| Objective: After preparing Folic Acid Modification of 3-bromopyruvate Liquid Crystalline Nanoparticles(3BP-LCNP-FA)and conducting 3BP-LCNP-FA prescription screening and preparation process optimization,the investigation Particle size and morphology;by simulating the tumor microenvironment p H 6.8 to investigate the cumulative release characteristics of 3BP-LCNP-FA in vitro;by culturing tumor cells in vitro to investigate the inhibitory effects of different drug dosage forms on cell proliferation and the effects on ATP levels in cells and cells;By establishing a tumor-bearing nude mouse model,the anti-tumor effect and toxic side effects of 3BP-LCNP-FA were investigated.Methods: 1.Screen 3BP-LCNP optimal prescription and best preparation process: use injection method combined with high-pressure homogenization method to prepare 3BP-LCNP,through orthogonal test design crystal type formed by liquid crystal and encapsulation rate index,screening auxiliary material glycerin The dosage and stirring time of acid ester(GMO),stabilizing agent Poloxamer 407(F127),dispersed phase pure water,to determine the best formulation prescription.Through single factor experiment,the influence of homogenization times and homogenization pressure on the average particle size and encapsulation rate of 3BP-LCNP was investigated.Observe the state of the crystal form under different prescription ratios through a polarizing microscope(PLM).The Malvern particle size analyzer was used to measure the average particle size distribution of 3BP-LCNP..2.Synthesis of folic acid(FA)and poloxamer 407(F127)(FA-F127)and preparation and characterization of 3BP-LCNP-FA: using differential scanning calorimetry(DSC)and nuclear magnetic resonance hydrogen spectroscopy(1H-NMR)Evaluate the synthesis of folic acid and poloxamer 407;prepare 3BP-LCNP-FA and characterize 3BP-LCNP-FA,observe the crystal forms of 3BP-LCNP and 3BP-LCNP-FA by PLM,observe by Malvern particle size analyzer The particle size distribution of 3BP-LCNP-FA;the morphology of 3BP-LCNP-FA nanoparticles was observed by cryo-transmission electron microscopy;the encapsulation rate of 3BP-LCNP-FA was determined by equilibrium dialysis..3.The cumulative release of 3BP-LCNP-FA in vitro and its effect on tumor cells,using phosphate buffer p H 6.8 as the release medium,using equilibrium dialysis to investigate the cumulative release of 3BP-LCNP-FA in vitro;live cell workstation to observe different drugs The dosage form acts on CNE2 Z with high expression of MCT1 and MDA-MB-231 cells with low expression of MCT1,and observes the changes of cell morphology and density;Inhibition of proliferation of MDA-MB-231 cells;ATP kit was used to detect changes in intracellular ATP levels after different drug dosage forms.4.3BP-LCNP-FA in vivo pharmacodynamic evaluation,establish a tumor-bearing nude mouse model,and evaluate the inhibitory effects and adverse reactions of different drug dosage forms on the tumor through the tumor volume and nude mouse body weight change trend during administration and the final tumor volume and tumor weight;Analyze the effect of different drug dosage forms on tumor tissue,lung,liver and kidney by H & E section;by analyzing serum alanine aminotransferase(ALT),aspartate aminotransferase(AST),creatinine(Cr)and urea nitrogen BUN)level to investigate the effect of different drug dosage forms on liver and kidney;finally PCNA immunohistochemistry and Tunel detection of tumor cell proliferation and apoptosis in tumor tissue.Result: 1.The optimal prescription prepared by injection method combined with high-pressure homogenization method is: the concentration ratio of GMO to F127 is 8: 1(total mass is maintained at 1 g),namely GMO 0.889 g,F127 0.111 g,pure water 25 m L,3BP 26.67 mg,Stir for 20 min.The prepared liquid crystal shows a dark field under a polarizing microscope,indicating that the prepared liquid crystal is a cubic phase liquid crystal.The optimized preparation process is 14900 psi,homogenized 9 times,that is to obtain drug-loaded nanoparticles with an average particle size of 192.3 nm,PDI of 