Study On Multifunctional Nanoparticles Based On Hyaluronic Acid For Anti-breast Cancer

In the world,the incidence of breast cancer is increasing year by year,seriously threatening the health of global women.There are many problems with traditional chemotherapeutic methods,such as rapid clearance of free drugs,non-selective distribution in the body,and inactivation of drugs in the body,which makes the results of chemotherapy less than expected,and even causes general systemic toxicity.In recent years,extensive research on Nano Drug Delivery System(NDDS)has found that it plays a huge advantage in increasing the solubility of poorly soluble drugs,achieving tumor targeting,improving the bioavailability of drugs,reducing side effects,and controlling drug release.NDDS brings new strategies for cancer treatment.Photodynamic therapy(PDT)has been applied in some tumor treatment.Combining chemotherapy and PDT is very valuable for the treatment of tumors.Accordingly,a multifunctional nanoparticle has been designed and developed which can deliver drugs to breast cancer cells with high expression of CD44 receptors,release drug responding to a highly reduced tumor environment,implement PDT under near infrared(NIR)light irradiation and achieve tumor imaging.Through a three-step amidation reaction,cystamine is used as a linker on the hyaluronic acid(HA),subsequently grafting docosahexaenoic acid(DHA)and chlorine6(Ce6),preparing a amphiphilic polymer—chlorin e6-hyaluronic acid-DHA(CHD).The polymer was characterized by FT-IR and 1H NMR and ultraviolet-visible absorption spectrum.The substitution degree of DHA was 8.8 and the content of Ce6 was 15.3±0.3%.Blank and drug-loaded nanoparticles were prepared by probe ultrasonic method.The average diameter of blank CHD nanoparticles was 214.1 ± 3.3nm,PDI was 0.255±0.016,and zeta potential was-29.4± 0.6mV;Prescription research was conducted on the selection of optimal solvent and the drug/carrier feeding ratio.Under the optimal prescription,the DTX drug loading was 9.28± 0.83%,and the encapsulation rate was 38.38± 3.78%.After loading drug,the particle size of DTX/CHD nanoparticles decreased to 181.4± 1.8nm,PDI was 0.241 ± 0.039,and zeta potential was-22.9± 0.7mV.Stability experiments show that DTX/CHD nanoparticles have little change in particle size and zeta potential within 72h,showing good stability.To investigate the reduction sensitivity of nanoparticles,detecting the fluorescence recovery of CHD nanoparticles in reducing environment,detecting reactive oxygen generation capacity using 9,10-Dimethylanthracene(DMA).The results showed that the amount of active oxygen produced increased in reducing environment.DTX release experiments from nanoparticles showed that the cumulative release amount at 48h was 84.48±1.68%,which was significantly higher than 65.84±2.67%in the low reducing environment.Nanoparticles were evaluated in vitro using human breast cancer MCF-7 cells with high expression of CD44 receptor.Using confocal microscopy and flow cytometry to explore the uptake behavior of MCF-7 cells to CHD nanoparticles.After 1h and 4h incubation,the intracellular fluorescence intensity of the CHD nanoparticle group was 9.7 and 13.9 times that of the free Ce6 solution group,or 1.7 and 1.9 times that of the HA pre-incubation group,respectively.It Demonstrated that the nanoparticles are taken up into cells by endocytosis mediated by CD44 receptors,showing the targeting of nanoparticles to tumor cells overexpressing CD44 receptors.Using cck-8 to study the cytotoxicity of nanoparticles:In the absence of NIR irradiation,the high concentration of CHD nanoparticles(the concentration of Ce6 was 40?g/mL)did not have obvious cytotoxicity.In the DTX/CHD+NIR group,combining PDT and chemotherapy,the cell activity was only 21.3±2.3%,showing the highest degree of inhibition of MCF-7 cell.The results of apoptosis experiments also showed that the combination therapy had the best effect.Observe the red fluorescence of Ce6 in CHD nanoparticles and the green fluorescence of lysosomes:The fluorescence of the two coincides when they are not irradiated by NIR,but they do not overlap after NIR irradiation,showing that NIR irradiation can help the nanoparticles to escape the lysosome.Fluorescent probe DCFH-DA was used to detect intracellular reactive oxygen species,the results showing that CHD under NIR irradiation had a higher active oxygen generation capacity than free Ce6;PI staining was used to detect the cell cycle:The DTX/CHD+NIR group blocked the cell cycle at the G2/M phase at 97.33%;The microtubules were stained with microtubule fluorescent probes,showing that the microtubules in the DTX/CHD+NIR group gathered to form vascular bundles,DNA fragmentation in the nucleus,and apoptotic bodies appeared,indicating that apoptosis was in progress;The above results indicate that the combined treatment of DTX and PDT shows a significant therapeutic effect;Using microtubule fluorescent probes to stain microtubules,the results showed that DTX inhibited microtubule disaggregation and played a role in promoting apoptosis;The above results showed that combined treatment with chemotherapy and PDT showed significant therapeutic effects.Balb/c mice were used for in vivo biological evaluation.In vivo imaging of mice showed that the fluorescence intensity of CHD nanoparticles in tumors is 4.2 times that of free Ce6,proving the targeting of nanoparticles to 4T1 cells at the animal level;In the in vivo anti-cancer activity,the tumor volume of DTX/CHD+NIR group was the smallest,showing a good tumor inhibition effect.The safety evaluation also showed that the DTX/CHD+NIR group was safe,demonstrating the superiority of chemotherapy combined with PDT.In summary,DTX?CHD nanoparticles have the advantages of CD44 receptor targeting,reduction-sensitive drug release,combining of chemotherapy and PDT and in vivo imaging.It shows good anti-tumor effect in vivo and in vitro biological evaluation,and has good biological safety,showing great potential in the treatment of breast cancer.