Synthesis And Evaluation Of The Novel Tumor-Targeting Folic Acid-o-Carboxymethyl Chitosans Superparamagnetic Oxide Iron Nanoparticles

Cancer has been one of the most serious threats to human health. up to several million of patients die from cancer, each year. It is a big burden not only for the patients and their families but also the whole society.Therefore, malignant tumors has become a major issue of concern to all mankind. The study on Superparamagnetic iron oxide nanoparticles were focused on MRI imaging from the initial to the current targeted drug delivery, treatment and imaging, etc.. superparamagnetic iron oxide nanoparticles with tumor targeting has been attracted the attention of many researcher because of their application in MRI diagnosis, hyperthermia and the potential for targeted drug delivery. In our study we synthesized a novel tumor targeting superparamagnetic iron oxide nanoparticles (SPIO-NPs) with three-step and we evaluate their tumor targeting and ability to evade the capture by macrophage in vivo and in vitro.1. To synthesize folic acid-o-carboxymethyl chitosan-superparamagnetic iron oxide nanoparticles for tumor targetings and optimize their prescription process.2. To characterize the property of the intermediate products (superparamagnetic iron oxide nanoparticles and o-carboxymethyl chitosan superparamagnetic iron oxide nanoparticles) as well as the end products (folic acid-o-carboxymethyl chitosan superparamagnetic iron oxide nanoparticles).3. To evaluate the tumor targeting, cytotoxicity and the ability to t of evade phagocytes phagocytosis of FA-OCMCS-SPIO-NPs in vitro.4. To preliminary evaluate the tumor targeting in vivo.Methods1. The FA-OCMCS-SPIO-NPs was synthesized by " three-step":At first, superparamagnetic oxide iron nanoparticles was synthesized by coprecipitation., then, the o-carboxymethyl chitosan and folic acid was covalently modified on the surface of SPIO-NPs in turn to prepare FA-OCMCS-SPIO-NPs.1.1 Superparamagnetic iron oxide nanoparticles were synthesized by the coprecipitation of ferric and ferrous salts in anaerobic conditions. We studied the effect of initial pH and temperature of iron salt solutions, the type of precipitation agent (NH3·H2O) and the amount of NH3·H2O on particles size and composition of SPIO-NPs during co-precipitation by one factor design. the Fourier transform infrared spectroscopy,X-Ray diffraction were used to confirm their synthesis, meanwhile, transmission electron microscope (TEM), dynamic light scattering (DLS), zeta-potential measurement and vibrating sample magnetometry (VSM) was applied to characterize their physicochemical property.1.2 o-Carboxymethyl chitosans superparamagnetic oxide iron nanoparticles (OCMCS-SPIO-NPs) composed of SPIO-NPs as "core" and OCMCS as "shell" were prepared by covalently modifying SPIO-NPs surface with OCMCS. In order to optimize the synthesis process, Firstly, we explore the effect of mass ratio between the OCMCS and SPIO nanoparticles on the particle size to determine the optimal ratio of SPIO and OCMCS. Then, On the base of initial preparative conditions, The optimum conditions were investigated by Lg (24) orthogonal design (carboxymethyl chitosan molecular weight, concentration of OCMCS in solution, ultrasonic time four factors and power of ultrasonic cell crushing instrument, two levels) through the index of particle size of nanoparticles as the dependent variable.the Fourier transform infrared spectroscopy,X-Ray diffraction were used to confirm their synthesis, meanwhile, transmission electron microscope (TEM), dynamic light scattering (DLS), zeta-potential measurement and vibrating sample magnetometry (VSM) was applied to characterize their physicochemical property. T2 relaxation rate was calculated according to MRI scanning. The iron content in dry SPIO-NPs and OCMCS-SPIO-NPs suspension was determined by o-Phenanthroline Method.1.3 The exposed amino group in OCMCS-SPIO-NPs was reacted with