targeted vascular therapeutics.

Background: We have previously defined mechanisms of intimal hyperplasia that could be targets for molecular therapeutics aimed at vascular pathology. However, biocompatible nanocarriers are needed for effective delivery. Cationic liposomes (CLPs) have been demonstrated as effective nanocarriers in vitro. However, in vivo success has been hampered by cytotoxicity. Recently, neutral PEGylated liposomes (PLPs) have been modified with cell-penetrating peptides (CPPs) to enhance cellular uptake. We aim to establish CPP-modified neutral liposomes as viable molecular nanocarriers in vascular smooth muscle cells. Methods: CLPs, PLPs, and CPP-modified PLPs (R8-PLPs) were assembled with short interfering RNA (siRNA) via ethanol injection. Characterization studies determined liposomal morphology, size, and charge. siRNA encapsulation efficiency was measured via RiboGreen assay. Vascular smooth muscle cells were exposed to equal lipid/siRNA across all groups. Rhodamine-labeled liposomes were used to quantify cell association via fluorometry, live/dead dual stain was used to measure cytotoxicity, and gene silencing was measured by quantitative polymerase chain reaction. Results: R8-PLPs exhibited increased encapsulation efficiency equivalent to CLPs. PLPs and R8-PLP-5 mol% and R8-PLP-10 mol% had no cytotoxic effect. CLPs demonstrated significant cytotoxicity. R8-PLP-5 mol% and R8-PLP-10 mol% exhibited increased cell association versus PLPs. R8-PLP-10 mol% resulted in significant gene silencing, in a manner dependent on lipid-to-siRNA load capacity. Conclusions: The negligible cytotoxicity and enhanced cellular association and gene silencing capacity exhibited by R8-PLPs reveal this class of liposomes as a candidate for future applications. Further modifications for optimizing R8-PLPs are still warranted to improve efficacy, and in vivo studies are needed for translational development. However, this could prove to be an optimal nanocarrier for vascular gene therapeutics. (C) 2017 Elsevier Inc. All rights reserved.