Paclitaxel-Loaded Targeted Magnetic Nanoprobe For Dual-Mode Imaging And Therapy Of Breast Cancer

Objective1 To prepare a kind of new smrat nanoprobe,paclitaxel -loaded magnetic nanoprobe decorated by Herceptin(PTX-USPIO-HER-NBs), and to detect their physical properties.2 To test the effects of ultrasound irradiation on uptake of PTX-USPIO-HER-NBs by the SK-BR-3 breast cancer cells, and to investigate the cytotoxicity of ultrasound irradiation of PTX-USPIO-HER-NBs on SK-BR-3 cells.Methods1 FTX-USPIO-HER-NBs were prepared by double emulsion and a carbodiimide technique, with different concentration of USPIO input in the oil phase to synthesize PTX-USPIO-HER-NBs with various USPIO-loaded. The morphology of the PTX-USPIO-HER-NBs was observed by the light microscope (LM), scanning electron microscope(SEM) and transmission electron microscope (TEM). The mean size and size distribution were measured by a laser instrument. The quality of the magnetic nanoprobe as a drug carrier was evaluated by the PTX encapsulation efficiency and the PTX loading efficiency by high performance liquid chromatography (HPLC) with absorption peak at 227nm. A typical PTX release behavior was observed for 30 days. USPIO encapsulation efficiency was determined using an atomic absorption spectrophotometer (AAS). The magnetization properties of PTX-USPIO-HER-NBs were detected by a vibrating sample magnetometer (VSM). Immunofluorescence method was performed to determine the localization of Herceptin covalently coupled to the PTX-USPIO-HER-NBs by observing the incorporation of FITC rabbit anti-human IgG with PTX-USPIO-HER-NBs under the confocal laser scanning microscope (CLSM). The amount of Herceptin bound to NBs was quantified using BCA Protein Assay Kit.2 Cellular targeting and uptake of non-targeted and targeted nanoprobe were investigated with MDA-MB-231 and SK-BR-3. The effect of ultrasound irradiation on uptake of PTX-USPIO-HER-NBs by the SK-BR-3 was observed by CLSM. The cytotoxicity of PTX-USPIO-HER-NBs on the cells was detected by CCK8 assay.Results1 The prepared PTX-USPIO-HER-NBs were well dispersed, uniform and spherical. The size of the PTX-USPIO-HER-NBs was (285.8±98.97)nm, the particle diameter was bigger than that of PTX-USPIO-NBs. The encapsulation efficiency of PTX in the PLGA matrix was 69.24±0.05%, and the PTX loading efficiency 6.92±0.13%. The release from drug loaded NBs triggered with ultrasound became significantly greater than the uninsonated controls. The images of TEM demonstrated a typical core-shell structure and USPIO were successfully encapsulated into the shell. With the increase of the USPIO input, the USPIO were distributed more and more to the shell of the nanospheres, but the distribution of USPIO was random. The VSM revealed that the magnetic nanoprobe possessed the superparamagnetic characteristic, which means that when the magnetic particles were removed from the field, they exhibited non-permanent magnetization. Immunofluorescence technique showed that Herceptin-conjugated nanoparticles glowed fluorescent green due to the specifically binding of FITC-labeled rabbit anti-human antibody, suggesting that the Herceptin antibodies were sufficiently conjugated to the particles surface. The amount of Herceptin bound to NBs was determined to be about 1.4±0.037mg in per gram of nanospheres using BCA Protein Assay Kit.2 After incubation for 2 h, a lot of PTX-USPIO-HER-NBs were observed around the SK-BR-3 cells. With ultrasound irradiation, more PTX-USPIO-HER-NBs were observed in the cytoplasmic. The results of cytotoxicity tests revealed that with PTX-USPIO-HER-NBs and ultrasound irradiation, the viability of SK-BR-3 cells was lowest. The effect of polymer materials and USPIO on the viability of SK-BR-3 cells can be ignored, suggesting that both of them had good security and biocompatibility.Conclusion The prepared magnetic probe exhibited excellent size distribution, superparamagnetic property, targeting ability, USPIO and drug loading. Herceptin-conjugated Paclitaxel-load PEGylated PLGA-based magnetic nanoprobes with suitable characterization were successfully prepared for targeted delivery of paclitaxel with low frequency ultrasound triggered. The fast drug release from the nanocarriers in vitro with ultrasonic irradiation indicated the great potential as a controlled release system for the anticancer drug. In vitro cytotoxicity studies of the smart probe showed greater cytotoxicity effects