10 mg of bombesin was dissolved by adding 2 ml of dmf . after the bombesin completely dissolved , 18 . 94 , of tea was added to the solution and stirred for 1 hr under nitrogen gas . on the other hand , 21 . 5 mg of dspe - peg - nhs was dissolved in 2 ml of dmf to completely dissolve and then dropped into the above solution to stir for 24 hr under nitrogen gas . the solvent was removed after the reaction finished . an excess of chloroform was added to the resultant solid product and the solution kept standing to carry out precipitation and was then filtered through a filter paper no . 42 . the precipitation was collected and dissolved in 2 ml water . the product was separated through a column of sephadex g - 25 with water as an eluent . the product was confirmed its location and purity by a bca protein assay and then collected by removing the solvent through a lyophilized . the product also was analyzed by hplc - elsd through a column of xterra msc18 ( 5 μm ) with 90 % water and 10 % methanol as an eluent and 10 minutes as analytic time . the retention time was 4 . 5 minutes . the product was analyzed average molecular weight by maldi - tof / tof as [ m + h ] + = 3751 da . 5 mg of bmeda and 0 . 5 ml of 0 . 17 mol / l glucohepatonate and 120 μl ( 10 μg / μl ) of stannous chloride were pipetted into a fresh vial , then flushing nitrogen gas for 2 minute to avoid the oxygenation of stannous chloride . 1 ml of highly specific activity of 188 re - sodium perrhenate were added , then sealed vial . the sealed vial was heated in an 80 ° c . water - bath for 1 h . the vial was cool down at room temperature , adjust ph to neutrality ( ph 6 ˜ 7 ) with 120 ˜ 150 μl of 5n naoh by slowly pipetting . the labeling efficiency of the 188 re - bmeda complexes was checked by paper chromatography with normal saline as the eluent . the labeling efficiency of 188 re - bmeda complexes was 90 ˜ 100 % ( rf : 1 , free 188 re ; rf : 0 , 188 re - bmeda ). 10 μl of dspe - peg 2000 - bbn ( 40 mg / ml ) and 188 . 5 μl dxr ( 10 mg / ml ) and 1 ml of liposomes encapsulating ( nh 4 ) 2 so 4 were mixed with 0 . 5 ml of 188 re - bmeda solution , and then incubated in a 60 ° c . water - bath for 30 mins . sephagrose cl - 6b column ( ge healthcare bio - sciences ab , sweden ) chromatography with normal saline was used to separate 188 re - dxr - liposome - bbn from free 188 re - bmeda and free dxr . eluted 188 re - dxr - liposome - bbn solution was collected in 0 . 5 ml into each tube for total 30 tubes . the yield of 188 re - dxr - liposome - bbn was calculated according to the following standard formula : labeling efficiency (%)=[ 100 ×( radioactivity of fractions with 188 re - dxr - liposome - bbn /( total fraction radioactivity + column residue )]. the yield of 188 re - dxr - liposome - bbn was 75 ˜ 85 % ( fig2 ). 1 ml acidic isopropanol ( 81 mm in isopropanol ) was mixed with 0 . 2 ml diluted dxr - loaded liposomes , the amount of doxorubicin trapped inside the liposome was determine with a spectrofluorometer ( fp6200 , jasco ) at an excitation wavelength of 475 nm and an emission wavelength of 580 nm . the concentration of liposomes was estimated by the phosphate assay ( bartlett , 1959 ). in this preparation ( n = 3 ), dxr - loaded liposomes contained 120 ˜ 160 μg / μmole phospholipid . particle size of liposome were measured by dynamic laser scattering with a particles analyzer ( nano zs90 , malvern , uk ). particle sizes ranged from 90 ˜ 110 nm in diameter ( table 1 ). condition microspin column with 2504 , normal saline ( g50 , ge healthcare bio - sciences ab , sweden ), then add 254 , liposome sample into the center of microspin column . the column was centrifuged with 3000 rpm for 2 mins and the eluted solution was collected in a fresh tube . then eluted the column with another 254 , normal saline and collected the eluting solution in the same tube . measured the amount of doxorubicin trapped inside the liposome . the dxr encapsulating efficiency of 188 re - dxr - liposome - bbn was calculated according to the following standard formula : encapsulating efficiency (%)= 100 ×{( the total volume of 188 re - dxr - liposome - bbn after purification )×( the concentration of 188 re - dxr - liposome - bbn after purification )/ 25 ×( the concentration of 188 re - dxr - liposome - bbn before purification )}. the dxr encapsulating efficiency of 188 re - dxr - liposome - bbn was larger than 85 %. cold competition receptor binding assay was studied using human bombesin 2 receptor expressed in hek - 293 cells as the source of grp receptor ( perkinelmer , boston , mass ., usa ). assays were performed using fc96 plates and the multiscreen system ( millipore , bedford , mass .). binding of 125 i - tyr 4 - bombesin ( perkinelmer , boston , mass ., usa ) to pc - 3 cell membranes ( 0 . 