| Background and ObjectivesActive targeted drug delivery system leads an increasing interest in cancer therapy. Among the formulations developed, novel Lipid-polymer hybrid nanoparticles(NP) based on liposomes and polymer nanoparticles have attracted more attention because of their special structure, properties and clinical applicability. MUC1Protein is an attractive target for anticancer drug delivery owing to its over expression in most malignant cell.In this work, anticancer drug Vinorelbine-loaded NP were constructed and were functioned with Aptamer S2.2(Apt) for site-specific targeting against tumor cells which over expresses MUC1protein. A series of targeted NP with increasing Apt densities were developed for studying the effect of Apt density on NP characteristics and targeted delivery.Methods and Results1. Construction and evaluation of Apt-NP/VRLNP/VRL were prepared by self-assembled using poly (lactic-co-glycolic acid, PLGA), lecithin and DSPE-PEG2000-COOH as carrier material, and hydrophobic anticancer drug VRL as moldered drug. The optimal formulation of NP/VRL was obtained by orthogonal design. The3’-NH2modified aptamer was conjugated to DSPE-PEG2000-COOH. And then fixed DSPE amount, by changing the ratios of DSPE-PEG2000-Apt and DSPE-PEG2000-COOH Synthesized different Apt densities Apt-NP/VRL. The characteristics of Apt-NP/VRL were evaluated.The obtained optimum experimental formulation was the ratio of lipid/PLGA was15%, the ratio of DSPE-PEG2000-COOH/lipid was60%, the ratio of VRL:PLGA was15:100, PLGA concentration was8mg·mL-1, the ratio of water:organic phase was2:1. Apt0-NP/VRL, AptO.5-NP/VRL, Apt1-NP/VRL, Apt2-NP/VRL, Apt5-NP/VRL Apt10-NP/VRL were recorded according to the percentage of DSPE-PEG2000-Apt in DSPE. XPS showed Apt was successfully conjugated to the surface of NP/VRL. TEM observation indicated a core-shell construction of the nanoparticle and the shell was10nm. Apt densities did not affect the NP characteristics. Apt-NP/VRL were about128.1-166.5nm, and the encapsulation efficiency was50.42-57.88%. The cumulative release rate of Apt-NP/VRL was less than50%within132h in PBS (pH7.4), which show slow release feature.2. Evaluation of the targeted cellular uptake of Apt-NP/VRLScreened breast cancer cells MCF-7as MUC1+cells and hepatic cancer cells HepG2as MUC1-cells by flow cytometry. Coumarin6-labeled nanoparticles (Apt-NP/Cou-6) were prepared. The targeted cellular uptake of Apt-NP/VRL and the influence of Apt density were evaluated qualitatively by fluorescence microscope and quantitatively by enzyme-labelling measuring instrument.The characteristics of Apt-NP/Cou-6were consistent with Apt-NP/VRL and less than5%of Cou-6released after24h which were optimal for its tracing nanoparticles in vitro. Fluorescence microscope show nanoparticles entered into cell in1h. The accumulation of Apt10-NP/Cou-6by MCF-7cell exhibited higher than AptO-NP/Cou-6owing to the conjugation between S2.2and MUC1. The accumulation of Apt10-NP/Cou-6by HepG2cell was lower than AptO-NP/Cou-6owing to the higher negatively charge. After1h incubation, the MCF-7cell intake of Apt0.5-NP/Cou-6, Aptl-NP/Cou-6, Apt2-NP/Cou-6, Apt5-NP/Cou-6, Apt10-NP/Cou-6were (15.7±1.4)%,(16.8±0.7)%,(18.8±2.3)%,(23.9±3.8)%,(24.6±4.6)%vs AptO-NP/Cou-6(11.6±1.2)%, meaning increasing Apt densities improved targeting capability.3. In vitro pharmacodynamic evaluation of Apt-NP/VRLThe viability of MCF-7cell and HpG2cell was evaluated by MTT assay to evaluate anti-tumor activity of Apt-NP/VRL. Compared with HepG2cell, Apt-NP/VRL showed an increased anti-tumor activity in MCF-7cell. By increasing Apt density, there were a1.3-.1.5-,1.6-,1.5-,1.7-fold increase in the inhibitory of MCF cell by AptO.5-NP/VRL, Apt1-NP/VRL, Apt2-NP/VRL, Apt5-NP/VRL, Apt10-NP/VRL compared with HepG2cell (p<0.05). The results presented promising results.The effect of AP-NP/VRL with specified concentrations on MCF-7cell viability was evaluated by MTT assay, The IC50of AptO.5-NP7VRL, Aptl-NP/VRL, Apt2-NP/VRL, Apt5-NP/VRL, Apt10-NP/VRL were (19.2±0.1),(15.4±0.2),(10.3±0.2),(8.7±1.0),(7.2±0.4)μg·mL-1, respectively, while the blank nanopartcle AptO-NPA/RL was (21.3±0.2)μg·-mL-1. The decorated S2.2enhanced cellular toxicity of VRL-loaded nanoparticles to MUCl+cell (p<0.05).ConclusionsMUC1-targeted drug delivery Lipid-polymer hybrid nanoparticles (Apt-NPATRL) was developed by self-assembled. Apt densities did not affect the nanoprticle characteristics. Apt-NP/VRL contained slow drug release feature. Apt-NP/RL can delivery drugs specifically to cancer cells overexpressed MUC1which enhanced cytotoxicity. By increasing the Apt density on the surface, target capability improved. |
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