The Preliminary Experiment Of Molecular Probe Labeled With ¹⁸⁸Re

Partâ… radiosynthesis and biological experiment of ¹⁸⁸Re-IGF-1ABackground The insulin-like growth factor-1 receptor (IGF-1R) is a cellular receptor found to be overexpressed in many tumor cell lines from different anatomical sites. It can be used as molecular targets by which radiolabeled insulin-like growth factor 1 analogue (IGF-1A) can localize cancers for peptide receptor radiation therapy.1. Investigations of labeling insulin-like growth factor 1 analogue with ¹⁸⁸ReObjective To establish a useful method for labeling IGF-1A with ¹⁸⁸Re. Methods Direct labeling method was adopted to label IGF-1A. Several labeling conditions were tested. The volume of Tween80 was changed from 2 to 10μL; the concentration of SnCl₂.2H₂O was changed from 0.75 to 25mg/mL; the amount of IGF-1A was changed from 20 to 100μg and the volume of ¹⁸⁸Re perrhenate was changed from 10 to 500μL. The labeling efficiency was analyzed from 15min to 8h after labeling. The in vitro stabilities of ¹⁸⁸Re-IGF-1A was analyzed in human serum or sodium chloride medium, and the labeling efficiency was determined from 2 to 24h after adding the medium. Results The optimum labeling condition was 100μL stannous chloride (10mg/mL) dissolved in sodium gluconate, 50μL IGF-1A (2mg/mL), 300μL Na3PO4, 10μL 0.1% Tween80, 50μL ¹⁸⁸Re perrhenate added and the incubation time was 30 min at room temperature, then added 500μL NaH₂PO₄ to adjust the pH about 7.0. The labeling efficiency of ¹⁸⁸Re-IGF-1A could reach (94.07±0.32)% and the amount of radiocolloid was (5.50±1.50)%. It was (85.50±1.21)% after incubation for 6h at room temperature, and was (76.57±9.96)% after incubation for 24h with human serum. Conclusion This method of labeling IGF-1A with ¹⁸⁸Re using SnCl₂.2H₂O is stable and high labeling efficiency can be obtained. 2. Experimental study of ¹⁸⁸Re-IGF-1A on cell combination and proliferation inhibition effect to pancreatic carcinoma cellObjective To investigate cell combination and proliferation inhibition effect of ¹⁸⁸Re-IGF-1A to Patu8988 human pancreatic carcinoma cell. Methods (1)The combinative efficiency of ¹⁸⁸Re-IGF-1A with Patu8988 cell was determined. (2)Patu8988 cell were seeded onto 96-well plate and divided into blank control group, IGF-1A group (1, 5, 10, 20μg), ¹⁸⁸ReO4- group (0.37, 1.85, 3.70, 7.40MBq) and ¹⁸⁸Re-IGF-1A group (0.37, 0.74, 1.85MBq). (3)The proliferation inhibition effect of ¹⁸⁸Re-IGF-1A group and ¹⁸⁸ReO4- group on cell growth was detected every day by MTT test from 1d to 7d after administration. The proliferation inhibition effect of IGF-1A group was detected every day from 1d to 6d, and inhibition rates were calculated. (4) At 3d after treatment with ¹⁸⁸ReO4- and ¹⁸⁸Re-IGF-1A (1.85, 3.70, 7.40 MBq), the cell's apoptosis was detected by flow cytometry. Results (1)The total combinative efficiency of ¹⁸⁸Re-IGF-1A with Patu8988 cell was (24.13±2.03)%, and the special combinative efficiency was 12.68%. (2) Inhibition rates of Patu8988 cell with ¹⁸⁸Re-IGF-1A group (0.37, 0.74, 1.85MBq) were (64.48±4.18)%,(66.89±1.39)% and (89.71±1.27)% after 3d, and the inhibition rates was higher when the radioactive dose higher. After 6d, inhibition rate of Patu8988 cell with ¹⁸⁸Re-IGF-1A group (1.85MBq) was (93.20±1.93)%. (3) Inhibition rates of Patu8988 cell with ¹⁸⁸Re-IGF-1A group were always higher than ¹⁸⁸ReO4- group and IGF-1A group. The difference was significant (P < 0.05). (4) At 3d after treated with ¹⁸⁸ReO4- and ¹⁸⁸Re-IGF-1A, float cell ratio were (16.58±3.57)%, (24.58±6.50)%, (34.12±7.39)% and (16.56±0.95)%, (33.39±5.93)%, (43.76±1.38)%, respectively. Float cell's apoptosis ratio of Patu8988 cell with ¹⁸⁸ReO4- and ¹⁸⁸Re-IGF-1A group were (9.27±1.80)%, (16.00±1.15)%, (15.47±0.65)% and (12.70±2.27)%, (17.80±1.51)%, (23.23±1.22)%, respectively. Conclusion Proliferation of human pancreatic carcinoma cell Patu8988 can be inhibited and apoptosis can be promoted with ¹⁸⁸Re-IGF-1A.3. Biodistribution characteristics and dosimetry measurement of ¹⁸⁸Re-IGF-1A in nude mice bearing the Patu8988 human pancreatic cancer xenograftsObjective To investigate the biodistribution characteristics and measure dosimetry of ¹⁸⁸Re-IGF-1A in nude mice bearing human pancreatic cancer xenografts. Methods (1)66 nude mice model bearing human pancreatic carcinoma cell Patu8988 xenografts were established and devided into the ¹⁸⁸Re-IGF-1A group (n=36) and the ¹⁸⁸ReO4- control group (n=30). (2)In the ¹⁸⁸Re-IGF-1A group, at 15min, 1, 4, 24, 72, and 120h after intratumor injection with ¹⁸⁸Re-IGF-1A, the mice were killed and the organs of interest were excised, weighted and counted on a gamma counter. The organ uptake was calculated as a percentage of the injected dose per gram of wet tissue (%ID/g) and the ratios of tumor to normal tissue (T/NT) were calculated. (3) In the ¹⁸⁸ReO₄^- control group, organs of interest were excised and counted. %ID/g and T/NT were calculated at 15min, 1, 2, 4 and 24h after intratumor injection. (4) Scintigraphy imaging for each nude mouse was performed at 15min after intratumor injection with radiopharmaceuticals,in which serial planar scintigraphy imaging were performed for 6 nude mice at 15min, 1, 4, 24, 72, and 120h after intratumor injection with ¹⁸⁸Re-IGF-1A. (5) Radiation dosimetry in organs of interest were estimated on the basis of the mean %ID/g. Results (1) ¹⁸⁸Re-IGF-1A was major distributed in tumors and the largest uptake of tumors was (41.22±23.88)% at 4h after injection. The uptake of tumor were (10.59±9.39)% and (5.30±2.28)% at 24h and 120h after injection, respectively. (2)¹⁸⁸ReO₄^- was major distributed in thyroid glands, stomachs, tumors and blood in nude mice after injection at first. Then uptake of tumor decreased rapidly, but slowly in stomachs and thyroid glands. The uptake of tumor were (0.09±0.03)% at 24h after injection. (3) The tumor in nude mice could be seen clearly in scintigraphy images until 120h after intratumoral injection with ¹⁸⁸Re-IGF-1A. Conclusion ¹⁸⁸Re-IGF-1A was major distributed in tumors after intratumor injection in nude mice bearing human pancreatic cancer xenografts, and we propose that ¹⁸⁸Re-IGF-1A may be a potential tumor transarterial radionuclide therapeutic agent used in clinic.Partâ…¡radiosynthesis and biological experiment of ¹⁸⁸Re-k-ras-AGPNABackground We hypothesized that antigene peptide nucleic acid (AGPNA) hybridization probes targeted k-ras 12 point mutation oncogene, with an IGF-1A peptide loop on the C-terminus and ¹⁸⁸Re chelator peptide on the N-terminus, could hybridize with k-ras 12 point mutation oncogene and decrease the multiplication of pancreatic cancer cell Patu8988.1. Experiment of k-ras-AGPNA on depress the k-ras gene expression in human pancreatic cancer cell Patu8988 Objective To investigate the effect of AGPNA on depress the k-ras gene expression and the k-ras protein expression of human pancreatic cancer cell Patu8988. Methods (1)AGPNA hybridizing k-ras 12 point mutation DNA was designed and synthesized. (2)Before and after transfected AGPNA, the expression level of k-ras mRNA in human pancreatic cancer cell Patu8988 were detected by reverse transcription- polymerase chain reaction (RT-PCR). (3)The expression ratio of k-ras protein in cell were detected by flow cytometry. Results (1)After transfected 1nmol/mL AGPNA and antigene oligonucleotides (AGON), the k-ras mRNA gray scale ratio were (1.00±0.39) and (1.22±0.31). Its was lower than (1.86±0.07) of the control group, and