A new acoustically-active delivery vehicle was developed by conjugating liposomes and microbubbles, using the high affinity interaction between avidin and biotin. Binding between microbubbles and liposomes each containing 5% DSPE-PEG2kBiotin was highly dependent on avidin concentration and observed above an avidin concentration of 10 nM. With an optimized avidin and liposome concentration, we measured and calculated as high as 1000 to 10,000 liposomes with average diameters of 200 and 100 nm, respectively, attached to each microbubble. Replacing avidin with neutravidin resulted in 3-fold higher binding, approaching the calculated saturation level. Highspeed photography of this new drug delivery vehicle demonstrated that the liposome-bearing microbubbles oscillate in response to an acoustic pulse similar to microbubble contrast agents. Additionally, microbubbles carrying liposomes could be spatially concentrated on a monolayer of PC-3 cells at the focal point of ultrasound beam. As a result of cell-vehicle contact, the liposomes fused with the cells and internalization of NBD-cholesterol occurred shortly after incubation at 37°C, with internalization of NBD-cholesterol substantially enhanced in the acoustic focus.
The current study presents an effective and selective multifunctional nanoparticle used to deliver antiatherogenic therapeutics to inflamed pro-atherogenic regions without off-target changes in gene expression or particle-induced toxicities. MicroRNAs (miRNAs) regulate gene expression, playing a critical role in biology and disease including atherosclerosis. While anti-miRNA are emerging as therapeutics, numerous challenges remain due to their potential off-target effects, and therefore the development of carriers for selective delivery to diseased sites is important. Yet, co-optimization of multifunctional nanoparticles with high loading efficiency, a hidden cationic domain to facilitate lysosomal escape and a dense, stable incorporation of targeting moieties is challenging. Here, we create coated, cationic lipoparticles (CCLs), containing anti-miR-712 (∼1400 molecules, >95% loading efficiency) within the core and with a neutral coating, decorated with 5 mol % of peptide (VHPK) to target vascular cell adhesion molecule 1 (VCAM1). Optical imaging validated disease-specific accumulation as anti-miR-712 was efficiently delivered to inflamed mouse aortic endothelial cells in vitro and in vivo. As with the naked anti-miR-712, the delivery of VHPK-CCL-anti-miR-712 effectively downregulated the d-flow induced expression of miR-712 and also rescued the expression of its target genes tissue inhibitor of metalloproteinase 3 (TIMP3) and reversion-inducing-cysteine-rich protein with kazal motifs (RECK) in the endothelium, resulting in inhibition of metalloproteinase activity. Moreover, an 80% lower dose of VHPK-CCL-anti-miR-712 (1 mg/kg dose given twice a week), as compared with naked anti-miR-712, prevented atheroma formation in a mouse model of atherosclerosis. While delivery of naked anti-miR-712 alters expression in multiple organs, miR-712 expression in nontargeted organs was unchanged following VHPK-CCL-anti-miR-712 delivery.
Synthesis of a radiolabeled diglyceride 3-[ 18 F]fluorodipalmitoyl-1,2-glycerol ( 18 F-fluorodipalmitin, [ 18 F]FDP) and its potential as a reagent for radiolabeling long-circulating liposomes were investigated. The incorporation of 18 F into the lipid molecule was accomplished by nucleophilic substitution of p-toluenesolfonyl moiety with a decay corrected yield of 43 ± 10% (n = 12). Radiolabeled long-circulating PEG-coated liposomes were prepared using a mixture of DPPC, cholesterol, DSPE-PEG2000 (61:30:9) and [ 18 F]FDP with a decay corrected yield of 70 ± 8% (n = 4). PET imaging and biodistribution studies were performed with free [ 18 Liposomes are vesicles composed of one or more concentric phospholipid bi-layers and such vesicles have been widely investigated as possible drug carriers [1,2]. Prolonged blood circulation of the liposomes is achieved with the addition of a polyethylene glycol (PEG) coating, which efficiently minimizes their removal by macrophages of the reticuloendothelial system [3][4][5][6]. Liposomes with various target-specific ligands attached to their surface are being investigated for targeted drug delivery [1,2,7]. Liposomes labeled with radioisotopes such as 99m Tc, 186 Re, 67 Ga, 111 In, and 18 F were previously employed to study the biodistribution of different types of liposomes in various animal models using scintigraphy, SPECT and PET. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access Materials and Methods GeneralThe solvents and chemicals were purchased from Aldrich (Milwaukee, WI). The 1 H and 13 C NMR spectra were recorded using a Bruker Avance 500 spectrometer and the chemical shifts are reported relative to TMS. Analytical reversed-phase HPLC was performed using a Phenomenex Jupiter 5μ C4 300A column ( . The size distribution of liposomes was determined using a particle size analyzer Nanotrac NPA150 purchased from Microtrac Inc. (North Largo, FL). The lipid concentration was determined using a Phospholipids B kit purchased from Wako Chemicals, Inc. (Richmond, VA). Synthesis of 3-tosyl-1,2-dipalmitoyl glycerol (2)The precursor 2 was prepared according to the previously published procedure [32] from 1,2-dipalmitoyl-sn-glycerol (1) and 4-toluenesulfonyl chloride. The crude product was subsequently purified by recrystalization from hexane. 1 mL). The content of the vial was dissolved in anhydrous acetonitrile (0.35 mL) and transferred into a suspension of 2 (5.5 mg) in anhydrous acetonitrile (0.5 mL). The reaction mixture was heated to 100 ºC for 20 minutes and allowed to cool to room temperature for 5 min...
