| The mRNA of breast carcinoma c-erbB2 oncogene was used as target and its antisense oligodeoxynucleotide (ASODN) as the carrying agent of radionuclide. By usage of c-erbB2 ASODN labeled with 99mTc complementarily binding to mRNA of target gene and then combination of epidermal growth factor receptor (EGFR) by nanotechnology, which were used to increase uptake of ASODN by cancer cells, a new method with high sensitivity and targeting for breast carcinoma radionuclide imaging was supposed to be constructed for the early diagnosis of breast carcinoma.The tissue distributions of EGFR and c-erbB2 oncogene were in consistency. Common overexpression of these two was simultaneously observed in breast carcinoma cells while their quantities in normal breast tissue were very low. So EGF was conjunct to albumin nanoparticle and then targeting nanoparticle was prepared. Consequently, its advantages were as follows: Firstly, the combination of EGF with its receptors on breast carcinoma cells can increase the aggregation of drug-carrying nanoparticle on the membrane of carcinoma cells, then expands the concentration difference between in and out of cells thus help the carcinoma cells uptake ASODN and raise the ratio of target/non-target; Secondly, the specific binding of receptor to its ligand can promote the carcinoma cells phagecytosis of nanoparticle and increase the velocity and quantity of ASODN entering carcinoma cells, thus raising its binding velocity and quantity of ASODN to mRNA of the target gene.Albumin is of safety and non-immunogenicity. It has outstanding biocompatibility and can be biodegraded. The nanoparticles prepared with albumin may improve the ability of ASODN entering cells and protect the ASODN against nuclease. In the present research, thio-chemical modification and albumin nanoparticles were used for the increase of the ASODN stability, and to keep it from being degraded by nuclease.METHODS:Part 1: Radiolabeling of c-erbB2 ASODN and identification.ASODN against c-erbB2 mRNA 5'terminal transcription initiation point, correlated sense sequence and non-sense sequence were synthesized. Oligodeoxynucleotides (ODN) were linked with L,L-ethylenedicysteine (EC) by chemical technology and EC-ODN were purified with Sep-Pak C18 reversed-phase column. SnCl2 and natrium tartaricum were used for 99mTc labeling. Purified by Sephadex G25 column chromatography, labeling yield, specific activity and radiochemical purity were tested by thin-layer chromatography. The radiochemical purity of 99mTc-EC-ODN at ambient temperature and incubated in fresh human serum at 37℃was determined for research on its stability. 99mTc-EC-ODN was incubated in plasma at 37℃and then plasma protein combination rate was measured. EC-ODN stored under -20℃were taken out at different time points for 99mTc labeling to assess their stability by the change of labeling yield. The activity of ASODN labeled with 99mTc was observed by the change of elutriant radiation peak in Sep-Pak C18 reversed-phase column after 99mTc-EC-ASODN combined with its complementary strand and by strips in polyacrylamide gel electrophoresis (PAGE).Part 2: Preparation and identification of targeting nanoparticle and its entrapment of ASODN labeled with radionuclideBovine serum albumin nanoparticles (BSANP) were prepared by twice ultrasonic emulsification and solvent volatilization technology. The particle diameter and their distribution were determined with laser particle size analyzer. Shapes were observed on transmission electron microscope. BSANP and EGF were connected by chemical technology. EGF-BSANP was separated and purified with Sephadex G50 column, and its particle diameter and distribution were determined. The linkage situation of EGF-BSANP was identified by SDS-PAGE. The linking quantity of EGF on EGF-BSANP was measured with 125I-EGF. Uptake of EGF-BSANP and BSANP by carcinoma cells was observed by radio-quantitive method. ASODN labeled with radionuclide were entrapped with targeting nanoparticle by packing and precipitated with 10% trichloracetic acid, and then drug-loading rate and drug-releasing rate at different time points were determined, drug-releasing curve was drawn. Changes of radiochemical purity in targeting nanoparticles loading 99mTc-EC-ODN under room temperature and in serum were determined by paper chromatography