| At present,cancer has gradually become one of the important clinical diseases commonly faced by the human economy and society,and it has become the main cause of the mortality of people under the age of 70 worldwide,which has caused serious adverse effects on both human health and social development.So far,no effective cancer diagnosis and treatment methods have been found.Current cancer treatment measures mainly include surgery,radiotherapy,chemotherapy and biological therapy.Taxanes are commonly used as first-line chemotherapeutic agents for the treatment of ovarian cancer,breast cancer and non-small cell lung cancer and other cancers.Cabazitaxel(CTX)is a new type of taxane anti-tumor drug which is similar to paclitaxel and docetaxel in structure.It was approved for marketing by the US FDA in 2010.The main site of action is tubulin,which promotes its aggregation into microtubules in the cell and forms a stable microtubule bundle in the cell,so that the mitotic spindle is no longer formed,thereby arresting the cell cycle and causing the cell to undergo apoptosis.Due to its structural methetherization,it has a lower affinity for p-glycoprotein than paclitaxel and docetaxel,and thus has a stronger inhibitory effect on tumor cells resistant to paclitaxel and docetaxel.However,cabazitaxel has poor water solubility,and the addition of solubilizer Tween-80 in clinical injection may lead to severe allergic reaction.Secondly,the lack of targeting of cabazitaxel in vivo has severely limited its clinical effects.The research purpose of this paper is to couple the polysaccharide and cabazitaxel through covalent bonds in order to improve its water solubility and to improve the targeting of macromolecular polysaccharide-cabazitaxel conjugates through enhanced permeability and retention effect(EPR)of tumors as well as to enhance the anti-tumor effect of cabazitaxel by connecting the sensitizer Docosahexaenoic acid(DHA).In recent years,the development of polysaccharide pharmaceutical polymers has increased due to their biocompatibility,biodegradability,high safety and low cost.Dextran is commonly used as plasma volume dilator in clinical practice.It has many advantages,such as cheap,easy to obtain,safe to use,good water solubility and narrow molecular weight distribution.Appropriate anions reduce the accumulation of dextran in the liver caused by nonspecific adsorption endocytosis and prolong the circulation.The carboxylated surface of dextran contains a variety of chemical groups,which is beneficial to drug loading.Therefore,in this paper,carboxylated dextran was used as the carrier of cabazitaxel,and the azide group was introduced for functional modification.At last,the conjugates were efficiently and specifically synthesized through Click reaction.Docosahexaenoic acid(DHA)has biological activities beneficial to human health,such as neuroprotection,anti-hypertension,anti-allergy and anti-arrhythmia.In addition,DHA has the effect of inhibiting tumor growth.Combined with chemotherapy drugs,DHA can enhance the sensitivity of tumor cells to drugs.In this study,DHA was conjugated with dextran to improve the antitumor efficiency of cabazitaxel.The molecular structure of cabazitaxel and functionalized polysaccharide carrier is complex.Due to the large steric hindrance of carrier,it is not easy to couple the drug with the carrier.In order to efficiently connect the drug and the carrier,as well as the DHA sensitizer,we have introduced a connecting arm.The link arm and its metabolites need to have no toxic and side effects,and can effectively release drugs in the body.The lysine structure contains three modifiable sites which are two amino groups and one carboxyl group.In addition,the water solubility of lysine is good,which is beneficial to improve the water solubility of the conjugate.Therefore,in this study,lysine is used as the structural basis of the connecting arm,and on this basis,different amino acids are further connected to investigate whether the length of connenting arm influence drug release and the anti-tumor activity of conjugates.Based on the above description,carboxymethyl dextran with ionic substitution degree of 0.58 was firstly synthesized as a polymer carrier which was connected with 0.30 mmol/g azide group.The functionalized dextran was named CDex which was used as a carrier to couple with cabazitaxel through click reaction.Then,in this paper,in order to study the effects of different lengths of link arms on the drug release and anti-tumor activity of the conjugate,linking arms containing 0,1,2,and 3 glycines were set up on the basis of lysine.However,the yield of the connecting arms containing 2 and 3 glycines is very low when esterified with cabazitaxel.In the end,only two link arms containing 0 and 1 glycine were obtained,and the link arms were then esterified