the difference was significant (P<0.01). Among the difference dose of AGPNA, AGON, and antisense oligonucleotides (ASON) group, the difference of the expression level of k-ras mRNA was not significant (P>0.05). (2)After transfected 1nmol/mL AGPNA, AGON, and ASON, the expression ratios of k-ras protein were (15.05±5.07)%, (10.20±2.63)%, and (8.80±4.31)%. Its was lower than (24.38±5.40)% of the control group, and the difference was significant (P<0.01). Compared with the difference dose of AGPNA, AGON, and ASON group, the difference of the expression ratios of k-ras protein was no significant (P>0.05). Conclusion AGPNA can depress the k-ras gene expression on the mRNA level and depress the k-ras protein expression of human pancreatic cancer cell Patu8988.2. Radiosynthesis of ¹⁸⁸Re labeled k-ras-AGPNA for binding with pancreatic carcinoma cellObjective To establish a method for labeling k-ras-AGPNA with ¹⁸⁸Re, and investigate its binding with pancreatic cancer cell Patu8988. Methods The directly labeling method was adopted, and several labeling conditions were tested, such as the concentration of SnCl2.2H2O and the amount of k-ras-AGPNA. The labeling efficiency was determined from 15min to 6h after labeling. The in vitro stability of ¹⁸⁸Re-k-ras-AGPNA was analyzed by using human serum or sodium chloride as medium, and the labeling efficiency was determined from 2 to 24h after labeling. The total binding efficiency of ¹⁸⁸Re-k-ras-AGPNA with pancreatic cancer cell Patu8988 and the bound of ¹⁸⁸Re-k-ras- AGPNA on the cellular nucleus were determined at various time points. Results The optimum labeling conditions were 100μL stannous chloride (20mg/mL) dissolved in sodium gluconate, and 20μL k-ras-AGPNA (2mg/mL), 100μL ¹⁸⁸Re perrhenate, the pH about 7.0, and incubated 30 min at room temperature. The labeling efficiency of ¹⁸⁸Re-k-ras-AGPNA could reach (89.61±0.91)% and the amount of radiocolloid was (9.40±0.55)%. The labeling efficiency was (89.14±0.63)% after incubated for 24h in human serum. The highest total binding efficiency of ¹⁸⁸Re-k-ras-AGPNA with Patu8988 cell was (38.16±2.17)%, and the highest binding efficiency on the nucleus was increased to (22.41±0.86)% at the 24th hour. Tumor in nude mice could be seen clearly in scintigraphy images until 7d after injection. Conclusion This method of labeling k-ras-AGPNA with ¹⁸⁸Re is stable and high labeling efficiecy can be obtained. ¹⁸⁸Re-k-ras-AGPNA can bind with the pancreatic cancer cell Patu8988 and penetrated into the Patu8988 cellular nucleus.3. Inducing apoptosis effect on Patu-8988 cell and biodistribution characteristics of the ¹⁸⁸Re-k-ras-AGPNA in nude mice bearing xenograftsObjective To investigate the inducing apoptosis effect on Patu8988 cell and biodistribution characteristics, determine the dosimetry in nude mice bearing xenografts of ¹⁸⁸Re-k-ras-AGPNA. Methods (1) At 3d to 5d after treatment with Patu8988 cell in culture flask with 925KBq ¹⁸⁸ReO₄^- or ¹⁸⁸Re-k-ras-AGPNA, the cell's apoptosis was detected everyday by flow cytometry. (2) 28 nude mice model bearing human pancreatic carcinoma cell Patu8988 xenografts were established. At 15min, 1, 4, 24, 72, 120 and 148h after intratumor injection with ¹⁸⁸Re-k-ras-AGPNA, the mice were killed and organs of interest were excised, weighted and counted on a gamma counter. The organ uptake was calculated as a percentage of the injected dose per gram of wet tissue (%ID/g) and the ratios of tumor to normal tissue (T/NT) were calculated. (3) Scintigraphy imaging was performed for each nude mice at 15min after intratumor injection with radiopharmaceuticals, in which serial planar scintigraphy imaging were performed for 6 nude mice at 15min, 1, 4, 24, 72, 120 and 148h after intratumor injection