BBN -assembled Gadolinium Oxide Nanoparticles As A Targeted Contrast Agent:Synthesis And Experimental Study

Background:Prostate cancer (PC) is one of the most common types of malignant tumor which is a major public problem, Prostate cancer alone will account for 27%(233,000) of incident cases in men. In its early stages, PC rarely causes symptoms and the majority of men have no specific symptoms, resulting in late diagnoses. The diagnosis of prostate cancer is often done through digital rectal exam and blood analysis of blood-serum levels of prostate-specific antigen (PSA), and diagnostic anatomical imaging generally with ultrasound (US), computed tomography (CT), or magnetic resonance imaging(MRI). The current methods of clinical determination for patients presenting with prostate cancer are considered to be inadequate for early diagnosis. For example, by the time a malignancy has been detected and a clinical diagnosis made, the disease has already metastasized. To improve the diagnostic sensitivity for early prostate cancer, attention has focused on the development of novel imaging strategies.Recent advances in molecular imaging and nanotechnology are providing new opportunities for biomedical imaging with great promise for the development of novel imaging agents. Nanoparticles are a potential artificial nanostructure for medical applications, such as cancer detection, diagnosis and treatment. The enhanced permeability and retention (EPR) effect caused by the unique vascular structure of tumors may allows the nanoparticles to extravasate and accumulate in tumor tissues. The imaging probes with better contrast enhancement, increased sensitivity, controlled biodistribution, better spatial and temporal information, multi-functionality is needed.The gastrin-releasing peptide receptor (GRPr) is G protein-coupled receptor that is overexpressed in several human tumors, particular pancreatic cancer, prostate cancer and breast cancer, making it ideal biomarkers for molecular targeting. The GRPr have been shown with high densities on the cell membranes of primary prostate cancer and invasive prostatic carcinomas, whereas normal prostate tissue and benign prostate hyperplasias are predominantly GRPr-negative. BBN is the 14 amino acid peptide analog of the 27 amino acid mammalian regulatory peptide GRP. BBN and GRP share a homologous,7 amino acid amidated N-terminus that is essential for high-affinity binding to GRPr. BBN and its analogues have been studied for diagnosis and therapy of GRPR-positive tumors.Gd2O3 has several potential applications in biomedicine, it is used in magnetic resonance imaging, since it exhibits paramagnetism and involves T1 relaxation, and can be useful as a multimodal contrast agent for imaging in vivo. It can also be easily doped with other element, In this study, GRPr-targeted fluorescent paramagnetic bimodal nanoparticles were synthesized to co-deliver paramagnetic and fluorescence agents for magnetic resonance (MR) and optical bimodal imaging contrast enhancement. Meanwhile, the physico-chemical properties, cytotoxicity effect and active targeting PC-3 cells for fluorescence imaging and MR imaging in vitro of Gd2O3-FI-PEG-BBN nanoparticles were evaluated. We hope to explore new ways for the early diagnosis of prostate caner and metastases, as well as real-time monitoring of drug efficacy and distribution.Objective:1. To synthesis a novel Bombesin-modified fluorophore-equipped gadolinium oxide nanoparticles (Gd2O3-FI-PEG-BBN NPs) as a targeted MR contrast agent, and to evaluate its physico-chemical properties.2. To explore the cytotoxicity effect and the cell target effect of Gd2O3-FI-PEG-BBN nanoparticles with GRP receptor expressing human prostate cancer cell PC-3 in vitro.3. Utilizing Nude mice beamg human prostate tumor xenograft to investigate the feasibility and characteristic of Gd2O3-FI-PEG-BBN for targeting prostate cancer imaging in vivo.Materials and Methods:1. Synthesis and Characterization1.1 Synthesis1.1.1 Synthesize of the Gd2O3 NPsBriefly, Gd (OAc)3 (670 mg,2 mmol) was dissolved in DMSO (30 mL). Then tetramethylammonium hydroxide (TMAH,200 mg,5.6 mmol) in EtOH (10 mL) was added dropwise. then, the mixture was stirred for 2h. The Gd2O3 nanoparticles were collected by centrifugation. The particles were washed thoroughly by three cycles of centrifugation/redispersion in EtOH.1.1.2 Synthesize of Gd2O3-FI NPsGd2O3 (400mg) dispersed in DMSO was mixed with 100mg 5(6)-carboxy fluorescein, constant