| Cancer,a serious threat to human health,is one of the main causes of death worldwide,and its treatment is still very challenging.The first-line treatments for solid tumors include surgery,radiotherapy and chemotherapy.Among them,chemotherapy is an important treatment approach that uses chemical drugs to kill tumor cells,thereby inhibiting the development of cancer.Paclitaxel,as one of the most successful drugs in the history of chemotherapy,is used to treat a variety of tumors such as ovarian cancer,breast cancer,and non-small cell lung cancer.Although it has a significant effect,the biggest disadvantage of paclitaxel is its poor water solubility.The preparation of paclitaxel injection with Cremephor EL and ethanol has achieved its clinical potential.However,Cremephor EL causes side effects such as peripheral neuropathy,hypotension and hypersensitivity.Therefore,in order to improve water solubility and reduce toxicity,the modification of paclitaxel structure or the optimization of formulations has always been a research hotspot in the global medical community.This thesis aims to improve the water solubility and targeting properties of paclitaxel by using the strategy of polymer macromolecule conjugated with paclitaxel.We synthesized a serious of paclitaxel-DHA-dextran macromolecular conjugates.The results showed that paclitaxel coupling with polysaccharide macromolecules improved the water solubility of paclitaxel,and can avoid toxic side effects caused by Cremephor EL and ethanol.At the same time,the EPR effect of macromolecular drugs in tumor tissues and the synergistic anti-tumor effect of DHA increased the accumulation of paclitaxel in tumor tissues,and ultimately enhanced the efficacy of paclitaxel.The charged nature of the macromolecular conjugate affects the metabolism and activity of the conjugates in the body.In order to compare the effect of the electronegativity of the PTX-DHA-dextran conjugates on the anti-tumor activity,this paper synthesized PTX-DHA-dextran conjugates with positive and negative ions.This thesis first used glycine and lysine as raw materials to synthesize a hydrophilic linker.The hydrophilic linker was connected to the C-2’position of paclitaxel to synthesize a paclitaxel modification.The compound THB-5 with both amino group and azide group was synthesized with DHA,tyrosine and 6-azido-n-hexylamine.The compound THC-7 with both carboxyl group and azide group was synthesized with DHA,tyrosine and 6-azido n-hexanoicacid.The polysaccharide was modified by chemical synthesis to carry positive amino groups and negative carboxyl groups.Finally,DHA,paclitaxel and dextran were linked into macromolecular conjugates through amide reaction and Click reaction.In this thesis,macromolecular conjugates were synthesized and their physical properties were characterized.The drug loading was determined by alkaline hydrolysis and 1HNMR.The drug loading of the conjugate determined by the 1NMR was 15.99%for the conjugate FB-2 and 11.56%for the BLC-2,respectively.The quantitative result of alkaline hydrolysis showed that paclitaxel content of the conjugate FB-2 was 15.82%.The particle size and Zeta potential were also measured to test its stability.The average particle size of FB-2 nanoparticles was 94.7±0.16 nm and Zeta potential was-25.27±0.42 mv.The average particle size of BLC-2 nanoparticles is 49.1±0.35 nm and Zeta potential is-16.20±0.30 mv.A drug release experiment in plasma was also carried out.The results of the experiment showed that both conjugates could smoothly release more than 80%of paclitaxel in the plasma for 72 hours,and the efficacy would not be affected by the inability to release the drug.Finally,a nude mouse tumor-bearing model of lung cancer cell H460 and a nude mouse tumor-bearing model of breast cancer cell MCF-7 were established for in vivo drug efficacy evaluation.In the lung cancer model,the tumor inhibition rate of the PTX(15 mg/kg)group was 43.9%;however,the tumor inhibition rates of the FB-2 conjugate at the dose 15 mg/kg and 22.5 mg/kg were 13.7%and 18.3%,respectively.FB-2 did not show comparable tumor inhibitory activity to that of the PTX group.The tumor inhibition rate of the BLC-2 conjugate(15 mg/kg)group was 31.9%,which failed to show the same tumor inhibition activity as the PTX group.BLC-2 conjugate(22.5 mg/kg)inhibited tumor growth by 44.5%,which was the same as the tumor suppressor activity in the PTX group.From the perspective of weight change,compared with the weight of the nude mice at the beginning of the administration,the weight of the PTX group decreased by about 10%,and the weight of the FB-2 and BLC-2 conjugate groups did not decrease.Although the toxicity of the conjugate is reduced,the anti-tumor activity of the two conjugates is not stronger than that of the paclitaxel.In the breast cancer model,PBS was used as a blank control,and Abraxane and PTX were used as positive controls to evaluate the antitumor activity and toxic side effects of the two conjugates at different doses.The tumor suppressor effect was observed 14 days after only one administration.The results showed that the tumor suppressor rate was 41.2%for PTX(15 mg/kg)and was 98.8%for Abraxane(30 mg/kg).The tumors in 3 nude mice disappeared completely in Abraxane group.FB-2 conjugate inhibited tumor growth by 33.1%and 95.6%at the dose of 15 mg/kg and 30 mg/kg,respectively.BLC-2 treatment at 15 mg/kg and 30 mg/kg caused 28.2%and 85.7%of tumor inhibition,respectively.Furthermore,a tumor in one nude mouse disappeared completely in the BLC-2(30 mg/kg)group.From the view of the weight change,the weights of each group increased because the nude mice were administrated only once in the duration of 14 days.The largest body weight increase was 4.9%in PBS control group,while the smallest increase was 0.2%in Abraxane group(30 mg/kg).The body weigh increased from 0.8%to 4.6%in another several groups.The results showed that in the nude mouse model of breast cancer,the 15 mg/kg dose of the conjugate groups had the same tumor inhibition rate as the paclitaxel,but the nude mice gained more weight than the PTX group.At the same dose,both FB-2 and BLC-2 were comparable to that of the Abraxane group.The weight of nude mice increased more than that of the Abraxane group,demonstrating that the toxicity induced by conjugates was less than that of Abraxane.In conclusion,the conjugates FB-2 and BLC-2 have stronger anti-tumor activity and lower toxicity in breast cancer.In addition,there is no significant difference between the conjugates FB-2 and BLC-2 in the activity of breast cancer,indicating that the amino-modified conjugate and the carboxy-modified conjugate have the same anti-tumor activity.In summary,the conjugates FB-2 and BLC-2 obtained in this paper can make tumors disappear or nearly disappear in nude mice with breast cancer,and have the same anti-tumor activity as Abraxane.Abraxane is a physically mixed nanoparticle of human albumin and paclitaxel.The conjugate FB-2 or BLC-2 is a nanoparticle formed by covalently linking the synergist DHA and paclitaxel to dextran.Compared with Abraxane,conjugates FB-2 and BLC-2 have stable structure,quality control,low cost,better solubility,and no foam when dissolved.The conjugates FB-2 and BLC-2 are composed of the nutrient DHA,the original drug paclitaxel and the clinically used dextran.They have strong pharmaceutical properties and low development risk.It is worth continuing to optimize and screen out conjugates with stronger activity than Abraxane. |
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