| Objective1. To synthesis the CREKA peptide and verify its binding activity to fibrin clot in tumor stroma, making preparation for next synthesis of targeted probe and tumor-targeting imaging.2. To prepare polylactic acid-coated superparamagnetic iron oxide nanoparticles (SPIO-PLA) and targeted molecular probe SPIO-PLA-CREKA, and investigate the ability of SPIO-PLA-CREKA as targeted contrast agent by physical-chemical characterization and in vitro MRI study of fibrin clots.3. To investigate the effectiveness of SPIO-PLA-CREKA for in vivo MRI imaging of tumor in a murine renal cell carcinoma model.Mehtods1. Synthesis of CREKA peptide and verification of its binding activity1) Synthesis of CREKA peptidePeptide CREKA was synthesized using standard solid phase peptide chemistry from Fmoc-protected amino acids on a Wang resin. Symmetric anhydride method was used for the coupling of the first amino acid at the carboxyl terminus to Wang resin. At the end of the reaction, sufficient pyridine and acetic anhydride were added to block remained active site on the resin. Amide bond coupling of next amino acid was achieved with HOBt/HBTU/DIEA as the peptide coupling reagent and repeated until the last amino acid was coupled to peptide resin. After the reaction was complete, trifluoroacetic acid as main cleavage reagent was used to harvest the peptide from the resin. Removal of the solvent by rotary evaporation gave a crude oil that was triturated with cold ether. The crude mixture thus obtained was centrifuged, the ether was removed by decantation, and the resulting white solid was purified by high performance liquid chromatogram (HPLC). The product were isolated by lyophilization and characterized by electrospray mass spectrometry.2) FITC-peptide conjugatesAt the end of the synthesis of CREKA peptide, that is to say, when the last amino acid was coupled to the peptide resin, an aminohexanoic acid spacer was added and coupled in the same way. Then FITC was attached on the amino termini through the ahx acid spacer by treating resin with FITC and diisopropylethyl amine in DMF for4h. After the reaction was complete, trifluoroacetic acid as main cleavage reagent was used to harvest the peptide from the resin. Removal of the solvent by rotary evaporation gave a crude oil that was triturated with cold ether. The crude mixture thus obtained was centrifuged, the ether was removed by decantation, and the resulting pallide-flavens solid was purified by HPLC. The product were isolated by lyophilization and characterized by electrospray mass spectrometry.3) FITC-Ahx-CREKA binding to clotted plasma protein in vitroHuman blood about10ml was anticoagulated with0.4%sodium citrate and centrifuged at2,500rpm for lOmin. The plasma was collected, spun again to remove remaining blood cells, and frozen at-80℃. Clotting was initiated by adding CaCl2to20mM, and incubated at37℃until clot formed. The clot was repeatedly washed and centrifuged with phosphate buffer saline (PBS) to remove various serum proteins. The freshly formed plasma clot was divided into6parts. The6little clots were incubated with FITC-Ahx-CREKA (n=3) and PBS (n=3), respectively. After30min of incubation in37℃, the clots were repeatedly washed in PBS. The clots were exposed to ultraviolet light for35seconds and the imaged with fluorescence microscope.4) FITC-Ahx-CREKA binding to tumor stoma in vitroA mass of formalin-fixed and OTC-embedded tumor tissue from xenogeneic graft ovarian cancer athymic mouse model was used. The frozen sections of3μm thickness were prepared and placed on clean glass slide. Slices were treated with FITC-Ahx-CREKA for30min at room temperature. Slides were rinsed with water for3times and viewed under a fluorescence microscopy. Then HE staining was performed to identify the location of fluorescence.5) Tumor modelRenca cells were revivaled and cultured in DMEM medium supplemented with10%fetal calf serum and maintained at37℃in a5%CO2, humidified incubator. For evaluation of cell number, cells were trypsinized after48hours incubation, stained with trypan blue and counted using a