| 1. Background and objective Molecular imaging with targeted contrast ultrasound is the use of ultrasound contrast agents (UC As) decorated with specific ligands like antibodies binding to specific target receptors for ultrasonic imaging through blood-circulation, which is high sensitive, widely available, real-time, repeatable, non-invasive, cost-effective, non-toxic and radiation free. Efficient UCAs for targeted molecular imaging require the ability of sensitively and selectively binding to the epitope of interest, stable and stealth enough to have a long circulating half-life at regions of interest, good acoustic properties and prominent contrast-to-noise enhancement, acceptable toxicity, ease of clinical use, and ideally applicable with standard commercially available imaging systems to lead to a more widespread clinical use. Nowadays the most commonly clinically used UCAs are micro bubbles (gas cores), such as commercial UCAs like SonoVueTM, of which the average diameter is2μm-4μm. They have no targeting ability but only a very short plasmatic half-life, and the micron-sized diameter of bubbles limits their use to the vascular space. Recently, liquid perfluorocarbons-filled nanocapsules (LNCs) have been developed. These new UCAs, consisting of poly (lactide-co-glycolide)(PLGA) encapsulating a liquid core of perfluorocarbon bromide (PFOB) which should be more resistant to pressure changes and mechanical stresses. Nowadays, studies of LNCs mainly focussed on the preparation of LNCs with different shell membranes and been applied mainly as blood pool agents. The factors that control particle size, the ability to decorate particle with target function, the impact of size distribution in physical properties, the effects of ultrasonic enhancement of the concentration and size distribution of particles, the potentialities of use to enhance the echogenicity of extra vascular tissues, and the biodistribution and biocompatibility of LNCs are still not clear to us.Objective:To prepare human vascular endothelial growth factor receptor2(VEGFR2) targeted LNCs with fitting diameter and concentration screened out, and to investigate its peculiarity of enhancing ultrasound imaging in mice bearing xenograft tumor with HepG2cells.2. Experimental methods2.1Rotary emulsion/evaporation techniques were used to prepare DSPE-PEG decorated LNCs. Size distribution of LNCs as a function of emulsification speed, sonication power and sonication time was evaluated through a single-factor experiment.2.2polycarbonate filters and density-gradient centrifugation method were used for LNCs separation and purification. The LNCs were split into4groups on the basis of particle sizes. Optical microscopy, scanning electron microscope, transmission electron microscope and laser-scanning confocal microscopy were used to observe the structure of micro/nanocapsules. Particle size, zeta potential and polydispersity index (PDI) was obtained using a Malvern Zetasizer Nano ZS (Malvern instrument. UK) based on quasi-elastic light scattering. The surface hydrophobicity of plain non-PEGylated-LNCs and LNCs was investigated by hydrophobic interaction chromatography (HIC). Cell viability of HUEVC was quantified with the MTT assays.2.3In vitro ultrasound imaging of LNCs. Quantitative and statistical analysis were performed to evaluate ultrasound contrast enhancement of LNCs with different concentration and size distribution in vitro. Stability of echogenicity and particle size of LNCs were also estimated.2.4In vivo ultrasound imaging of LNCs. The concentration of LNCs screened out, which is of the best size based on the above experiments, was injected into New Zealand rabbits via the ear vein; ultrasound scanning was performed instantly or24h after injection to evaluate the ultrasound contrast enhancement.2.5Preparation and identification of V2-LNCs. Prepare DSPE-PEG-biotin decorated LNCs of proper size. VEGFR2-tageted LNCs (V2-LNCs) was further made through conjugating the ligand (biotin labeled anti-VEGFR2antibody) to the surface of LNCs by biotin-avidin system. Immunofluorescent staining assay was used to identify the decoration of biotin and the combination of ligand to the nanocapsules.2.6Physical properties of V2-LNCs. The structure, appearance, distribution, size, polydispersity index (PDI), zeta-potential, surface hydrophobicity, Cell viability properties of V2-LNCs were evaluated and In vitro ultrasound contrast enhancement of V2-LNCs and LNCs were performed to assess the impact of targeted decoration in echogenic properties of LNCs. 2.7In vitro targeting assay. Targeting properties of V2-LNCs were evaluated under a fluorescence microscope in vitro. HUVEC cells and HepG2cells were fixed by4%PFA followed by incubation with PBS, V2-LNCs, LNCs or IgG-LNCs for2h. Cells preprocessed with anti VEGFR2antibodies before incubating with V2-LNCs were utilized to perform a blocking-test. The combine efficiency with different incubation time of V2-LNCs was also evaluated via incubate LNCs with fixed cells for0.5h and1.5h.2.8In vivo ultrasound imaging and histological analyses. In vivo ultrasound imaging was performed using a clinical imaging system (Esaote mylab90). Animals (planting HepG2tumor model in nude mice) were injected with LNCs, V2-LNCs or PBS as a blank group (intravenous injection or intra-tumoral injection).24h after the in vivo ultrasound imaging, mice injected intravenously were sacrificed to perform histological analyses.2.9Biodistribution and biocompatibility of LNCs and V2-LNCs in vivo. ICR mice were used for assessing the biodistribution and biocompatibility of LNCs and V2-LNCs Mice injected with V2-LNCs or LNCs via tail vein (5mg/kg) were sacrificed. Slices