with ¹⁸⁸Re-k-ras-AGPNA. (4) According the mean %ID/g of organs in different time point, the absorbed dose of organs were calculated. Results (1)At 3d to 5d after treated with ¹⁸⁸ReO₄^- and ¹⁸⁸Re-k-ras-AGPNA, float cell ratio were (5.68±0.82)%, (8.14±0.12)%, (11.87±0.17)% and (5.99±3.59)%, (25.66±8.51)%, (29.59±4.92)%, respectively. Float cell's apoptosis ratios of ¹⁸⁸ReO₄^- and ¹⁸⁸Re-k-ras- AGPNA group were (3.88±2.10)%, (8.75±3.11)%, (16.87±5.85)%, and (5.28±1.12)%, (26.30±7.45)%, (27.90±10.38)%, respectively. (2) ¹⁸⁸Re-k-ras-AGPNA was major distributed in tumors and the largest uptake of tumors was (53.23±16.64)% at 15min after injection. The uptake of tumor were (35.96±7.80)% and (15.46±4.93)% at 24h and 148h after injection, respectively. (3) Tumor in nude mice could be seen clearly in scintigraphy images until 148h after intratumoral injection with ¹⁸⁸Re-k-ras-AGPNA. (4) The absorbed dose of tumor was 15569mGy/MBq. Conclusion ¹⁸⁸Re-k-ras-AGPNA was major distributed in tumors after intratumor injection in nude mice bearing human pancreatic cancer xenografts, and we propose that ¹⁸⁸Re-k-ras-AGPNA may be a potential tumor transarterial radionuclide therapeutic agent used in clinic.Partâ…¢The biodistribution characteristics of ¹⁸⁸Re-tin sulfur colloid after transhepatic arterial embolization in rabbits bearing VX2 liver tumorObjective To investigate the method for labeling tin sulfur colliod (TSC) with rhenium-188 and its biodistribution after transhepatic arterial embolization in rabbits bearing VX2 liver tumor. Methods The direct method was adopted to label TSC or macroaggregated albumin(MAA) with ¹⁸⁸Re-perrhenate, and labeling efficiency was determined at the different time point. The in vitro stabilities of ¹⁸⁸Re-TSC and ¹⁸⁸Re-MAA were analyzed by in the medium of sodium chloride or human serum (v:v=1:10), and the labeling efficiency was determined by Xinhua 1# paper chromatography (PC) analysis from 30min to 72h after added to medium. Particle size of ¹⁸⁸Re-TSC was measured (n=32) by laser scatterance/diffraction method used a Mastersizer 2000. 31 rabbits bearing VX2 liver tumor were performed transcatherter hepatic arterial injection of ¹⁸⁸Re-TSC (n=12), ¹⁸⁸Re-MAA (n=15), and Na¹⁸⁸ReO₄^- solution (n=4), respectively. Planar scans with IRIX3 SPECT (PHILIPS, Holander) were obtained at 1 and 24h after administration. The biodistribution characteristics of ¹⁸⁸Re-TSC, ¹⁸⁸Re-MAA, and ¹⁸⁸Re-perrhenate in rabbits at 1 and 24h after administration were evaluated. Organs were excised, washed with saline, weighed, and counted on a gamma counter. The organ uptake was calculated as a percentage of the injected dose per gram of wet tissue (%ID/g). Tumor retention was calculated and compared between ¹⁸⁸Re-TSC and ¹⁸⁸Re-MAA. Results The labeling efficiencies of ¹⁸⁸Re-TSC and ¹⁸⁸Re-MAA were reached (99.94±0.04)% and (99.95±0.03)%, and radiopharmaceuticals were stable for 72h in human serum. The particle size of ¹⁸⁸Re-TSC was 10.83±6.60μm (D10), 44.91±14.46μm (D50), and 235.29±126.61μm (D90), respectively. In 17 of the 27 rabbits, TAE was performed successfully. The radioactive ratio of tumor/liver in ROI of SPECT images was 2.15±0.80 and the tumor can be seen clearly in planar imaging. Tumor uptake of ¹⁸⁸Re-TSC at 1h and 24h were (24.32±11.93)% and (21.88±18.29)%, and the radioactive ratio of tumor/liver were 70.89±19.58 and 17.42±13.96, respectively. Tumor uptake of ¹⁸⁸Re-MAA at 1h and 24h were (38.78±30.23)% and (15.98±26.64)%, and the radioactive ratio of tumor/liver were 39.71±25.06 and 8.13±4.61, respectively. The retention ratio of 24h to 1h of ¹⁸⁸Re-TSC in tumor was 89.95%, and it was 41.22% of ¹⁸⁸Re-MAA. Conclusion ¹⁸⁸Re-TSC may be a potential radiopharmaceutical for the therapy of tumors.