Folate-targeted SPIO-PBCA As Novel MR Contrast Agents: Synthesis And Experimental Study

Objective1. To prepare folate-targeted and non-targeted polybutylcyanoacrylate (PBCA) nanoparticles loaded with SPIO under neutral condition successfully;2. To investigate the cell target effect of folate-targeted SPIO-PBCA-NP compared with folate-free SPIO-PBCA-NP in cell experiment with human cervical carcinoma Hela cell and human pulmonary adenocarcinoma A549 cell, and to explore its clinical potential.3. To investigate the tumor target effect of SPIO-PBCA-FA-NP by intravenous injection of SPIO-PBCA-NP, compare the effect of imaging of folate-targeted SPIO-PBCA-NP, folate-free SPIO-PBCA-NP and negative control in animal experiment, and to explore its clinical potential.Materials and MethodsⅠ. Preparation and characterization1. Synthesis and analysis of FA-PEG-NH2 The folic acid and the PEG-(NH2)2 were mixed at the molar ratio of 1:1 as that 13.2g folic acid was dissolved in 1ml DMSO and a certain amount of DCC and NHS and 120g PEG-(NH2)2 was added under mechanical agitation. The reaction was carried out at room temperature for 4h. The 5 mL water was added and the insolubles were removed by filtration. The supernatant was free-dried and the Freeze-dry was washed for several times. At last, the yellow solid FA-PEG-NH2 was obtained which is detected by Infrared spectrophotometry.2. Synthesis and morphology of the targeted blank carrier FA-PEG-PBCAAccording to the references,2.7ml diluted BCA (1.5% v/v) was added in 20ml deionized water dissolving 2.0g synthetic FA-PEG-NH2 (MW=4000Da) with 5ml syringe in dropwise under mechanical agitation. The BCA was diluted by dichloromethane to 1:5. The polymerization process was carried out under mechanical agitation at room temperature for 4h. After vacuum filtration, the colloid solution of FA-PEG-PBCA was obtained which filtered with 0.45μm membrane, and stored in 4℃refrigerator.A little of the diaylsed solution was dilluted with distilled water (1:4) and applied to metallic sample plate following negative staining with sodium phosphotungstate solution. The sample was dried absolutely and examined by transmission electron microscopy.3. Analysis FA-PEG-PBCA with infrared spectrophotometryThe blank carrier FA-PEG-PBCA and the non-targeted blank carrier were detected by infrared spectrophotometry to compare the IR spectra between FA-PEG-PBCA and PEG-PBCA, FA-PEG-PBCA and FA-PEG-NH2.4. Non-targeted blank PBCA-NP hydrophilic surface modification with PEG.As above,2.7ml diluted BCA (1.5% v/v) was added in 20ml deionized water dissolving 2.0g NH2-PEG-NH2 (MW=4000Da) with 5ml syringe in dropwise under mechanical agitation. The BCA was diluted by dichloromethane to 1:5. The polymerization process was carried out under mechanical agitation at room temperature for 4h. After vacuum filtration, the colloid solution after PEG hydrophilic modified PBCA was obtained which filtered with 0.45μm membrane, and stored in 4℃refrigerator.5. The synthesis and Malvern detection of targeted agent FA-PEG-PBCA-SPIO and non-targeted PEG-PBCA-SPIO2.7ml diluted BCA (1.5% v/v) containing 10 ml SPIO was added in 20ml deionized water dissolving 2.0g FA-PEG-NH2 (MW=4000Da) with 5ml syringe in dropwise under mechanical agitation. The BCA was diluted by dichloromethane to 1:5. The polymerization process was carried out under mechanical agitation at room temperature for 4h. After vacuum filtration, the colloid solution after PEG hydrophilic modified PBCA was obtained which filtered with 0.45μm membrane, and stored in 4℃refrigerator.As mentioned above,2.7ml diluted BCA (1.5% v/v) containing 10ml SPIO was added in 20ml deionized water dissolving 2.0g NH2-PEG-NH2 (MW=4000Da) with 5ml syringe in dropwise under mechanical agitation. The BCA was diluted by dichloromethane to 1:5. The polymerization process was carried out under mechanical agitation at room temperature for 4h. After vacuum filtration, the colloid solution after PEG hydrophilic modified PBCA was obtained which filtered with 0.45μm membrane, and stored in 4℃refrigerator.NP size and size distribution measured:The NP size and size distribution were measured using dynamic light scattering (a Malvern Zetasizer 30000HS). A sample volume of 5μl was diluted with distilled water then measured under 633nm laser and a temperature of 25.00±0.05.6. Electron microscope detection of targeted agentsSEM:A small amount of dialysis samples was freeze-dried into powder. The doubled-sided tape was fixed on the SEM's object plate and small amount of powder was put on the tape at the center of the plate. The powder was blown gently towards the radial outward direction of plate using inflatable rubber ball to make it uniformly distributed on the plate. And the rickety powder was also blown off to avoid contamination of the spectacle. (The mouth was forbidden to use so as not to blow saliva in the sample. Any tools were disabled to dial the powder to prevent damage to the morphology pattern). The conductive silver