for observation on the stability of 99mTc-EC-ODN.Part 3: Targeting performance of targeting nanoparticles loading 99mTc-EC-ODN on breast carcinoma cells in vitro.1. MDA-MB-231 cells were used as control. Uptake of targeting nanoparticles loading 99mTc-EC-ODN and 99mTc–ODN not entrapped with nanoparticles by SK-Br3 cells was observed. The uptake rate was calculated after cultured for different time under the corresponding treatment factors.2. Intracellular stagnation rate were calculated after cultured for different time with corresponding treatment factors cleared.3. Effects of targeting nanoparticles loading 99mTc–ODN on the vigour of SK-Br3 cells and MDA-MB-231 cells were determined by MTT method.4. Influence of targeting nanoparticles loading 99mTc–ODN on the c-erbB2 mRNA expression in SK-Br3 cells was determined by RT-PCR semiquantitative analysis.5. Influence of targeting nanoparticles loading 99mTc–ODN on the c-erbB2 protein expression in SK-Br3 cells was measured by Western blot. Part 4: Distribution of c-erbB2 targeting nanoparticles loading ASODN labeled with 99mTc in Jimpy mice and imaging in bearing cancer nude mouse by SPECT.Distributions of targeting nanoparticles loading 99mTc–ASODN in normal Jimpy mice and bearing human breast carcinoma nude mice were carried out. SPECT imaging research on targeting nanoparticles loading 99mTc–ASODN in bearing human breast carcinoma nude mice was carried out.RESULTS:Part 1: Radiolabeling of c-erbB2 ASODN and identification. Labeling yield of EC-ASODN with 99mTc was 81.2%±7.9% and specific activity, (3.1±0.7)MBq/μg.The first radioactive peak after separated and purified with Sephadex G25 column was 99mTc-EC-ASODN. Radiochemical purity (average more than 80%) subtly declined after 24h. Incubated in fresh human serum at 37℃, radiochemical purity determined by paper chromatography at 4h (average more than 85%) was slightly lower than that at 1h.Plasma protein combination rate of 99mTc-EC-ASODN in fresh human plasma at 1h was (8.2±2.1)%; at 6h, (11.7±2.5)%. As for 99mTc-EC-ODN, at 1h, (8.6±2.4)%; at 6h, (12.8±3.1)%. 99mTc-EC-NSODN, at 1h, (8.3±1.7)%; at 6h, (10.2±3.6)%.Labeling yield of EC-ODN with 99mTc stored at -20℃1d later was (81.0±6.4)%; 15d later, (80.2±5.7)%; 30d later, (78.0±5.2)%; 90d later, (75.2±5.5)%.The polarity of 99mTc-EC-ASODN binding to its complementary strand declined after incubated in human serum at 37℃. After hybridizated with its complementary strand, ASODN was treated by PAGE and then two clear strips were observed.Part 2: Preparation and identification of targeting nanoparticle and its entrapment of ASODN labeled with radionuclideObserved on transmission electroscope, the nanoparticles were spherical and of uniformity. 90% of the BSANP particle diameters determined with laser particle size analyzer were smaller than 38nm with an average of 34nm while 90% of EGF-BSANP smaller than 39nm with an average of 36nm, a little larger than that of BSANP.SDS-PAGE displayed that the BSANP strip was near to Marker 68kDa while EGF-BSANP near to Marker 75kDa.Linkage rate of EGF and BSANP in five groups with different quantities of EGF ranged from 92.3% to 98.9%.Uptake rate of EGF-BSANP at concentration of 0.1mg/ml～2.0mg/ml by SK-Br3 cells at 4h ranged from 83.7% to 99.2% while BSANP, from 25% to 30%. As culture time went on, quantity of EGF-BSANP uptaken by SK-Br3 cells increased with value of (50.3±3.7)% at 0.5h, (97.2±5.5)% at 4h. However, the uptake rate of BSANP remained at 25%～35%. With imaging agent at same concentration, the entrapment rate and drug-carrying quantity of three targeting nanoparticles carrying c-erB2 ASODN were not of significant difference. However, with the increase of marker concentration, entrapment rate gradually decreased while drug-carrying quantity increased.Trend of the three nanoparticles carrying ASODN was almost similar, which displayed the slow release of the entrapped drugs.The radiochemical purity of targeting nanoparticles carrying 99mTc-EC-ASODN 24h after stored in neutral solution at room temperature was (88.6±6.3)%. 