stirring for 12h at room temperature. The obtained product collected by centrifugation was washed respectively with DMSO, EtOH and DCM three times. Then, Gd2O3-FI nanoparticles were obtained.1.1.3 Synthesize of the Gd2O3-FI-PEG NPsa-carboxyl, w-hydroxy polyethylene glycol (PEG-2000) (100mg) added to DMSO containing 200mg Gd2O3-FI under constantly stirring for 12h at room temperature then centrifugation. The obtained product collected by centrifugation was washed respectively with DMSO, EtOH and DCM three times. Then, Gd2O3-FI-PEG nanoparticles were obtained.1.1.4 Synthesize of the Gd2O3-FI-PEG-BBN NPs:BBN (7-14) were synthesized according to a traditional Fmoc chemistry solid phase peptide synthesis method。Bombesin(7-14) (20mg), 1-[3-(Dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (EDC.HC1) (20mg), and 4-dimethylaminopyridine (10 mg) were mixed with 100mg Gd2O3-FI-PEG which was dispersed in DMSO. The mixture was stirred for 12h at room temperature then centrifuged. Precipitation was collected by centrifugation and washed with DMSO, EtOH and DCM three times respectively.1.2 Characterization1.2.1 The size and morphology of the synthesized NPs were characterized using a transmission electron microscope (TEM)Get one drop Gd2O3-FI-PEG-BBN suspension on amorphous carboncovered copper grids.and leave them for about five minutes, and then absorb redundant suspension from the edge of the gdds with filter paper. The morphology of Gd2O3-FI-PEG-BBN was observed with transmission electron microscopy when they were totally dried.1.2.2 The size and dispersity of Gd2O3-FI-PEG-BBN nanoparticles were characterized using Malvern Zetasizer 30000HS.Diluted samples of Gd2O3-FI-PEG and Gd2O3-FI-PEG-BBN nanoparticles suspension were measured with Malvem Zetasizer 30000HS using 633nm laser at the temperature of 25.00±0.05℃.1.2.3 The analysis of Fourier Transform Infrared Spectroscopy (FTIR) of Gd2O3 and Gd2O3-FI-PEG-BBN nanoparticlesIR spectroscopy studies were carried out on a FT-IR instrument using SeZn tablets. A few drops of sample were placed on a SeZn disk and dried with N2 to remove the solvent. Measurements were performed in the range 4000-400 cm-11.2.4 The thermogravimetric analysis of Gd2O3-FI-PEG-BBN nanoparticles:Thermal analysis was performed using a Netzsch TG 209 F1 Iris instrument. The temperature was increased to 710℃ at a rate of 10℃ min-1 (in 100 mL min-1 Nitrogen flux).1.2.5 In vitro MRI relativity measurementThe NPs were prepared previously in 0,0.25,0.5,1.0,2.0,4.0mmol/L of Gd concentrations respectively in 7 eppendorf tubes. T1WI were Acquired using a 3.0 T Philips Achieva whole-body scanner and a standard 8 channel head coil. A T1 weighted Image based on an TSE sequence (TR:100,200,400,600, 800,1000ms,TE:10ms., slice thick:4mm, gap:0.5mm, fov:120×100×60mm, NSA:2).2. In Vitro cell studiesGRPr positive PC-3 cells were from American Type Culture Collection and were cultured with complete growth medium (DMEM media containing 10% FBS) in a humidified incubator at 37℃ and 5% CO2. Cells in logarithmic growth period were used for assay.2.1 The Cytotoxicity assayCell viability of PC-3 cells were determined by MTT assay.3000 viable PC-3 cells/150μL media were added in each well of a 96-well plate.and then allowed to adhere to the plates overnight. Cells in 96-well plates were treated with different concentrations of Gd2O3-FI-PEG or Gd2O3-FI-PEG-BBN nanoparticles (0.125,0.25, 0.5,1,2,4,8mmol/L of Gd concentrations), compared with untreated (culture media) and blank control (solvent).PC-3 cells were then cultured for 24 or 48 h. At the end of incubation,20μL of MTT was added, and the cells were incubated for another 4 h. The culture medium was then carefully removed, and 150μL dimethyl sulfoxide (DMSO) was added to each well to dissolve the for mazan crystals for 10 min. The absorbance at 490 nm was measured by a microplate reader. The relative cell viability(%) related to control wells containing no NPs was calculated by [[A]test-[A]blank control]/[[A]untreated-[A]blank control]×100%.2.2 Binding and internalization Assay2.2.1 Visualization of the cellular uptake of NPs in vitro by fluorescence microscopy4×105 viable cells were seeded in each well of a 6-well culture plate and incubated for 18 hours. Removed the media and added in 2ml DMEM culture solution containing Gd2O3-FI-PEG-BBN or Gd2O3-FI-PEG suspension (0.8 mmol/L of Gd concentrations). The wells without NPs were used as blank. After 4 hours incubation, the cells were