with cabazitaxel 2’-OH to obtain linking fragments LI-8 and LI-11 containing DHA and cabazitaxel.Finally,the linker fragments were coupled with the functionalized carboxylated dextran CDex through the Click reaction.LI8-DHA-CDex and LI11-DHA-CDex conjugate were obtained and then were quantified by proton NMR spectroscopy.The drug loading of cabazitaxel in the two conjugates were 12.2%and 16.3%,respectively.The drug loading of cabazitaxel can be controlled by controlling the feeding ratio in the reaction system.Based on the content of cabazitaxel in the conjugates,the solubility of the two conjugates in water was 37.2 mg/mL and 44.7 mg/mL,respectively.The conjugates significantly improved the solubility of the parent cabazitaxel which dissolves in water in the level of 4 μg/mL.Transmission electron microscopy(TEM)results showed that LI8-DHA-CDex and LI11-DHA-CDex conjugates self-assembled into nanoparticles with uniformLy spherical in water.The dynamic light scattering(DLS)measurement showed that the particle size of the conjugate was 88.04±2.23 nm and 41.78±0.63 nm,and the Zeta potential was-43.18±3.01 mV and-23.73±5.39 mV,respectively,indicating that the nanoparticles have good stability.In the in vitro release of cabazitaxel in plasma,the release rates of LI8-DHA-CDex and LI11-DHA-CDex conjugates were 54.5%and 11%,respectively.The conjugate LI11-DHA-CDex has higher stability in systemic circulation.The amino acid in the connecting arm increases drug release time.The results of cytotoxicity experiments showed that the anti-tumor activity of the conjugate on tumor cell proliferation was stronger than that of the prototype drug cabazitaxel at the same concentration when cells were treated at lowe concertrations.At low concentrations of cabazitaxel,the cytotoxicity of the LI11-DHA-CDex conjugate is significantly higher than that of LI8-DHA-CDex;however,there is no significant difference in the cytotoxicity of the two conjugates at high concentrations.Compared with the apoptotic rate of the control group(5.3%),the apoptotic rate of the cabazitaxel(14%)and conjugate LI8-DHA-CDex(20.2%)and LI11-DHA-CDex(20.4%)groups increased.In addition,the effect of conjugate group on cell apoptosis was better than that of cabazitaxel group,which is consistent with the results of cytotoxicity experiment.The results of the Transwell cell migration experiment showed that the number of cell migration in all the treatment group was significantly reduced as compared with the control group.Compared with the cabazitaxel(397.7±70.9)group,the conjugate LI8-DHA-CDex(124.7±27.7)and LI11-DHA-CDex(71.0±8.5)significantly reduced the migration of tumor cells,indicating that both conjugates had improved inhibitory effect on the migration of tumor cells.Finally,the H460 tumor model of lung cancer cells were established for therapeutic efficacy and safety evaluation in vivo.Preliminary experiments in nude mice of this paper showed that cabazitaxel injection at 7.5 mg/kg once every 4 days decreased the body weight of nude mice by more than 20%after the administration for four times.Therefore,only the dosage of 5 mg/kg of cabazitaxel was set in this study.The results of anti-tumor activity in nude mice showed that the conjugate LI8-DHA-CDex group(39.2%)had a significantly higher tumor inhibition rate than cabazitaxel(24.3%)when administered at a low dose(5 mg/kg).The tumor inhibition rate of LI11-DHA-CDex(19.6%)was not significantly different from that of cabazitaxel.At high dose(7.5 mg/kg),both conjugates LI8-DHA-CDex and LI11-DHA-CDex dramatically inhibited tumor growth.The safety evaluation results showed that the weight loss of the conjugate groups was higher than that of cabazitaxel and that the conjugate LI11-DHA-CDex treatment resulted in a less weight loss than LI8-DHA-CDex,suggesting further nude mouse experiments were needed to verify or continue to optimize the structure of the conjugates.The above results were consistent with the data from biochemical and pathological analysis.The H&E staning showed that the conjugates induecd the lung and liver inflammation at lower dose,and induced spleen inflammation at higher dose.The degree of liver inflammation induced by LI11-DHA-CDex was lower than that of LI8-DHA-CDex,demonstrating that the conjugate of LI11-DHA-CDex with 1 more glycine in the link arm has lower toxicity than LI8-DHA-CDex.In conclusion,carboxylated and azide functionalized dextran was firstly developed;then we synthesized the conneting arms containing DHA and cabazitaxel.Finally,both conjugates were obtained by coupling the arms with the dextran therough a Click reaction.Both conjugates improved the water solubility of cabazitaxel and enhanced the anti-tumor effect,demonstrating its value for further optimization and development. |
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