paste was overlaid on the tape edge in order to connect the sample with object plate. The plate was put into the ion sputtering instrument after the silver paste was dry. When the degree of vacuum was below 0.2Torr, the timer was opened (the steaming golden time was set to 5min) and the current was adjusted to around 4mA so that the sample can be uniformly coated with a surface layer of 20nm gold membrane. Then the object plate was put into the sample holder to observe.TEM:A little SPIO and SPIO-PBCA-NP was dilluted with distilled water (1:4) and applied to metallic sample plate following negative staining with sodium phosphotungstate solution. The sample was dried absolutely and examined by transmission electron microscopy.7. Measurement of entrapment rate and drug-loading rate5ml undialysis SPIO-PBCA-NP was centrifugation at 1,2000 rpm for 30min and the supernatant and the precipitation were collected to measure entrapment rate and drug-loading rate, so as to the iron concentration of the domestic SPIO by Phenanthroline-spectrophotometry.8. Measurement of the T2 time of the targeted agent10ml purified water, blank nanoparticle and targeted contrast agent with the concentration of 0.1,0.12,0.14,0.16 and 0.18 mmol/L was put into seven test tubes (diameter 1.0cm). T2 map sequence with 8 echo was applied to get cross section images of the tubes (TE=20ms,40ms,60ms,80ms,100ms,120ms,140ms and 160ms, TR=2000ms, NEX=4, FOV=75×75cm, scanning thickness=2mm, interval=2mm), and T2 value of each tube could be measured through T2 mapping analysis software of Functool software package using the initial data of T2 mapping.Ⅱ. Cellular experiment1. Culture and morphological observation of tumor cells.Human Cervical Carcinoma Line Hela and Human Lung Adenocarcinoma Line A549 was continuously cultured and passaging into RPMI1640 medium containing 100μg/ml bovine serum under condition of 37℃,5% CO2 and saturated humidity to keep them adherent. Before using, the cells in logarithmic phase was washed with folate-free RPMI1640 medium for 3 times, and cultured in folate-free RPMI1640 medium for 1 week. When using, the cells was washed with serum and folate-free RPMI1640 medium for one time, collected and counted. The concentration of cell suspension was adjusted to the desired levels to reserve.Both types of cells undergo HE staining and Prussian Blue staining, and cell morphology are observed.2. Phagocytosis test of tumor cellFolate-targeted and non-targeted contrast agents were incubated with Human Cervical Carcinoma Line Hela and folate-targeted agent was incubated with Human Lung Adenocarcinoma Line A549 for 2 hours. Cellular iron incorporation and detention were observed by Prussian Blue staining to detect the iron absorption of the tumor cells.3. Folic acid competitive examinationFree folic acid was incubated with Human Cervical Carcinoma Line Hela for 30min first, and then the targeted contrast agents with the Fe concentration being 100μg/ml for 2 hours. The intracellular iron was obserbed by Prussian blue staining.Ⅲ. The study of SPIO-PBCA-NP targeted tumor-bearing nude mice imaging.1. Culture of tumor cellsHuman Cervical Carcinoma Line Hela and Human Lung Adenocarcinoma Line A549 was continuously cultured and passaging into RPMI1640 medium containing 100μg/ml bovine serum under condition of 37℃,5% CO2 and saturated humidity to keep them adherent.2. Establishment of nude mice models bearing subcutaneously transplanted human cervical carcinoma and lung adenocarcinoma18 BALB/C-nu/nu nude mice (Experimental Animal Center of Southern Medical University,4 to 6 weeks old, weighing 18～24g, female) were raised in the Southern Medical University Animal Center meeting the SPF (specific-pathgen free) conditions in the nude mouse room.Steps of nude mice models bearing subcutaneously transplanted human cervical carcinoma and lung adenocarcinoma establishment were as follows:(1) After disinfection,0.2ml Hela human cervical carcinoma cell suspension was put (2×105/ml) using syringe with 7# needle into the subaxillary subcutaneous tissue and the subcutaneous tissue of thgh of mice;(2) The nude mice was raised in standard ultra-clean environment for 4 to 6 weeks until the diameter of implanted tumors reached 1～1.5cm;(3) One week before the following experiment, all nude mice were given folate-free food instead of the normal diet.3. Subgroup and treatment of experimental animalDivide 18 nude mice bearing subcutaneously transplanted human cervical carcinoma and lung adenocarcinoma into three groups with 6 mice in each group (1) Experimental group:The mice were injected folate-targeted contrast agent (5mg Fe/kg) into nude mice bearing Hela human cervical carcinoma through caudal vein;(2) Non-targeted control group:The mice were injected nontargeted contrast agent (5mg Fe/kg) into nude mice bearing Hela human cervical carcinoma through caudal vein;(3) Negative control group:The mice were inject folate-targeted contrast agent (5mg Fe/kg) into nude mice bearing A549 lung adenocarcinoma