Active Targeting Nanoparticles Loaded With Tetremethylprazine As An Agent Against Multidrug Resistance In Cancer Cells

Objective:To investigate the effect that folate modified chitosan nanoparticles loaded withtetremethylprazine (FA-CS-TMP-NPs) reverses cancer multidrug resistance in vitro,and reserch its reversal mechanism.Method:1. Ionic gelation method was put to use in preparing the folate modified chitosantetremethylprazine nanoparticles. With tetremethylprazine as the target drug, folatemodified chitosan as carrier material, sodium polyphosphate anion as Physical inducer,and ionic gelation method was put to use in preparing folate modified chitosantetremethylprazine nanoparticles. At the same time, preparing tetremethylprazinenanoparticles (passive targeted nanoparticles) and empty nanoparticle for controlgroup.2. Determine the cytotoxicity of FA-CS-TMP-NPs. Human breast carcinomacells MCF-7/ADM and human leukemia cells K562/ADM which grow in logarithmicphase were treated with different concentration of FA-CS-TMP-NPs, after24,48,72h of incubation,0.5%MTT solution were added, the cells were then incubated foranother4h, and then100μL DMSO were added. The absorbance at492nm weremeasured by enzyme-labeled instrument. Origin software was put to use in designingthe growth curve of both the two cells, further more, respectively calculated the IC95(low dose)、IC85(middle dose) and IC80(high dose) in MCF-7/ADM cell line and IC95in K562/ADM cell line.3. Experimental groups and dosing regimenLow dose （safe dose）of tetremethylprazine in company with safe dose of ADM (0.5μg·mL-1) was exposed to each group. All the experiments grouped as follows:Control group: Blank medium group;Experimental Group: active targeting nanoparticles group;Experimental control group: passive targeting nanoparticles group; TMP solutiongroup; empty nanoparticles group; active targeting nanoparticles group (WithoutADM); ADM group;Positive control group: verapamil solution (10μg·mL-1)4. Pharmacodynamic evaluation of the reverse effect belong to FA-CS-TMP-NPs.The reverse effect of FA-CS-TMP-NPs was detected by MTT method. MCF-7、MCF-7/ADM、 K562、K562/ADM cell lines were divided into8groups as the3rditem. After exposed to the drug for24h,0.5%MTT solution were added, the cellswere then incubated for another4h, and then100μL DMSO were added. Theabsorbance at492nm were measured by enzyme-labeled instrument. Calculated thegrowth rate of cell lines in each group. Further more, compare the reverse effect amongeach group.5. Determination of intracellular fluorescence intensity of ADM. Intracellularfluorescence intensity of ADM was detected by flow cytometer. MCF-7/ADM cellswere divided into8groups according to the3rd item. After exposed to the drug for24h, cells were washed3times by cold PBS. Flow cytometer was put to use in detectingintracellular fluorescence of ADM. On the other hand, buried plate method was put touse, logarithmic growth phase MCF-7/ADM cells were divided into8groupsaccording to the3rd item, after exposed to the drugs for24h, laser scanning confocalmicroscope was put to use in photographing the cell morphology, and compare thefluorescence intensity of ADM among each group.6. Determination of Intracellular accumulation of ADM. Intracellularaccumulation of ADM was calculated by Fluorescence spectrophotography method.Preparing different concentration of ADM standard hydrochloric acid (3mol·L-1) inethanol (60%) solution, and preparing the standard curve, further more, calculate it’sLinear regression equation. On the other hand, Logarithmic growth phase MCF-7/ADM cell line was divided into8groups according to the3rd item. After exposed tothe drug for24h, the cells were collected, and washed3times by PBS. Fluorescencespectrophotography was put to use in detecting Intracellular accumulation of ADM ineach group. 