0.113,and an encapsulation rate of 68.9%.2.The DSC spectrum shows that the phase transition temperature of the synthesized FA-F127 disappears;the 1H-NMR spectrum shows that the hydrogen spectrum peak shows the unique hydrogen spectrum peak of FA-F127,and there is no other impurity peak.The above proves that FA and F127 are chemically combined Way to connect,not a simple physical mixture.3BP-LCNP-FA also shows a dark field under PLM,indicating that FA-F127 does not change the crystal structure of the cubic liquid crystal;3BP-LCNP-FA has a nanoparticle size of 201.7 nm and a PDI of 0.102.The similar particle size distribution curve of LCNP shows that FA-F127 has no significant effect on the particle size of the cubic liquid crystal;the particle morphology of 3BP-LCNP-FA observed under TEM is smooth and round,non-adhesive spherical particles;3BP-LCNP-The encapsulation rate of FA reached 70.23%,and the RSD was less than 4%.3.The cumulative release of 3BP-LCNP-FA in vitro showed that the cumulative release of 3BP in 3 hours reached 96.89%,while that of 3BP-LCNP-FA was only 21.99%.It reached 79.62 % in 24 h and 90.76 % in 48 h,indicating that 3BP-LCNP-FA has a certain sustained-release effect.In CNE2 Z cells,different drug dosage forms have similar changes in cell morphology and density,while in MDA-MB-231 cells,3BP-LCNP-FA can significantly reduce cell density,make cell morphology change,nuclear division and enlargement.MTT results show that the blank carrier has no effect on CNE2 Z and MDA-MB-231 cells The cell survival rate was more than 90 % and had good cell compatibility.In CNE2 Z,when the concentration of 3BP was 50 mmol / L,the inhibition rates of 3BP and 3BP-LCNP were 4.87 % and 7.83 %.The inhibition rate of 3BP-LCNP-FA was 86.3 %.3BP,3BP-LCNP and 3BP-LCNP-FA had similar effect at 125 mmol / L with the increase of 3BP concentration.However,in MDA-MB-231 cells,with the increase of 3BP concentration,the inhibition rate of 3BP and 3BP-LCNP was 10.23 % and 32.4 % at 125 mmol / L,while the inhibition rate of 3BP-LCNP-FA was 73.37 %.The results of ATP level detection showed that 3BP,3BP-LCNP and 3BP-LCNP-FA significantly reduced the ATP level in CNE2 Z cells,but in MDA-MB-231 cells.3BP did notsignificantly reduce the intracellular level,3BP-LCNP and 3BP-LCNP-FA could significantly reduce the intracellular ATP level.4.In vivo pharmacodynamics experiment of 3BP-LCNP-FA showed that during the administration,the change trend of tumor volume in the 3BP-LCNP-FA group was significantly smaller than that in the 3BP group,and the final tumor volume and tumor weight were significantly different from that in the 3BP group,and the tumor inhibition effect was superior to that in the 3BP group.During the administration of 3BP,the body weight of nude mice showed a downward trend,which was not seen in other groups;the levels of AST,ALT,Cr and BUN in different administration groups were all within the normal range;the damage morphology of liver and kidney tissue was not observed by H&E staining.The results of PCNA immunohistochemistry showed that there were less brown granules in the group of 3BP-LCNP-FA administration,and the proliferation activity of tumor cells was weak;TUNEL staining showed that the green fluorescence of 3BP-LCNP-FA positive granules showed that the apoptosis activity of tumor cells in the group of 3BP-LCNP-FA administration was strong.3BP-LCNP-FA has a stronger inhibitory effect on tumor tissue than 3BP.Conclusion: To sum up,the formulation of 3BP-LCNP-FA is reasonable,the preparation process is feasible,the particle size of the prepared nanoparticles meets the requirements,the size is uniform,there is no adhesion,and has a certain slow-release effect.3BP-LCNP-FA has excellent inhibitory effect on CNE2 Z and MDA-MB-231,and is MCT1 dependent.3BP-LCNP-FA has a good therapeutic effect in nude mice bearing tumor,and has no obvious side effects.The above results show that folic acid modification of liquid crystal nanoparticles can be used as a new delivery system,which has potential clinical significance. |
PDF Full Text Request