Repeated administration of chemotherapeutics is typically required for the effective treatment of highly aggressive tumors and often results in systemic toxicity. We have created a copper-doxorubicin complex within the core of liposomes and applied the resulting particle in multi-dose therapy. Copper and doxorubicin concentrations in the blood pool were similar at 24 hours (~40% of the injected dose) indicating stable circulation of the complex. Highly-quenched doxorubicin fluorescence remained in the blood pool over tens of hours, with fluorescence increasing only with the combination of liposome disruption and copper trans-chelation. At 48 hours after injection, doxorubicin fluorescence within the heart and skin was one-fifth and one-half, respectively, of fluorescence observed with ammonium sulfate-loaded doxorubicin liposomes. After 28 days of twice per week doxorubicin administration of 6 mg/kg, systemic toxicity (cardiac hypertrophy and weight and hair loss) was not detected with the copper-doxorubicin liposomes but was substantial with ammonium sulfate-loaded doxorubicin liposomes. We then incorporated two strategies designed to enhance efficacy, mTOR inhibition (rapamycin) to slow proliferation and therapeutic ultrasound to enhance accumulation and local diffusion. Tumor accumulation was ~10% ID/g and was enhanced approximately two-fold with the addition of therapeutic ultrasound. After the 28-day course of therapy, syngeneic tumors regressed to a pre-malignant phenotype of ~ (1 mm)3 or could not be detected.
The development of treatment protocols that result in a complete response to chemotherapy has been hampered by free drug toxicity and the low bioavailability of nano-formulated drugs. Here, we explore the application of temperature-sensitive liposomes that have been formulated to enhance stability in circulation. We formed a pH-sensitive complex between doxorubicin (Dox) and copper (CuDox) in the core of lysolipid-containing temperature-sensitive liposomes (LTSLs). The complex remains associated at neutral pH but dissociates to free Dox in lower pH environments. The resulting CuDox-LTSLs were injected intravenously into a syngeneic murine breast cancer model (6 mg Dox/kg body weight) and intravascular release of the drug was triggered by ultrasound. The entire tumor was insonified for 5 min prior to drug administration and 20 min post drug injection. A single-dose administration of CuDox-LTSLs combined with insonation suppressed tumor growth. Moreover, after twice per week treatment over a period of 28 days, a complete response was achieved in which the NDL tumor cells and the tumor interstitium could no longer be detected. All mice treated with ultrasound combined with CuDox-LTSLs survived, and tumor was undetectable 8 months post treatment. Iron and copper-laden macrophages were observed at early time points following treatment with this temperature sensitive formulation. Systemic toxicity indicators, such as cardiac hypertrophy, leukopenia, and weight and hair loss were not detected with CuDox-LTSLs after the 28-day therapy.
Interest in ultrasound contrast agents (lipid-shelled microbubbles) as delivery vehicles is increasing; however, the biodistribution of these agents remains uncharacterized, both with and without ultrasound. In this study, an 18 F-labeled lipid ([ 18 F]fluorodipalmitin), incorporated in microbubble shells, was used as a dynamic microPET probe for quantitative 90-minute biodistribution measurements in male Fischer 344 rats (n = 2). The spleen retained the highest concentration of radioactive lipid at ~2.6 percent-injected dose per cubic centimeter (% ID/cc) and the liver demonstrated the largest total accumulation (~17 % ID). The microbubble pharmacokinetic profile differed from free lipid, which is rapidly cleared from blood, and liposomes, which remain in circulation. Additionally, region of interest (ROI) analysis over 60 minutes post-ultrasound treatment quantified the delivery of lipid by therapeutic ultrasound from microbubbles to kidney tissue (n = 8). The ultrasound sequence consisted of a 200 kPa, 5.3 MHz radiation force pulse followed by a 1.6 MPa, 1.4 MHz fragmentation pulse and was applied to one kidney, while the contralateral kidney served as a control. ROI-estimated activity in treated kidneys was slightly but significantly greater at 0 and 60 minutes than in untreated kidneys (p = 0.0012 and 0.0035, respectively). This effect increased with the number of microbubbles injected (p = 0.006). In summary, [ 18 F]fluorodipalmitin was used to characterize the biodistribution of contrast microbubble shells and the deposition of lipid was shown to be locally increased after insonation.
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