carboxyl groups in folic acid to synthesize FA-OCMCS-SPIO-NPs., Fourier transform infrared spectroscopy,X-Ray diffraction were used to confirm their synthesis and the influence of the grafting process of folic acid on the crystal structure of Fe3O4 to determine the best process to modify the OCMCS-SPIO-NPs with FA. The FA-OCMCS-SPIO-NPs were characterized according to the methods 1.2 for characterization,then The FA-OCMCS-SPIO-NPs, OCMCS-SPIO-NPs and SPIO-NPs suspension was stored in 4℃to observe the property of the suspension, preliminary study of their solution stability was studied.2. Dextran superparamagnetic iron oxide nanoparticles (dextran-SPIO-NPs) was synthesized by Co-precipitation of ferric, ferrous salts and dextran solution in anaerobic conditions. The synthetic dextran-SPIO-NPs were characterized according to the Method 1.2 too.3. Evaluation the cytotoxicity, folate receptor tumor targeting and the ability to evade the capture by macrophage cells in vitro. 3.1 CytotoxicityLO2 cells (normal liver cells), KB cells (human oral epithelial cells, a folate receptor over-expressing cell line) and A549 cells (small cell lung adenocarcinoma cells, folate receptor deficient cell line),routinely culture in full media. Then,the cells were seeded in 96 wells and continued incubating with full media containing FA-OCMCS-SPIO-NPs, OCMCS-SPIO-NPs, dextran-SPIO-NPs and non-coated SPIO-NPs with different concentrations respectively. with after 24h incubation, the viability of the KB, A549 and LO2 cells was determined by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.3.2 Tumor Targeting3.2.1 Quantification of cellular uptake by ferrozine assay:A549 cells were routinely cultured in full RPMI1640 culture medium, Hela cells (human cervical cancer cells, FR moderately over-expressed) and KB cells were routinely cultured in full folate free RPMI1640 culture medium. Then the cells were seeded in 24-well plates, after 24h, the cells were washed three times with PBS, Then, the full media containing different concentrations of FA-OCMCS-SPIO-NPs and OCMCS-SPIO-NPs were added, after 24h incubation, the cells were washed thoroughly three times with PBS and dispersed in 0.5ml of 0.1M hydrochloric acid,the intracellular iron content was determined by ferrozine Assay3.2.2 Prussian blue staining:A549 cells were routinely cultured in full RPMI1640 culture medium, Hela cells (human cervical cancer cells, FR moderately over-expressed) and KB cells were routinely cultured in full folate free RPMI1640 culture medium. Then the cells were seeded in 6-well plates, after 24h, the cells were washed three times with PBS, Then,2 ml of the full media containing FA-OCMCS-SPIO-NPs and OCMCS-SPIO-NPs (iron concentration was 0.4mgFe/ml)were added, after 24 incubation,the above cells were stained with Prussian blue and observed with an inverted microscope..3.3 Capture by macrophage cells in vitro3.3.1 Quantification of cellular uptake by ferrozine assay: RAW 264.7 cell were routinely cultured in full DMEM culture medium. cells were seeded in 24-well culture plates with approximately 2.5×105 cells/well. After incubation at 37℃for 24 h, the cells were washed three time with PBS. 1ml of the full DMEM culture medium containing SPIO-NPs, dextran-SPIO-NPs, OCMCS-SPIO-NPs or FA-OCMCS-SPIO-NPs(iron concentration ranging from 50μg/ml to 800μg/ml) was added to RAW264.7 cell respectively. the intracellular iron content was determined by ferrozine Assay.3.3.2 Prussian blue staining:RAW 264.7 cell were routinely cultured in full DMEM culture medium. Then the cells were seeded in 6-well plates, after 24h, the cells were washed three times with PBS, Then,2 ml of the full DMEM culture medium containing SPIO-NPs, dextran-SPIO-NPs, OCMCS-SPIO-NPs or FA-OCMCS-SPIO-NPs were added, after 24 incubation,the above cells were stained with Prussian blue and observed with an inverted microscope.4 Preliminary studies of FA-OCMCS-SPIO-NPs tumor targeting and distribution in