| Objective: To investigate the labeling method of antisense peptidenucleic acid (PNA) probe that targets epidermal growth factor receptor（EGFR）mRNA using radionuclide99Tcmmediated by cationic liposome,detect the radiolabeling rate and evaluate the stability in vivo and vitro.Inquire into the differences of target cell uptake in vitro and its utility fortumor imaging and biodistribution in vivo of99Tcm-EGFR-PNA mediated bycationic liposome or not. Explore new methods to promote antisense probesushering into cells for further improvement of tumor oncogene imaging.Methods: A single-strand antisense PNA targeting EGFR mRNA (thesequence from503to519(Gene Bank No:201283.1) was synthesized. On the5’ terminus of the PNA, a four amino-acid (Gly-(D)-Ala-Gly-Gly) linkerforming a N4structure could serve as a chelator that strongly binds to a-singlephoton emitting radionuclide99Tcm. As a spacer to eliminate the sterichindrance, one4-aminobutyric acid (Aba) was introduced between the PNAsequnce to the chelator. PNA has no electrical charge,so all we have to do is make it negatively charged in favor of liposomemediation. The oligonucleotide with the sequences of bases (5′-CTGGTTAT-3′)complementary to eight bases on3’ terminus of PNA is hybridized in ananti-parallel orientation to PNA mentioned above. The hybridizationcomplexes are negative, which could help PNA to be coated by cationicliposome. The assembly of lipofectamine and99Tcm-labeled heteroduplex wasachieved by electrostatic interactions. The labeling rate and radiochemicalpurity were assessed by reverse-phase high performance liquidchromatography (RP-HPLC). To evaluate the labeling stability in vivo, weincubated lipofectamine-mediated99Tcm-EGFR-PNA and99Tcm-EGFR-PNA in normal saline or fresh human serum at37℃for1,2,4,6,12and24hrespectively and the radiochemical purity at each time point were determinedby RP-HPLC. The SKOV3cells overexpressing EGFR and MDA-MB-435Scells with low expression of EGFR were incubated by standing adherent. Thecells in logarithmic growth phase were harvested and were injectedsubstaneously into the right anterior superior limbs of the mice with2×107SKOV3cells or MDA-MB-435S cells in0.2ml. The tumors were allowed togrow to a diameter of1cm. The metabolic stability of lipofectamine-mediated99Tcm-EGFR-PNA or99Tcm-EGFR-PNA was tested by analysising theradiochemical purity of urine within2-3h after injecting the probes into lateraltail vein of BALB/c nude mice bearing SKOV3tumor xenografts. Thedisparities of cellular uptake and retention kinetics in SKOV3cells andMDA-MB-435S cells were analyzed aiming at studying the pharmacokineticsof probes. For biodistribution studies, thirty-two BABL/c nude mice withSKOV3xenografts were divided into two groups with16mice in each groupas random digits table (one group for injection of lipofectamine-mediated99Tcm-EGFR-PNA and the other for injection of99Tcm-EGFR-PNA). SixteenBALB/c nude mice with MDA-MB-435S xenografts injectedlipofectamine-mediated99Tcm-EGFR-PNA were used in biodistributionstudies too. At1,2,4and6h after injection, four mice of each group wereselected as random digits table and sacrificed by cervical dislocation. Tumorand other tissues were weighed, and radioactivity was obtained by γ-wellcounter. Calculate the percentage of injected dose per gram of organ (%ID/g)of each issue and the ratios of tumor to contralateral muscles (T/M).Lipofectamine mediation effects on distribution in animal models of probewere analyzed. For imaging studies, ten BABL/c nude mice with SKOV3xenografts were divided into two groups of five mice each as random digitstable (one group for injection of lipofectamine-mediated99Tcm-EGFR-PNAand the other for injection of99Tcm-EGFR-PNA). Five BABL/c nude micewith MDA-MB-435S xenografts injected lipofectamine-mediated99Tcm-EGFR-PNA were also used in imaging studies. At1,2,4,6,8and10h, the mice were anesthetized and imaged. The ratio of radioactive