hemocytometer. The survival rate was over98%. For in vivo implantation, Renca cells were washed in saline by twice centrifugalization and injected subcutaneously at105cells in0.1ml in left back in6-week-old Balb/c mice. A total of4tumor models were generated after the tumors were grown for14days.6) FITC-Ahx-CREKA binding to tumor stoma in vivoBalb/c mice bearing Renca tumor were used.200μg FITC-Ahx-CREKA in0.1ml physiological saline was injected intravenously to the mouse through a tail vein when the tumor grown up to1.0cm in diameter. The tumor-bearing mice were sacrificed at6houres after injection. Tumor tissues were obtained and divided into two parts. One part was frozen in liquid nitrogen for frozen section and the other was fixed with formalin and cut into slices after paraffin embedding. Frozen sections were analyized with fluorescence microscopy. HE staining was performed on paraffin section.2. Synthesis of SPIO-PLA-CREKA and in vitro imaging1) Synthesis and characterization of SPIO-PLA-CREKAPreparation of polylactic acid-coated superparamagnetic iron oxide nanoparticles (SPIO-PLA):0.39g FeCl2·4H2O and1.08g FeCl3·6H2O were dissolved in20ml of deionized water with fixed Fe2+to Fe3+molar ratio of1:2. The PLA were then added to reach specific polymer concentrations. To initiate iron oxide formation, sodium hydroxide solution (5M) was added to the intensely stirring mixture drop-wise until the pH of the system reached10. Close the reaction container tightly and vigorously stirred overnight at room temperature. Gradient centrifugation from1000to15000rpm was used to remove large particles. The resultant suspension was dialyzed against phosphate buffer with dialysis membrane (molecular weight cutof,10,000) for3d to remove free PLA and iron salts. Dialysis was carried out at4℃and dialysate was changed every24h. SPIO-PLA was harvested by filtration with0.22μm micropore film and stored at4℃Preparation of SPIO-PLA-CREKA conjugation:1.20mg of EDC-HC1was dissolved in100μl buffer bicarbonate (pH8.3). The solution was added drop-wise to lml of SPIO-PLA suspension under stirring condition. Then the solution of1.27mg sulf-NHS dissolved in100μl buffer bicarbonate was added drop-wise to the reaction systerm. The solution was left under magnetic stirring for10min. Then5.0mg of CREKA peptide in500μl buffer bicarbonate (pH8.3) was added and the reaction mixture was stirred at room temperature for2h. The reaction mixture was purified by gel permeation chromatography using a Sepharose4Fast Flow column. SPIO-PLA-CREKA was harvested by filtration with0.22μm micropore film and stored at4℃.Nanoparticle morphology was examined by using a transmission electron microscope (TEM). The particle size distribution of the magnetic nanoparticles was determined by HYL-1080laser particle size distribution. The average hydrodynamic diameter and polydispersity index were evaluated. The lyophilized magnetic nanoparticles were grounded with KBr for FT-IR measurement by Fourier Transformed Infrared Spectrometer.Magnetic resonance imaging studies and T2measurement were carried out on a1.5T MRI scanner. Concentration series from0.05to1.6mmol/L of SPIO-PLA-CREKA and SPIO-PLA were prepared in6tubes. T2-weighted images were acquired with spin echo sequence (TR/TE527.3/50ms, field of view230x230mm, matrix256×205, thickness5mm). For the determination of the T2relaxation times, a Carr-Purcell-Meiboom-Gill sequence was used (TR2,000ms; TE range,30-960ms,32echoes; field of view230×230mm, matrix,256×205, slice thickness,2mm; band width,40; number of excitations was3).2) In vitro MRI study of fibrin clotsThirteen fibrin clots were prepared.10ml of anticoagulant plasma was added to each of the13tubes. Clotting was initiated by adding CaCl2to20mmol/L. Clots formation was achieved with4h’s incubation at37℃. The clots were repeatedly washed and centrifuged with PBS to remove various serum proteins.Plasma fibrin clots were randomly divided into two groups:SPIO-PLA group and SPIO-PLA-CREKA group (n=6), the remaining1as a blank control. SPIO-PLA group was randomly divided into two