vivo4.1 Tumor targeting in vivo4.1.1 Experimental Animals:SPF- nude mice 20 were bought animal center of Southern Medical University, age 4-6 weeks, male and female in half, randomly divided into two groups: KB tumor cell group (n= 10), and A549 tumor cell group (n= 10).4.1.2 Cell culture and tumor animal model develop:A549 were routinely cultured in RPMI-1640 full culture medium, KB were routinely cultured in FFPRMI-1640 full culture medium, then were detached by 0.25% trypsin-EDTA solution and washed three time by PBS s, followed by diluting in serum-free medium with 1.0×107 cells/ml,0.2 ml of cell suspension and were injected subcutaneously in the neck of nude mice. Two weeks later, when tumor diameter≥1.0 cm, the mouse with successful tumor model (16,8 for each group) were selected for animal experiments to study tumor targeting.4.1.3.The study of FA-OCMCS-SPIO-NPs tumor targeting imaging4.1.3.1.AnimalsIn this experiment, the 16 mice with successful tumor model (8 for both A549 and KB tumor group) were subdivided into four group at random. namely groupⅠ:A549, OCMCS-SPIO-NPs (n= 4);Ⅱgroup:A549, FA-OCMCS-SPIO-NPs (n= 4);ⅢGroup:KB cells, OCMCS-SPIO-NPs; IV Group:KB cells, FA-OCMCS-SPIO-NPs group (n= 4). (5.54 mg Fe/ml) or OCMCS-SPIO-NPs (5.62 mg Fe/ml) suspension, a dose of 0.25ml/nude mouse was intravenously injected.4.1.3.2 MR imagingAll MR imaging was performed before and after FA-OCMCS-SPIO-NPs or OCMCS-SPIO-NPs suspension were injected 3h at field strength of 1.5 Tesla MR scanner4.1.3.3 MR imaging analysisAverage signal intensity (SI) over region-of-interest (ROI) drawn on tumor were measured on MR images4.1.4. Histopathology: After the completion of MR imaging,tumors were collected Mice were deeply anesthetized with an intraperitoneal injection of chloral hydrate (400 mg/kg). The tumor tissues were excised and stored in 4% paraformaldehyde. Paraffin-embedded histological slices (3 mm thick) were stained with hematoxylin-eosin (H&E) or Prussian blue stain to identify the iron in the histological sections of the tumor. The images obtained on a OLYMPUS DMR inverted microscope4.2 Preliminary evaluation the distribution of OCMCS-SPIO-NPs. FA-OCMCS-SPIO-NPs and dextran-SPIO-NPs in vivo. SPF level obtained 20 Kunming mice were bought from the Southern Medical University were divided into four groups, n= 5:I group-OCMCS-SPIO-NPs group; II group- FA-OCMCS-SPIO-NPs group;Ⅲgroup -- dextran-SPIO-NPs (5.76mgFe/ml) group, IV group is the control group. The mice in all the four groups were injected with OCMCS-SPIO-NPs (5.44 mg Fe/ml), FA-OCMCS-SPIO-NPs (5.62 mg Fe/ml), dextran-SPIO-NPs (5.76mgFe/ml) or saline 0.25ml respectively, 12h later, the mice were killed, the hearts, livers, spleens, lungs and kidneys of all the mice were collected and the these tissues were excised and stored in 4% paraformaldehyde. Paraffin-embedded histological slices (3 mm thick) were stained with Prussian blue stainning to identify the iron in the histological sections of,while the HE staining in blank group were used to histologically identify the tissue. The images obtained on a OLYMPUS DMR inverted microscope5. Statistical analysis: SPSS 13.0 was used as analyzing software. Orthogonal factorial experiment analysis, factorial analysis and Paired-Samples T Test was applied for statistical analysis, P<0.05 considered significant statistical difference.Results1.Based on the initial preparative conditions of SPIO-NPs by one factor design,Physicochemical factors such as the PH, temperature in iron salt solution and the ripening temperature for have important effect on the size and size distribution as well as composition of SPIO-NPs.The optimal process-formulation was as follows:molar ratio of Fe+:Fe2+is 2:1, the initial pH and temperature of iron salt solutions were pH 3.0 and 30℃respectively, the precipitant agent was ammonia solution, the volume of 25% ammonia solution was 48ml, the ripening temperature is 80℃.2 Orthogonal experimental design