against SK-BR-3 cell line with the highest HER2 expression level.Objective To investigate the potential of PTX-USPIO-HER-NBs as contrast agent for enhanced ultrasonography(US) and magnetic resonance imaging(MRI) in vitro; to observe the influence of PTX-USPIO-HER-NBs on SK-BR-3 xenograft tumor-bearing nude mice US/MR imaging in vivo, and investigate the enhanced capacity and the principle.Methods1. MR imaging and R2* measurement were carried out on a 1.5T MR scanner. The prepared PTX-USPIO-HER-NBs with different iron concentrations were applied to perform MR imaging with FSPGR-T2*WI sequence in vitro. The inherent echogenicity of PTX-USPIO-HER-NBs was determined by using the ultrasonic imaging system of the Biosound Esaote MyLab 70 with a 7.5 MHz transducer. Acoustic analysis was carried out by exposing a series of fractions with gradient NBs concentration in a self-made gel phantom. The mean gray scales of each US image were measured by using a ultrasound image analysis software DFY.2.15 SK-BR-3 xenograft tumor-bearing nude mice were randomly divided into three equal groups with the injection of saline, PTX-USPIO-NBs and PTX-USPIO-HER-NBs via tail vein respectively (iron dose about 0.49ug/g body weight). MR imaging was performed with a small animal coil on a 3.0T MR scanner. And T2WI images were obtained at different time points. Signal intensity of the tumor (SItumor) and thigh muscle(SImuscie)were measured before and after enhancement on the same level at different time point. The relative signal intensity (Sir) were calculated by SItumor divided by SImuscie. Ultrasonic contrast enhancement experiment was carried out by high frequency ultrasound imaging systerm PanoViewβ1500.100 frames were acquired as the reference image without contrast agents. After injection of contrast agent,1000 frames were obtained within more than 60 min. Determine the tumor ROI where the contrast image is present with green colors, and draw up the time intensity curve (TIC) and video intensity curve (VIC). TIC displays the enhanced signal intensity as a function of time whereas VIC displays the percentage of the presence of microbubbles as a function of time. The values of GSI and VI% in different groups before and after contrast mode were analysized by statistical methods.Results1. The capability of PTX-USPIO-HER-NBs as the contrast agents for ultrasound imaging was assessed in vitro using the typical B mode and contrast mode. The results showed that the ultrasound signals gradually increase with increasing of NBs concentrations. Compared with pure degassed water background, the mean gary scale of PTX-USPIO-HER-NBs with concentration gradient had significant difference (p<0.05). The gary scale of the image of the USPIO-integrated NBs was brighter than that of the PTX-HER-NBs. To validate the feasibility of PTX-USPIO-HER-NBs as an MRI contrast agent, the relaxivity of PTX-USPIO-HER-NBs in aqueous media was examined. The results indicated that PTX-USPIO-HER-NPs displayed a signal reduction in the in-vitro T2*-weighted images and a signal increase in transverse relaxation rate (R2*) map with the increase of Fe concentration. The specific transverse relaxivity value of r2 was calculated to be 11.47mM-1s-1 for transverse relaxation-weighted sequence through the curve fitting of R2* VS Fe concentration.2. In vivo MR imaging findings suggested that signal intensity of the tumor did not change after injection of saline, there was no significant difference for PSIL at different time point(p>0.05). After injection of PTX-USPIO-NBs and PTX-USPIO-HER-NBs via tail vein, the overall signal in liver was immediately negatively enhanced. As scanning time prolonged, signal intensity of tumor gradually reduced. Stastistical analysis showed that PSIL in PTX-USPIO-HER-NBs group was increased significantly compared with PTX-USPIO-NBs group(p<0.05). The enhancement of breast cancer could be detected after injected with PTX-USPIO-HER-NBs by contrast mode with PanoViewβ1500 While the tumor in control group injected with saline did not show any increase in echogenicity over the duration of the imaging period. Compared with the control group, contrast enhancement can be observed in the other two groups. Stastistical analysis showed that GSI and VI% in PTX-USPIO-HER-NBs group were increased significantly compared with PTX-USPIO-NBs group(p<0.05).Conclusion The prepared PTX-USPIO-HER-NBs can be successfully acted as dual-modality biological