counts in thetumor to that in the contralateral equivalent region (T/NT) was calculated bydrawing regions of interest at each time point. The changes ofradioactive concentration in tumor site were observed and differences of thethree groups were analyzed. All experimental variables are expressed asaverage±SD and were performed with Two-sample t (or t′) test and Wilcoxonrank sum test using SAS9.1.Results: The labeling rates of lipofectamine-mediated99Tcm-EGFR-PNAand99Tcm-EGFR-PNA were（95.17±0.55）%(n=6) and （98.75±1.09）%(n=6)within6h, which can meet the demand for in vivo imaging without furtherpurification. RP-HPLC gave single peak at retention time of19min and12.5min respectively which was consistent with its peak of absorbance at260nm.The probes incubated in normal saline or fresh human serum at37℃for1,2,4,6,12and24h respectively. RP-HPLC indicated that the labeling rates wereall above90%within6h and the radiation peaks were stable without drift.RP-HPLC analysis of urine of mice bearing SKOV3xenografts within2-3hafter administering the probes revealed two peaks. The peak detected beforemain peak that the retention time was consistent with that of labeled probes,may be the degradation fragments of the PNA rather than free99TcmO-4. Theradiochemical purity of lipofectamine-mediated99Tcm-EGFR-PNA or99Tcm-EGFR-PNA in urine was89.3%and90.0%respectively. Lipofectaminecan increase celluar uptake of99Tcm-EGFR-PNA in vitro notablely（t′=47.11-58.67，Z=2.80，all P<0.05），up to a maximum of8.8times.Aftermediated by lipofectamine, the cellular uptake peak was deferred from6huntil12h. The uptake of lipofectamine-mediated99Tcm-EGFR-PNA in SKOV3cells was significantly higher than that in MDA-MB-435S cells(t=20.54-30.16,t′=9.45, Z=2.80, all P﹤0.05). Lipofectamine can increase celluar retentionobservably of99Tcm-EGFR-PNA in SKOV3cells（t′=7.25-11.55, Z=2.80, all P<0.05）. The result of biodistribution in mice bearing SKOV3xenograftsdemonstrated that high accumulation of radioactivity could be seen mainly intumor, liver and kidney. The accumulation of radioactivity of tumor was more obvious than contralateral muscles, and radioactive concentration intumor increased clearly with the extension of time whether mediated bylipofectamine or not. The ratios of tumor to contralateral muscles (T/M) werealso elevated after lipofectamine treatment（t=11.24, t′=3.96-11.94, all P<0.05）. The radioactive concentration of lipofectamine-mediated99Tcm-EGFR-PNA in SKOV3tumor xenografts was more obvious than that inMDA-MB-435S tumor xenografts. The imaging confirmed thatafter lipofectamine-mediated99Tcm-EGFR-PNA or99Tcm-EGFR-PNA wasinjected into mice bearing SKOV3tumor xenografts1h, the tumor could bevisualized in both groups, but lipofectamine-mediation made99Tcm-EGFR-PNA visualizing in tumors more clearly. The degree of radioactiveconcentration increased over time which peaked after6h. A decline thatfollowed could be observed, but the decline of99Tcm-EGFR-PNA was morerapidly than that of lipofectamine-mediated99Tcm-EGFR-PNA.Lipofectamine-mediation could raise the ratio of radioactive counts in thetumor to that in the contralateral equivalent region (T/NT) at each time point(t=3.96, t′=12.65-14.69, Z=2.83-5.29, all P<0.05). After administration oflipofectamine-mediated99Tcm-EGFR-PNA, no significant localization of theimage was seen in MDA-MB-435S tumor xenografts within theexperimental time.Conclusions: Lipofectamine-mediated99Tcm-EGFR mRNA antisensePNA which has the characteristics of high labeling efficiency and goodstability can be specifically taken up by EGFR-positive cells and can be usedin the imaging of EGFR mRNA overexpressing malignant tumors in vivo.Lipofectamine-mediation can obviously promote the intracellular delivery of99Tcm-EGFR-PNA and can greatly improve the imaging effect on tumors thatEGFR overexpressed which will be helpful to oncogene imaging of tumors. |
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