subgroups:SPIO-PLA1h and SPIO-PLA6h each had three tubes. Also, SPIO-PLA-CREKA group was randomly divided into two subgroups:SPIO-PLA-CREKA1h and SPIO-PLA-CREKA6h each had three tubes.Three tubes of SPIO-PLA1h were respectively incubated with SPIO-PLA in concentration of1.6、0.8、0.4mmol/L at37℃for1h. Three tubes of SPIO-PLA6h were respectively incubated with SPIO-PLA in concentration of1.6、0.8、0.4mmol/L at37℃for6h. Three tubes of SPIO-PLA-CREKA1h were respectively incubated with SPIO-PLA-CREKA in concentration of1.6、0.8、0.4mmol/L at37℃for1h. Three tubes of SPIO-PLA-CREKA6h were respectively incubated with SPIO-PLA-CREKA in concentration of1.6、0.8、0.4mmol/L at37℃for6h. The control tube was not treated with contrast agents. All clots were washed and centrifuged with PBS for several times to remove unbounded contrast agents. T2-weighted images were acquired with1.5T MRI scanner and turbo spin echo sequence (TR,4015.3ms, TE110ms, field of view230×230mm, matrix256X205, thickness2mm).Binding inhibition:Nine clots were prepared using the same method as above and equally divided into A, B and C groups.5mg of free CREKA peptide in1.5ml of PBS was added to the tubes of group A, each0.5ml. Tubes of group B and group C were added with0.5ml of PBS respectively. Then SPIO-PLA-CREKA SPIO-PLA-CREKA and SPIO-PLA were added to A, B, C groups, respectively. After1h’s incubation at37℃, clots were washed with PBS for several times. To view the binding activity, T2-weighted images were obtained in the same way as above.3. In vivo MRI study of tumor model1) Tumor modelIn this study, Renca cells were cultured in DMEM medium supplemented with 10%fetal calf serum and maintained at37℃in a5%CO2, humidified incubator. For evaluation of cell number, cells were trypsinized after48hours incubation, stained with trypan blue and counted using a hemocytometer. The survival rate was over98%. For in vivo implantation, Renca cells were washed in saline by twice centrifugalization and injected subcutaneously at106cells in0.1ml in left back in6-to8-week-old female Balb/c mice. A total of12tumor models were generated after the tumors were grown for14-18days. The diameter of the subcutaneous tumor mass was approximately1.5cm.2) In vivo imaging protocolTwelve Renca tumor model were randomly divided into two groups: SPIO-PLA-CREKA group and SPIO-PLA group (n=6), and treated with SPIO-PLA-CREKA and SPIO-PLA respectively. Firstly, mice were anesthetized by peritoneal injection of pentobarbital sodium and a plain T2-weighted scan was performed. Mice of two groups were injected with SPIO-PLA-CREKA and SPIO-PLA at the dose of5mg/kg respctively. MRI study was performed using a Philips Achieva1.5T clinical scanner and an articular genu coil. T2-weighted images pre-injection and at60min,120min、80min after injection were obtain using turbo spin echo sequence (TR3047ms, TE100ms, field of view230x230mm, matrix256×205, thickness5mm).For quantification of enhancement over time, Signal-to-noise ratio (SNR) was determined for tumor tissue following equation:SNR=SIt/SIn, where SIt is the signal intensity of the tumor, SIn is the signal intensity of noise.3) Statistical analysisAll data were analyzed using the Statistical Package for Social Sciences version13.0(SPSS Inc., Chicago, Illinois, USA). SNR were expressed as mean±SD. Comparesion of SNRs at different time points were perfomed using Repeated measures ANOVA. Multiple comparesion of SNRs at different time points in each group were perfomed using Bonfferoni method. Two-sample t-test was employed to compare SNR in two groups at each time point before and after enhancement. P values less than0.05were considered statistically significant.4) Histologic examinationThe tumor-bearing mice were sacrificed after the completion of MRI scans. Tumor tissue were collected and fixed with formalin. Tissue sections of3μm thickness were prepared and placed on clean glass slide. HE and Prussian blue staining were performed routinely.Results1. Synthesis of CREKA peptide and verification of its binding activity1) Synthesis of CREKA peptideThe obtained CREKA peptide by