results shows:The molecular weight of o-carboxymethyl chitosan, concentration of OCMCS and the power of ultrasonic have significant influence on the hydrophilic size of OCMCS-SPIO-NPs, while the ultrasonic time have no significant on the hydrophilic of OCMCS-SPIO-NPs. The optimal formulation-process was A1B1C1D2:namely,carboxymethyl chitosan concentration was 2%, MW of OCMCS was 1-2 million, ultrasonic time was 30minutes, the power of ultrasonic was 600W.3 Fourier transform infrared spectroscopy and X-Ray diffraction indicated that the best process to modify the OCMCS-SPIO-NPs with FA was as follows. The mass ratio of folic acid active ester and OCMCS the 4:1 ratio,the reaction temperature was 50℃under the conditions of anhydrous dimethyl sulfoxide solution with the nitrogen protecting from oxygen.4 Characterizations of resulting SPIO-NPs, OCMCS-SPIO-NPs, FA-OCMCS-SPIO-NPs and dextran-SPIO-NPs4.1TEM showed the resulting SPIO-NPs, OCMCS-SPIO-NPs,FA-OCMCS-SPIO-NPs and dextran-SPIO-NPs are almost spherical or ellipsoidal. The size of SPIO-NPs, OCMCS-SPIO-NPs, FA-OCMCS-SPIO-NPs and dextran-SPIO-NPs were present as x±s, whose particle size were 12.5±3.0nm,13.7±3.6nm,15.4±4.5nm and 17.5±4.8nm, respectively.Hydrophilic size of SPIO-NPs, OCMCS-SPIO-NPs, FA-OCMCS-SPIO-NPs and dextran-SPIO-NPs The average intense size was 201nm and the polydispersity was 0.234 for SPIO-NPs The average intense size was 38.2 nm and the polydispersity was 0.132 for OCMCS-SPIO-NPs, The average intense size was 41.4nm and the polydispersity for FA-OCMCS-SPIO-NPs; The average intense size was 125 nm and the polydispersity was 0.119 for dextran-SPIO-NPs.4.3.Magneitc properties The results of magnetic properties of the samples which were study by VSM indicated that the SPIO-NPs, OCMCS-SPIO-NPs, FA-OCMCS-SPIO-NPs were superparamagnetic, The saturation magnetization values for the SPIO-NPs, OCMCS-SPIO-NPs, FA-OCMCS SPIO-NPs were:98.0,71.4 and 69.6 emu/gFe.4.4.T2 rate of the resulting SPIO-NPs:OCMCS-SPIO-NPs, FA-OCMCS-SPIO-NPs and dextran-SPIO-NPs in the T2 rate was 0.1685×106mol/s,0.1512×106mol/s and 0.139×106mol/s, which were higher than 0.062×106mol/s minimum standards.The transverse relaxation time (T2) shorten with the iron concentration increase in SPIO-NPs suspension.4.5. Zeta potential of FA-OCMCS-SPIO-NPs, OCMCS-SPIO-NPs and dextran-SPIO-NPs were-21.36±1.15mV,-27.88±0.73m and-18.1±1.01lmV respectively. 4.6. The iron content in resulting SPIO-NPs was as follows:OCMCS-SPIO-NPs, FA-OCMCS-SPIO-NPs and dextran-SPIO-NPs solution iron content were 5.44 mg Fe /ml,5.62mgFe/ml and 5.76mgFe/ml respectively; iron content in SPIO-NPs, OCMCS-SPIO-NPs and FA-OCMCS-SPIO-NPs dry powder was 663.89mg/g, 615.61mg/g, and 601.40mg/g respectively.4.7. X-Ray diffraction results showed that the powder crystals of resulting SPIO-NPs,OCMCS-SPIO-NPs and FA-OCMCS-SPIO-NPs, were consistent with that of standards Fe3O4, more important, the entire preparation process of nanoparticles have no obvious impact on those powder crystal. FTIR results confirmed that OCMCS and the FA were conjugated on the surface of SPIO-NPs by covalent.5 Evaluation of resulting SPIO-NPs In vitro5.1 In vitro cytotoxicityThe cytotoxicity of FA-OCMCS-SPIO-NPs, OCMCS-SPIO-NPs, dextran-SPIO- NPs and SPIO-NPs against LO2, A549 and KB cells showed the cytoto xicity increase with the increase of iron concentration in culture media. The modification with OCMCS and FA could significantly decrease the cytotoxicity against all of the three cell. The cytotoxicity of FA-OCMC-SPIO-NPs against KB cell (FR over-expressed) was much higher than OCMCS-SPIO-NPs and dextran-SPIO-NPs(P < 0.05). While there is no significant difference among the cytotoxicity of FA-OCMCS-SPIO-NPs, OCMCS-SPIO-NPs and dextran-SPIO-NPs against LO2 and A549(FR negative) (P> 0.05).5.2 Evaluation folate receptor tumor targeting in vitroFerroine assay showed that the intracellular iron content of both KB cells and Hela cells in FA-OCMCS -SPIO-NPs group was significantly higher than that in OCMCS-SPIO-NPs (PHela= 0.000<0.05; PKB= 0.000<0.05), while,the intracellular iron content in A549 cells, there was no significant difference between FA-OCMCS-SPIO-NPs group and OCMCS-SPIO-NPs group(P= 0.106> 0.05).Prussian blue staining results showed both KB cell and Hela cell cultured with FA-OCMCS-SPIO-NPs were have dark prussian blue staining positive, which was much darker than those cells cultured in OCMCS-SPIO-NPs. While, A549 cultured in either FA-OCMCS-SPIO-NPs or OCMCS-SPIO-NPs, both of them were almost prussian blue staining negative in intracellular. 5.3 Macrophage uptake in vitroIntracellular iron content in RAW264.7 determined by Ferroine assay cells and Prussian blue staining showed that the iron content in RAW264.7 cultured in media containing FA-OCMCS-SPIO-NPs, OCMCS-SPIO-NPs, dextran-SPIO-NPs was much lower than that cultured in media containing SPIO-NPs group, moreover, the intracellular iron content of RAW264.7 culture in media containing the FA-OCMCS-SPIO-NPs were significantly lower than the that cultured in media containing dextran-SPIO-NPs.6. Evaluations in vivo6.1. Tumor targeting in vivo6.1.1 MRI scanT2-weighted spin echo images of the tumor-bearing mice showed that the average intensity of the entire KB cell subcutaneous tumors decreases about 27.23% between preinjection and postinjection images injected with FA-OCMCS-SPIO-NPs (p=0.01<0.05). However, the signal intensity had no significant difference between preinjection and postinjection images of subcutaneous tumors for other groups(p>0.05).6.1.2 HistopathologyHE and Prussian blue staining of tumor tissue slice showed only the bearing KB cell tumor mice were intravenously injected with FA-OCMCS-SPIO-NPs can be seen scatter SPIO-NPs aggregation and Prussian blue staining positive.while in other group there were no SPIO-NPs aggregation in HE staining or Prussian blue positive6.2 Histological evaluation of the distribution of nanoparticles in vivoPrussian blue staining results suggested that the mice that were intravenously injected with FA-OCMCS-SPIO-NPs and OCMCS-SPIO-NPs, there were no stainable iron was observed in the heart, liver, spleen, kidney and lung tissue. While the mice that were intravenously injected with dextran-SPIO-NPs, the stainable iron was observed in KCs in hepatic sinusoid, in splenic cord of the perimeter section of follicle and in splenic red pulp. however there were no stainable iron in the other tissuesConclusions1. we have successfully synthesized the FA-OCMCS-SPIO-NPs with a typical "core-shell" structure, those nanoparticles has good hydrophilicity and strong superparamagnetic with a hydrophilic size e<50nm.2. In vitro cytotoxicity results showed that covalently modify the SPIO-NPs with OCMC and folic acid could significantly reduce the cytotoxicity of SPIO-NPs. The cytotoxicity of FA-OCMCS-SPIO-NPs on cells (FR positive) were higher than that of OCMCS-SPIO-NPs, There was no significant difference on the cytotoxicity of FA-OCMCS-SPIO-NPs and OCMCS-SPIO-NPs against LO2(normal cell line) and A549(FR negative)3. Tumor targeting results showed the synthesized FA-OCMCS-SPIO-NP had a strong targeting folate receptor, the higher folate receptor expressed on the surface, the higher the intracellular iron content4. Uptake of all the synthesized SPIO-NPs by RAW264.7 in vitro indicated that the OCMCS surface modification can significantly reduce uptake of superparamagnetic iron oxide nanoparticles by the RAW264.7 cells, while the further modification had no influence on the uptake by RAW264.7 cells.5.In vivo evaluation results showed that FA-OCMCS-SPIO-NPs had a high folate receptor tumor targeting and could avoid capture by reticuloendothelial systemThe innovation of the paperIn this article, the OCMCS was used as a conjugate to associate the SPIO core and tumor target ligand (folic acid) to develop a novel active tumor-targeting SPIO-NPs system-FA-OCMCS-SPIO-NPs.