of each organ were put under laser confocal microscopy to observe the existence or accumulation of nanocapsules and evaluate whether there was any acute toxicity existing in these organs. In addition, the whole blood and separated serum were also collected from the mice and the aspartate aminotransferase (AST)/alanine aminotransferase (ALT) levels were analyzed to monitor acute toxicity reactions.3. Results3.1Capsules were obtained by a solvent emulsification-evaporation process with phospholipids incorporated in the organic phase before emulsification. The mean diameter of LNCs decreased with the increase of emulsification speed, sonication power and sonication time.3.2The LNCs displayed sphere with a hydrophilic surface and low cytotoxicity. The core-shell structure remains unchanged as size distribution of LNCs verified.3.3In vitro ultrasound imaging of LNCs. The LNCs with size of (220nm-450nm) was screened out to perform in vivo experiments which show efficient ultrasound contrast enhancement at different concentrations (0.05,0.5,5,50mg/mL). In low concentration (0.005mg/mL), the ultrasound contrast enhancement increased with the increase of particle size, while in high concentration layer the decay of echogenicity emerged, the degree of ultrasonic signal attenuation accompanies with the increase of particle size. Interestingly, LNCs with the size of less than100nm did not show ultrasonic signal attenuation at the concentration of50mg/mL but a relatively low ultrasound contrast enhancement. The observation and analysis of echogenicity and particle size indicate a perpetual stability of LNCs.3.4In vivo ultrasound imaging of LNCs. Size and concentration of LNCs screened out based on the above experiments significantly enhanced the blood vessel, liver and renal parenchymal echogenicity of New Zealand rabbits after being injected via an ear vein.3.5Biotin-LNCs were obtained by a solvent emulsification-evaporation process with DSPE-PEG-biotin incorporated in the organic phase before emulsification. The LNCs are uniform and stabilized with the size of about350nm. VEGFR2-tageted LNCs (V2-LNCs) was further made through conjugating the ligand (Biotin anti-VEGFR2antibody) to the surface of biotin-LNCs by biotin-avidin system. As to ligand ligation with nanoparticles, the V2-LNCs were positive in immunofluorescent straining assay. 3.6Targeted decorations indicate no impact of nanocapsules in physical properties like structure, appearance, distribution, size, polydispersity index (PDI), zeta-potential, the surface hydrophobicity and cell viability properties. The statistical significance and the ultrasound images proved both V2-LNCs and LNCs can significantly enhanced ultrasound imaging contrast agent effect but with no statistical differences in vitro.3.7In vitro, the targeting ability research showed the V2-LNCs could actively adhere to HUEVC cells and HepG2cells, while the control groups were negative. At the same time, utilizing anti-VEGFR2antibody to pre-incubation with HUVEC cells and HepG2cells could effectively block the interaction between V2-LNCs and cell. The cell label fixed efficiency associated with incubating time.3.8In vivo ultrasound imaging was assessed in mice bearing xenograft tumor with HepG2cells, either after an intra-tumoral or intravenous injection of LNCs or V2-LNCs. Before injection tumors appeared dark whereas after intra-tumoral injection (50μL,5mg/mL) it presented significant enhancement for both LNCs and V2-LNCs on tumors. No significant difference could be detected between LNCS and V2-LNCs. After intravenous injection, no significant contrast was observed in tumors neither directly after injection nor later (0.5h, lh,2h,4h,12h and24h). In histological analysis, only a weak and sparse fluorescent signal could be observed in control tumors and in tumors of mice injected with LNCs. By contrast areas fluorescent signals of high intensity (red spots) were observed in the tumor tissue of mice injected with V2-LNCS. Confocal microscopy therefore confirms the presence and the accumulation of V2-LNCS in tumor tissue by Enhanced Permeation and retention (EPR) and active targeting effect demonstrating that tissue distribution is not the unique property required for ultrasound contrast agents to be efficient. It also confirms that the tumor vasculature is leaky and that V2-LNCs can adhere to blood vessel endothelium and are small enough to escape the vascular compartment.3.9Biodistribution and biocompatibility of V2-LNVs and LNCs. Both types of capsules were unavoidably captured and accumulated in liver, spleen, lung and kidney by the reticulo-endothelial system (RES).In histological analysis on various organs (liver, spleen, lung, and kidney) of mice injected with V2-LNCs and LNCs to observe the probable acute toxicity reaction, there were no pathological changes in sections of above organs. Furthermore, there was no significant difference on ALT/AST levels between V2-LNCs-injected or LNCs-injected mice and untreated control.4. Conclusions4.1The median diameter of LNCs decreased with the increase of emulsification speed, sonication power and sonication time.4.2There is a definite correlation between echogenic properties and particle size&concentration of LNCs in vitro. The experiment using LNCs with diameter of350nm, at a concentration of5mg/mL that was screened out, revealed significant ultrasound contrast enhancement in vivo.4.3A stable VEGFR2-targeted LNCs with excellent acoustic properties and ideally biocompatibility was prepared successfully, which can be sensitively and selectively binding to tumor tissues through active targeting function. |
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