washed with PBS solution three times again, then fixed by 2 mL of 4% paraformaldehyde solution for 15 minutes. Rewashed three times after fixation, using 100ul diaminophenyl indole (DAPI) dye for nuclear staining lasting for 3-5 minutes, rewashed again. We can check the labeling effect with the fluorescence microscope.2.2.2 Visualization of the cellular uptake of NPs in vitro by flow cytometry4×105 viable cells were seeded in each well of a 6-well culture plate and incubated for 18 hours. Removed the media and added in 2ml DMEM culture solution containing Gd2O3-FI-PEG-BBN or Gd2O3-FI-PEG suspension (0.8 mmol/L of Gd concentrations). The wells without NPs were used as blank. After 4 hours incubation, the cells were washed with PBS solution three times again, de-attached with EDTA-trypsin at 37℃ for 1 minite.The samples were centrifuged for 3 min respectively, the supernatant was removed, and the cells washed twice and re-suspended in 500μL of PBS until analyzed by flow cytometry2.2.3 Visualization of the cellular uptake of NPs in vitro by MRIThe PC-3 cells were seeded equally in six 10cm Petri dishes, until their confluence of 60%, Gd2O3-FI-PEG-BBN or Gd2O3-FI-PEG suspension (0.8 mmol/L of Gd concentrations) was added to the dishes and incubated at 37℃ for 4h. The cells were then washed with PBS for 3 times and de-attached with EDTA-trypsin at 37℃ for 1 minite. Then cell pellets collected respectively were rewashed and resuspended in 500ul of 1% agarose solution in eppendorf tubes for further MR scan. A tube of 500ul of 1% agarose solution was used as control. The T1-weighted MR images were produced on a 3.0 T Philips Achieva whole-body scanner by using a 8 channel head coil. Images of PC-3 cells suspension in 500ul of 1% agarose solution were collected using a TSE sequence. Slice thickness was 4 mm and the stacks was 1mm. Other parameter settings were TR=500ms, TE=10ms, flip angle was 90°, field of view =100×60×20mm, NSA was 2. Then average T1 signal intensity and noise of each sample were measured. SPSS 16.0 statistical package was used as analyzing software. One way ANOVA Was used to analyze the difference of SNR among three group. SNK method was used to analyze the difference between two groups.P<0.05 was considered as significant difference.3.in vivo study3.1 Establishment of tumor-bearing nude miceMice were injected subcutaneously with 6×106 PC-3 cells suspended in 150μL phosphate buffered saline (PBS) in the right flank. When the tumor reached 1.0 to 2.0cm in diameter, the mice were randomized into two groups of targeted Gd2O3-FI-PEG-BBN and non-targeted Gd2O3-FI-PEG and prepared for imaging respectively.3.2 MRI of the tumor-bearing nude MiceTumor-bearing mice were injected through the tail vein with Gd2O3-FI-PEG-BBN (500uL,4mmol/L of Gd concentration). The control group was injected with 500μl Gd2O3-FI-PEG solution.We performed MRI on tumor-bearing mice before injection and 0.5h, 1h,2h,4h, 8h, and 24 h post-injection. In all animals, TSE T1 coronal were used.(repetition time [TR]=500ms, echo time [TE] range=10 ms, field-of view [FOV]=100×100× 17mm, slice=8, slice thickness=2 mm, gap=0mm, NSA= 3).The MR data were processed with Image J software and Philips workstation, the tumor ROI mask was copy to each time point. Signal intensity was measured. Paired-Samples T Test was used to analyze the difference of enhancement ratio after injecting with Gd2O3-FI-PEG and Gd2O3-FI-PEG-BBN solution. P<0.05 was considered as significant difference.3.3 Fluorescence immunohistochemistryTumor-bearing mice were sacrificed after MR imaging studies, The tumor was separated. The frozen section were observed for the fluorescence imaging in the tumor.Results:1. Synthesis and Characterization1.1. The morphology of Gd2O3-FI-PEG-BBN nanoparticles under transmission electron microscope (TEM)Gd2O3-FI-PEG-BBN nanoparticles were approximately spherical with uniform size, and the mean particle diameters were 50-60nm.1.2. The size and dispersity of Gd2O3-FI-PEG-BBN nanoparticles under Malvem Zetasizer 30000HSThe physical characteristics of particle sizes, hydrodynamic diameters, and polydispersity indices were obtained by Malvem Zetasizer 30000HS. It was shown that the average particle size with narrow size dispersity. the mean particle diameter was 90 nm.1.3 The analysis of Fourier Transform Infrared Spectroscopy (FTIR) of Gd2O3 and Gd2O3-FI-PEG-BBN nanoparticlesThe most prominent peaks of Gd2O3 were found in the range 1400cm-l-1600 cm-land are mainly