solid phase synthesis was white powder. The molecular mass (m/z:[M+H]+) determined by Mass chromatographic analysis (ESI) was606.0, which was in accordance with calculated604.71, proved the correct synthesis. The final product was purified by preparative HPLC. The purity of CREKA peptide was99.65%as determined by analytical HPLC.2) FITC-Peptide conjugatesA FITC was attached through an ahx acid spacer on the amino termini of CREKA. The final product FITC-Ahx-CREKA was pallide-flavens solid powder. The molecular mass (m/z:[M+H]+) determined by Mass chromatographic analysis (ESI) was1108.5, which was in accordance with calculated1107.61, proved the correct conjugation. The final product was purified by preparative HPLC. The purity of FITC-Ahx-CREKA peptide was98.13%as determined by analytical HPLC. 3) FITC-Ahx-CREKA binding to clotted plasma protein in vitroThe plasma clots incubated with FITC-Ahx-CREKA and washed with PBS after incubation exhibited green fluorescence under fluorescence microscope. The clots which treated with PBS as the contrast did not show visible green fluorescence. This result suggested that CREKA peptide bind to plasma clots specificly.4) FITC-Ahx-CREKA binding to tumor stoma in vitroFrozen sections of SKOV-3tumor tissue incubated with FITC-Ahx-CREKA peptide showed obviously green fluorescence under fluorescence microscope in a round manner. HE staining verified that localization of green fluorescence was the wall of tumor vessel and the stroma around it.5) Tumor modelFour tumor models were generated by subcutaneous injection of Renca cells in the left back of6Balb/c mice.14days after inoculation, tumors were grown up to about1.Ocm in diameter. No obvious mass were grown in the other two mice.6) FITC-Ahx-CREKA binding to tumor stoma in vivoFluorescence microscopic analysis showed that FITC-Ahx-CREKA diffusely distributes in the tumor tissue, but no fluorescence was detected in normal tissue. HE staining of paraffin section showed that heteromorphious tumor cells with big deep stained nucleus with multipule pathological karyokinesis status, which is in accordance with histopathology appearance of murine renal cell carcinoma. This result confirmed that FITC-Ahx-CREKA bind to tumor stroma in vivo specifically.2. Synthesis of SPIO-PLA-CREKA and in vitro imaging1) Synthesis and characterization of SPIO-PLA-CREKAThe resulted SPIO-PLA and SPIO-PLA-CREKA suspension were brownish-black in color. Fe concentration was182.8mmol/L and167.4mmol/L respectively. TEM revealed that particles had typical core-shell structure. The particle size distribution analysis showed that:the size of SPIO-PLA nanoparticles were approximately133nm average by volume and85nm average by area, with a median of124nm; the size of SPIO-PLA-CREKA nanoparticles were approximately150nm average by volume and88nm average by area, with a median of136nm. Infrared spectrum indicated that the colloidal particles consist of iron oxide crystals covered with a PL A layer, and SPIO-PLA was successfully conjugated with CREKA.T2-weighted images of SPIO-PLA-CREKA and SPIO-PLA showed that T2signal intensity decreased significantly with the increase of Fe concentration. T2signal intensity of SPIO-PLA-CREKA targeted contrast agent was slightly lower than SPIO-PLA with the same concentration. T2time measurement results show that:the T2time is gradually reduced with the increased concentration of contrast agent. SPIO-PLA-CREKA had a shorter T2than SPIO-PLA with the same concentration. These results indicated that SPIO-PLA-CREKA and SPIO-PLA can be used as contrast agents, and their contrast effect can be enhanced by elevating concentration.2) In vitro MRI study of fibrin clotsOn T2-weighted image, the signal intensity of clots of SPIO-PLA group had no significant difference from the control clot; the signal intensity of clots of SPIO-PLA-CREKA group was obviously lower than the control, furthermore, the signal intensity decreased with the increase of Fe concentration. Of the two subgroups, SPIO-PLA-CREKA6h had lower signal intensity than SPIO-PLA-CREKA1h.In competitive