7. Determination of time and dose dependent of FA-CS-TMP-NPs. MCF-7/ADM cells were divided into3groups, respectively exposed to into low, middle, highdose of FA-CS-TMP-NPs. At the same time, each group was exposed to safe dose ofADM. After1,6,12,24,48,72h, Fluorescence spectrophotography was used to detectintracellular ADM.8. Determination of P-gp and GST-π level by Western Blotting. MCF-7/ADMwere divided into7groups as above. After exposed to the drug for24h, the wholeprotein was extracted, electrophoresed, electrotransfered, incubated by first antibodyand second antibody and then stained by ECL, photographed under UV light, theexpression of P-gp and GST-π was detected by gray analysis. β-actin level was put touse as a protein loading control in western blotting.9. Determination of the MDR-1and GST-π level by Real Time PCR. MCF-7/ADM were divided into7groups as above. After exposed to the drug for24h, themRNA was extracted, MDR-1and GST-π level was detected by Real Time PCR,β-actin level was put to use as control.Result1. After exposed to FA-CS-TMP-NPs for24、48、72h, both of the two cellssuffered little cytotoxicity, with time extended the growth rate of cells decreased. WhenMCF-7/ADM exposed to FA-CS-TMP-NPs for24h, it’s IC95、IC85and IC80are0.261mg·mL-1,0.503mg·mL-1and0.737mg·mL-1respectively. And when K562/ADM exposed to FA-CS-TMP-NPs, it’s IC95is0.238mg·mL-1.2. After exposed to safe dose FA-CS-TMP-NPs in company with ADM for24h,the growth rate of MCF-7/ADM was dramatic decline. And the declination indicatesstatistical significant capered with CS-TMP-NPs and TMP solution. However, whenthe same treatment carried out on K562/ADM, on which the expression of FR is low,there was no statistical significantce between FA-CS-TMP-NPs and CS-TMP-NPs.3. The fluorescence intensity of intracellular ADM in ADM group、active andpassive targeting nanoparticles respectively were175.27±11.25、237.38±13.46and212.78±15.85. The result of FCM detection indicates that folate modified acitivetargeting nanoparticles could significantly enhance the fluorescence intensity ofintracellular ADM in MCF-7/ADM cell lines. What’s more, statistical significantceexist between active and passive targeting nanoparticles. But for MCF-7cell line, thereis no statistical significantce between active and passive targeted nanoparticles. The cell photographed by laser scanning confocal microscope indicates the same resultwith FCM.4. Folate modified tetremethylprazine acitive targeting nanoparticles couldsignificantly increase intracellular concentration of ADM. For MCF-7/ADM cell line,after the treatment for24h, the intracellular concentration of ADM in ADM group、active and passive targeted nanoparticles group respectively were0.343±0.013μg·mL-1、0.716±0.016μg·mL-1and0.630±0.036μg·mL-1.Compared with ADMgroup, both active and passive targeting nanoparticles can increase intracellularconcentration of ADM. What’s more, the comparisons between active and passivetargeting nanoparticles indicates statistical significant.5. Within24h, the concentration of intracellular ADM in MCF-7/ADM can beincreased with the time of treatment by FA-CS-TMP-NPs extended, that is, to someextent, the reversal effect of FA-CS-TMP-NPs is time dependent. After exposed toFA-CS-TMP-NPs for24h, the concentration of intracellular ADM in MCF-7/ADMincreased with the dose of FA-CS-TMP-NPs increased, that is, to some extent, theeffect of FA-CS-TMP-NPs is dose dependent.6. Compare with group ADM, Both active and passive targeting nanoparticlescould significantly down-regulated the activities of resistance-associated protein P-gp、GST-π and their gene MDR-1、GST-π mRNA. Further more, significant differenceexited between active and passive targeting nanoparticles.ConclusionAll the results demonstrate that compared with CS-TMP-NPs and TMP solution,FA-CS-TMP-NPs can reverse MDR in MCF-7/ADM by inhibit the expression ofP-gp and GST-π protein and it’s gene level.