16 g per well ) was determined in the presence of increasing concentrations ( 0 . 001 nmole / l to 1000 nmole / l ) of bombesin - finer , dspe - peg - bbn and liposome - bbn in a buffer solution ( 20 mmol / l hepes , ph 7 . 4 , 3 mmol / l mgcl 2 , 1 mmol / l edta and 0 . 3 % bsa ) with a total volume of 250 μl per well . after incubation for 120 min at rt , membranes were filtered and washed with ice - cold tris - hcl buffer ( 50 mmol / l ). the filters containing membrane - bound radioactivity were counted using a cobra ii gamma - counter ( packard , meriden , conn .). the inhibitory concentration of 50 % ( ic 50 ) was calculated using a 4 - parameter curve - fitting routine using the excel software . as shown in fig1 , for the receptor binding with grpr , the ic 50 of bombesin - iner , dspe - peg - bbn and liposome - bbn was 0 . 186 , 0 . 627 and 4 . 480 nm respectively . the ki of bombesin - iner , dspe - peg - bbn and liposome - bbn was 0 . 146 , 0 . 494 and 3 . 527 nm respectively . the cytotoxic activity assay of 188 re - dxr - liposome - bbn on pc - 3 human prostate cancer cell line was measured with an countess ™ cell counter ( invitrogen , carlsbad , calif ., usa ). adherent pc - 3 cells were seeding on 25t flasks . after growth overnight , pc - 3 cells were treated with a medium containing 188 re - bmeda ( 30 . 5 μci / ml ), 188 re - liposome - bbn ( 188 re - lb , 30 . 5 μci / ml ), dxr - liposome - bbn ( ldb , 32 μg / ml ), 188 re - dxr - liposome - bbn ( 188 re - ldb , 32 μg / 30 . 5 μci / ml ) or control ( normal saline ) at 37 ° c . for 1 h . additional normal groups were performed without any addition of drugs in medium . after washing with cold pbs , cells were additionally incubated at 37 ° c . for 2 days . cells were stained with trypan blue and analyzed for cell viability using a countess ™ cell counter . the cell viability was calculated using the following formula : as shown in fig2 , the results of cytotoxic activity assay demonstrated that 188 re - ldb have the superior cytotoxic activity on pc - 3 human prostate cancer cell line . the cell viability of 188 re - ldb in this study is 28 . 6 ± 3 . 7 %. microspect imaging and images semi - quantification analysis of targeted 188 re - liposome - bbn imaging was acquired using low - energy , high - resolution collimators at 1 , 24 , 48 and 72 hr after intravenous injection of 188 re - liposome - bbn . when the imaging acquisition , the mice were anesthetized with 1 ˜ 2 % isoflurane in 100 % o 2 . the energy window was set at 155 kev ± 10 ˜ 15 %, the fov ( field of view ) was 12 . 5 cm . spect imaging was followed by ct image acquisition ( x - ray source : 50 kv , 0 . 4 ma ; 256 projections ) with the animal in exactly the same position . images were calibrated to standardized uptake values ( suv ). for calculate standardised tumor uptake value ( stuv ), known radio activity re - 188 was performed as reference . the suv was determined from the regions of interest ( roi ) on the tumor with uptake . the suv was calculated according to the following standard formula : as shown in fig3 , the images revealed a high uptake in tumors at 1 and 24 h after intravenous injection . the suv of 188 re - liposome - bbn in tumor was 1 . 54 and 1 . 25 at 1 and 4 h after injection , respectively .
one pot process of preparing multifunctional liposome drug is provided . in this one pot process , liposome reacted with radionuclide labeled solution , chemotherapy drug , and targeted ligand at appropriate temperature . the product in this invention for preparation multifunctional liposome drugs in for imaging , delivery and targeting in cancer diagnosis and therapy has proved to be more simple , convenient , effective and easier than the prior art is .