inhibition study, the clot incubated with SPIO-PLA-CREKA showed significantly decreased signal intensity when compared with SPIO-PLA. The signal intensity of the clot incubated with free CREKA and SPIO-PLA-CREKA decreased slightly. The results suggested that the binding of SPIO-PLA-CREKA to fibrin clot was inhibited by free CREKA peptide.3. In vivo MRI study of tumor model1) Tumor modelTwelve tumor models were generated by subcutaneous injection of Renca cells in the left back of15Balb/c mice. The achievement ratio was80%(12/15).14-18days after inoculation, tumors were grown up to about1.5cm in diameter. The mean of maximum diameter determined by sliding caliper was1.45±0.43cm.2) In vivo imagingTo investigate the tumor stroma targeting ability and MRI visibility of SPIO-PLA-CREKA conjugates, in vivo T2-weighted MRI scan was performed with mice bearing murine Renca tumor. On plain scan, tumors of both SPIO-PLA-CREKA group and SPIO-PLA group ahowed high signal intensity. After the injection of contrast agent, the signal intensity of tumors of SPIO-PLA-CREKA group decreased gradually; the signal intensity of tumors of SPIO-PLA group had no obvious change compared with plain scan.Statistical analysis suggested that tumors had significant difference in SNR before and after the injection of contrast agents (Ftime=88.249, P<0.001). Contrast enhancement has reciprocation effectiveness with the time (Fcontrastxtime=59.462, P<0.001). Tumors of SPIO-PLA-CREKA group had significant lower SNR than that of SPIO-PLA group (Fcontrast=33.097, P<0.001).Comparison of the two groups at different time ponts before and after contrast: there was no significant difference between SPIO-PLA-CREKA group and SPIO-PLA in tumor SNR at plain scan and60min after injection (30.08±0.66vs.30.13±0.76,t=0.122, P=0.905;29.48±0.82vs.29.97±0.85, t=1.002, P=0.340); At120min after injection, tumor SNR in SPIO-PLA-CREKA group was significantly lower than that of SPIO-PLA (27.37±1.14vs.29.62+0.68,7=4.159, P=0.002); At180min after injection, SPIO-PLA-CREKA group also had significant lower tumor SNR than SPIO-PLA group (24.22±0.48vs.29.53+0.56; t=17.652, P<0.001).3) Histologic analysisIn the tumor tissue slices injected with SPIO-PLA-CREKA, dark brown particles were observed along the wall of tumor vessel before staining. These particles turned blue after Prussian blue staining. When the tumor cell turned red by eosin staining, the blue spots along and around the wall of tumor vessels were highlighted by the red background. This result confirmed that SPIO-PLA-CREKA specifically bind to tumor stroma and accumulate at the wall of tumor vessels and the stoma around it.Conclusions1. We successfully synthesized the homing-peptide CREKA with its purity of99.65%, which can fulfill the requirement for next study.2. FITC-Ahx-CREKA is successfully prepared with the purity of98.13%, which can be used for next study in vitro and in vivo.3. The specific binding activity of CREKA to fibrin of tumor stoma is verified through in vitro or/and in vivo binding study of FITC-Ahx-CREKA to plasma fibrin clots and tumor tissue.4. SPIO-PLA is successfully synthesized; SPIO-PLA nanoparticles have core-shell structure, good dispersivity and are uniform in size as well as negative enhancement effect on T2WI.5. The prepared SPIO-PLA-CREKA also has good dispersivity and core-shell structure as well as negative enhancement effect on T2WI, which yield a novel targeted contrast agent for molecular magnetc resonance imaging.6. Targeted probe SPIO-PLA-CREKA has specific binding activity to fibrin clots, suggests that the conjunction of SPIO-PLA to CREKA do not impact the biological activity of CREKA.7. The tumor model of Balb/c mice bearing subcutaneouly murine renal carcinoma (Renca).8. T2signal intensity in tumors of animal model is significantly decreased after the injection of SPIO-PLA-CREKA.9. Pathology examination confirms that the probe SPIO-PLA-CREKA binds to tumor stoma, indicating that SPIO-PLA-CREKA can be used for tumor-targeting imaging in vivo. |
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