assigned to originate from the antisymmetric, vs (COO-), and symmetric, vs (COO-), carboxylate stretching modes. The observed absorption frequencies characteristics the -OH stretching and the C-0 stretching vibration near 3427cm-1、1110cm-1, and the C-H stretches weakly at 2922 cm-1, the C=O stretches near 1600cm-1. After BBN and PEG molecules have coordinated to the Gd2O3 NPs, there was little change,-SH stretch of Met was hardly to detect.1.4.The TGA of Gd2O3-FI-PEG-BBN nanoparticlesA total weight loss of 51% was observed on heating Gd2O3-FI-PEG-BBN to 700℃. The curve tended to be flat in the range of 400℃-700℃.1.5 MRI measurementMR TIWI signal intensity of NPs samples increase gradually according to the increasing of Gd concentration.2. In Vitro cell studies2.1 The Cytotoxicity assayThe short-term cell viability (24h or 48h) of the Gd2O3-FI-PEG-BBN and Gd2O3-FI-PEG NPs were all above 80% in test dosage range, the decline trend seemed to be not obvious. It is an indication of their excellent biocompatibility in vitro.2.2 Binding and internalization assay2.2.1 Visualization of the cellular uptake of NPs in vitro by fluorescence microscopyThere was strong green fluorescence in the cytoplasm of PC-3 cells with strong blue nuclues in the Gd2O3-FI-PEG-BBN group, and whereas there was no significant binding in the Gd2O3-FI-PEG group. There was no green fluorescence in the cytoplasm of PC-3 cells with strong blue nuclues in the blank control group. These results clearly indicate that the binding of Gd2O3-FI-PEG-BBN nanoparticles was specifically mediated by the GRP receptor on their surface.2.2.2 Visualization of the cellular uptake of NPs in vitro by flow cytometryThe fluorescence intensity tested by flow cytometry could reflect the cell labeling effect. In the Gd2O3-FI-PEG-BBN group, The fluorescence intensity increased from 296 to 15326, while in the Gd2O3-FI-PEG group, The fluorescence intensity was only 2067. This experiment in vitro confirmed that Gd2O3-FI-PEG-BBN nanoparticles had the ability to target PC-3 cells mediated by GRP receptors on their surface.2.2.3 The MR images of cellular uptake of NPs in vitroThe signal noise ratio of the PC-3 cells incubated with Gd2O3-FI-PEG-BBN was significantly higher than the control groups, the blank control group and 1% agarose solution. The difference was statistically significant (P<0.05).while the difference between the non-targeted control groups and the blank control was not statistically significant (P>0.05). Overall, the in vitro cell imaging experiments demonstrated that Gd2O3-FI-PEG-BBN could realize in vitro MR image enhancement, and the Gd2O3-FI-PEG-BBN could be used for targeted MR imaging of GRPr-expressing cancer cells.3. in vivo study3.1 Establishment of tumor-bearing nude mice:We established the tumor-bearing nude mice successfully. After 30-40 days, the tumor reached 1.0 to 2.0 cm in diameter, They were randomly divided into two groups.n=5.3.2 MR imaging in vivoThe MR imaging was performed at different time points after administered Gd2O3-FI-PEG-BBN or Gd2O3-FI-PEG via tail vein. It could been observed increased T1 signal intensity in the tumor area, and peak value was appeared at 2h post-injection. A relatively significant enhancement of T1 signal intensity was appeared in the tumor region of mice administrated Gd2O3-FI-PEG-BBN, In comparison, the T1 signal enhancement of tumor in mice received Gd2O3-FI-PEG was not so significant. The enhancement ratio of the targeted group in 2h post-injection was 27.95+5.86%, while that of the non-targeted group in 2h post-injection was 4.02+2.24%. The difference between two group was statistically significant (P<0.05).These results indicated as expected that the GRPr-targeted NPs could actively accumulate in tumors, meanwhile, induced the enhancement of MR signals in the tumor region.3.3 Fluorescence immunohistochemistryThe results showed that Gd2O3-FI-PEG-BBN with green fluorescence was observe in tumor cells, which confirmed that Gd2O3-FI-PEG-BBN had been uptaked and accumulated in tumor cells.Conculsion:We synthesized and evaluated Bombesin-assembled fluorophore-equipped gadolinium oxide nanoparticles (Gd2O3-FI-PEG-BBN NPs) with relative good water dispersibility,favorable biocompatibility and efficient T1 relaxivities, the nanoparticles could accumulate in prostate tumor tissues selectively.These favorable properties make Gd2O3-FI-PEG-BBN nanoparticles a suitable molecular probe for tumor-targeted MR and fluorescence imaging.