through caudal vein.4. MRI scanning of the nude mice:On basis of T2 negative reinforcement effect of SPIO, reflect the iron containing in tumor tissue was reflected by measuring the MRI signal intensity on T2WI.All MR imagings were performed before and after different enhancement protocols at field strength of 3.0 Tesla MR scanner (Signa Excite, GE, USA) with use of a elbow coil for transmission and reception of the signal. MR sequences included T1-weighted spin echo images (repetition time [TR] mess/echo time [TE] mess, 600/16) and T2-weighted turbo spin echo images (TR/TE,4000/107.2). The thickness was 2.0mm, the space was 0.5mm, the matrix was 256 X 256, and FOVwas 75～100mm.MRI Examination steps of nude mice were as follows:(1) Nude mice were anesthetized by intraperitoneal injection of 4.3% chloral hydrate (430mg/kg);(2) Each nude mice underwent a plain scan as baseline before enhancement;(3) According to the grouping request, folate-targeted contrast agent (5mg Fe/kg) and non-targeted contrast agent (5mg Fe/kg) were intravenously injected in the nude mice by tail vein;(4) Immediately after contrast medium injection, dynamic data of different group were aqcquired at 0h, 1h,4h,8h and 24h;(5) When MRI scanning was completed, the data were transferred to post-processing workstation for the follow-up MRI image analysis.5. MR image analysisAverage signal intensity (SI) over region-of-interest (ROI) drawn on tumors and muscles were measured on MR images. Background noise was measured in each image and its ROI was placed adjacent background outside mice. Signal to noise ratio (SNR) of tumors and muscles in each time points were calculated on T1WI imaging and T2WI in three groups.6. Statistics analysisSPSS 16.0 was used as analyzing software. Repetitive measurement ANOVA analyzed the difference in SNR of the tumors and muscles before and after enhancement. One-way ANOVA analyzed the difference in SNR of the tumors and muscles among different groups and time points. P>0.05 was regarded as no statistical difference; P<0.05 was regarded as statistical significant difference.7. Pathology examinationAfter MR examination, the animals were put to death and the tumors were cut off to observe. Formalin-fixed, paraffin-embedded sections (1.5μm) were prepared. Two series slices were taken from each specimen to undergo Hematoxylin-Eosin staining, immunohistochemical staining and Prussion Blue Staining to observe the tumor appearance through light microscope, FR expression and iron content.Results:1. Folate-targeted and folate-free PBCA-NP were successfully prepared which was certifid by infrared, TEM and and SEM. It appeared as round-shaped uniform particle. 2. After adding liposoluble SPIO, folate-targeted and folate-free SPIO-PBCA-NP appeared as core-shell structure particle under TEM.3. Measured by dynamic light scattering, the average size of targeted SPIO-PBCA-NP was 202.2nm and the index of size distribution was 0.254, the average size of folate-free SPIO-PBCA-NP was 210.7nm and the index of size distribution was 0.336.4. The iron content of the targeted angent was 2.01mgFe/ml, entrapment rate was 76.91%, drug-loading rate was 7.04%; The iron content of the non-targeted angent was 2.18mgFe/ml, entrapment rate was 83.42%, drug-loading rate was 7.63%; The iron content of the domestic SPIO was 7.84mg/ml.5. The Hela cells incubated with folate-targeted SPIO-PBCA-NP showed much higher intracellular iron density than the Hela cells with non-targeted agents and the A549 cells with folate-targeted SPIO-PBCA-NP.6. Folic acid competitive examination and Prussian blue staining showed that intracellular iron density in Hela cells was significantly lower than non-folate incubated Hela cells.7. On MRI, the signal intensity of tumors in nude mice with Hela human cervical carcinoma reduced significantly after intravenous injection of targeted contrast medium and lasted for a long time. The signal of tumors reduced insignificant compared with targeted group after intravenous injection of non-targeted agent. While the signal of the tumors in nude with human pulmonary adenocarcinoma A549 reduced insignificant after intravenous injection of targeted contrast medium. The signal of the muscles changed insignificant of these three group.8. In histopathologic examination, the inmmunohistochemisty certified that Hela cells were positive expression of FR and A549 were negative expression; there were lots of blue iron particle in tumors of targeting gproup, while there were little in the non-targeting group and negative control group.Conclusions:1. PBCA-NP can be prepared successfully under neutral condition.2. Folate-targeted SPIO-PBCA-PEG-FA can be prepared by means of surface modification PBCA-NP packed with SPIO using PEG linking falate.3. SPIO-PBCA-FA-NP has good targeting tropism to the cells with folate receptor on their surface.4. After intravenous injection, SPIO-PBCA-FA-NP has good targeting tropism to folate receptor-positive tumors in tumor-bearing mude mice.