imaging contrast agents for US and MR imaging in vitro. In-vivo experiment showed that in contrast to non-targeted nanoprobe, Herceptin-conjugated magnetic nanoprobe can concentrate in the tumor, thus have the capacity of markedly enhanceing dual-mode US/MR imaging of the breast cancer. As a safe and effective delivery carrier for paclitaxel, PTX-USPIO-HER-NBs can further improve imaging-guided therapies, which may give a novel strategy in the detection and treatment of breast cancer.Objective To explore the effect of ultrasound irradiation on the distribution of PTX-USPIO-HER-NBs in tumor-bearing nude mice. To explore the antitumor effect of PTX-USPIO-HER-NBs combined with US triggered in situ drug release for SK-BR-3 xenograft tumor-bearing nude mice.Methods1. The 10 SK-BR-3 xenograft tumor-bearing nude mice were divided into 2 groups:Herceptn-targeted group (Dil/PTX-USPIO-HER-NBs) and non-targeted group (Dil/PTX-USPIO-NBs). The nanoprobes were injected through the tail vein at a dose of 0.5 ml(10mg/ml).Continue to raise for 24 hours after injection, targeting performance of targeted and non-targeted nanoprobes to SK-BR-3 xenograft tumor were investigated by the fluorescence imaging systems of small animals in vivo. The fluorescence intensity was quantified and analyzed by imaging post-processing software.2. The 5 nude mice tearing two SK-BR-3 xenograft tumors were selected as the animal modes. The DiI labeled PTX-USPIO-HER-NBs were injected through the tail vein, and only the right tumor was sonicated by 1.00-1.10MHz CW ultrasound, intensity of 3.5W and exposure time of 20min. The mice were sacrificed after 24h and the tumor, liver, spleen, lung, kidney and brain were collected for frozen sections to investigate the distribution of DiI-labled nanoprobes in different tissues by CLSM. Integrated optical density (IOD) in the fluorescent images of different organs was analysized by imaging post-processing software.3. The 25 nude mice bearing two SK-BR-3 xenograft tumors were selected and randomized into five groups(5 mice every group):control group (injection of saline), paclitaxel group (PTX), non-targeted nanoprobe group(PTX-USPIO-NBs), Herceptin-targeted nanoprobe group (PTX-USPIO-HER-NBs) and Herceptn-targeted nanoprobe combined with US group (PTX-USPIO-HER-NBs+US). The mice were treated by three systemic injections (20 mg/kg as PTX), given at intervals of three days. The volume of tumor was measured at designed time. After 21 days of treatment, tumors were excised and weighed. Tumor masses were fixed in 4% paraformaldehyde, paraffin embedded and sliced for histological analysis, cell apoptosis was detected by TUNEL and the expression of PCNA, CD34 was detected by immunohistochernistry.Results1. The images of the fluorescence imaging systems of small animals revealed that the xenograft tumor showed red fluorescence in Herceptn-targeted nanoprobe group and weak red fluorescence in non-targeted nanoprobe group. The fluorescence intensity was significantly different between the two groups. It showed that the retention of Herceptin-targeted nanoprobe was more than non-targeted nanoprobe in the tumor site.2. The results of frozen sections of the different organs showed that different contents of fluorescence were detected in the tumor, liver, spleen, lung, kidney and brain of the nude mice. Fluorescence intensity of the right tumors with ultrasound irradiation was higher than that of the left ones, there was significant difference between the two groups (P<0.05). There was no significant difference of fluorescence intensity between the right tumor and liver (P>0.05). The fluorescence intensity in the brain was lowest.3. From the tumor growth curve, the group of PTX-USPIO-HER-NBs+US exhibited a notably strong antitumor effect than the other four groups, the tumor in this group grew the most slowly(P<0.05) and TIR was highest (70.67%). AI of PTX-USPIO-HER-NBs+US group was highest among all groups (P<0.05) and PI was lowest (P<0.05). MVD of PTX-USPIO-HER-NBs+US group was lowest among all groups (P<0.05).Conclusion PTX-USPIO-HER-NBs combined with ultrasound irradiation could markedly enhanced the distribution in the breast cancer, and PTX would slowly release in the tumor which provides evidence for further inhibiting proliferation and promoting apoptosis. Therefore, the application of these prepared PTX-USPIO-HER-NBs was a potential technique for multimodal imaging and imaging-guided therapy.