99mTc was uneasy to fall off 99mTc-EC-ODN. These ASODN were more stable than those not entrapped by nanoparticles at room temperature. The radiochemical purity in serum at 4h was (92.7±6.1)% while at 0.5h, (95.5±8.1)%.These two were of not significant difference (P>0.05). These ASODN were more stable than those not entrapped by nanoparticles in serum.Part 3: Targeting performance of targeting nanoparticles loading 99mTc-EC-ODN on breast carcinoma cells in vitro.1. Uptake rate of targeting nanoparticles carrying 99mTc-ASODN by SK-Br3 cells was (16.92±1.91)% at 30min, (21.70±2.10)% at 60 min and then gradually declined to (7.11±2.76)% at 240min. The uptake rate of 99mTc-ASODN not entrapped with nanoparticles by MDA-MB-231 cells was (12.27±2.76)%, up to peak (13.19±2.34)% at 60min and (3.70±1.91)% at 240min. As time went on, the accumulation uptake rate of SK-Br3 cells and MDA-MB-231 cells both gradually increased but the two rates were of significant difference.2. Stagnation rate of targeting nanoparticles carrying 99mTc–ASODN in SK-Br3 cells gradually declined: (92.26±9.19)% at 15min; (73.70±6.10)% at 60 min; (43.70±3.10)% at 360min. In targeting nanoparticles carrying 99mTc–SODN group: (93.18±8.02)% at 15min; (54.91±5.96)% at 60min; (5.24±1.27)% at 360min. In targeting nanoparticles carrying 99mTc–NSODN group: (92.62±9.50)% at 15min; (54.91±5.96)% at 60min; (4.91±1.88)% at 360min. The stagnation rate of 99mTc–SODN group and 99mTc–NSODN group both displayed quick decline while their stagnation rate were significantly different with 99mTc–ASODN group.3. Determined by MTT, inhibition rate of SK-Br3 cells and MDA-MB-231 cells by targeting nanoparticles carrying 99mTc–ASODN was respectively (26.5±6.4)% and (6.5±3.1)%.The difference was of significance (P<0.01). Inhibition rate of SK-Br3 cells and MDA-MB-231 cells by 99mTc–ASODN was (14.8±3.4)% and (5.1±3.6)% respectively. The difference was of significance (P<0.01). Inhibition rate of SK-Br3 cells by targeting nanoparticles carrying 99mTc–ASODN and 99mTc–ASODN was of significant difference (P<0.05).4. c-erbB2 gene mRNA level determined by RT-PCR was as follows: In targeting nanoparticles carrying 99mTc–ASODN group, expression of c-erbB2 gene mRNA (gray scale value: 60.63±3.3) was weaker than that in 99mTc–ASODN not entrapped with nanoparticles group (96.82±5.1) and the difference was of obvious significance (P<0.01).5. c-erbB2 protein expression level in SK-Br3 cells determined by Western blot was as follows: inhibition rate of c-erbB2 protein expression in SK-Br3 cells in targeting nanoparticle-mediating 99mTc–ASODN group was obviously higher than that in 99mTc–ASODN group and the difference was of significance (P<0.01).Part 4: Distribution of c-erbB2 targeting nanoparticles loading ASODN labeled with 99mTc in Jimpy mice and imaging in bearing cancer nude mouse by SPECT.1. Distribution of 99mTc–ASODN with and without entrapped with targeting nanoparticles in normal Jimpy mice: The former was quickly removed after administered into serum while the latter slowly cleared out of serum. Stagnation quantity of these two was obviously different.2. Distribution of 99mTc–ASODN and targeting nanoparticles loading 99mTc–ASODN in bearing cancer nude mouse: Radioactivity ratio of tumor/serum and tumor/muscle of the latter gradually rose, up to its peak (3.31±0.61 and 14.87±6.06 respectively) at 2h and then subtly declined. Ratio trend of these two was similar but the ratio value of the former was comparatively lower.3. SPECT static imaging was launched in bearing cancer nude mouse at 2h. Radioactivity was aggregated in tumor location and tumor imaging was clearly observed.CONCLUSION:In the present research, EC was used as chelator of labeling of ASODN with 99mTc. EC and ASODN were connected by chemical technology. This method is of high labeling yield, of high radiochemical purity, of suitable specific activity and of low plasma protein-combining rate. ASODN in 99mTc-EC-ASODN was of high hybridization activity. EGF and BSANP are linked by chemical synthesis for preparation of targeting nanoparticles which are of targeting activity and entrapment efficiency. Entrapment of ASODN with targeting nanoparticles improved its stability in serum, which provides suitable vector for the introduction of ASODN into tumor cells with high proportion. Antisense imaging is a promising method for tumor early diagnosis. Furthermore, albumin targeting nanoparticle vector affords a new way for early diagnosis, particular therapy and prognosis judgment of breast carcinoma.
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