The Synthesis And Antitumor Activity Of Dextran-unsaturated Fatty Acid-cabazitaxel Conjugates

Cancer is one of the major threats to public health,and a great deal of effort has been invested in research at the molecular,cellular and clinical levels in the search for a complete cure for cancer;however,most treatments have failed to completely cure the disease.Chemotherapy is one of the important methods to treat cancer.However,many chemotherapeutic drugs currently used in clinic have problems such as poor water solubility,non-specific distribution,low targeting and easy to produce drug resistance.In recent years,the emergence of various new drug delivery systems provides a new idea to solve these problems.Through rational design of drug delivery systems,the water solubility of hydrophobic drugs can be increased,and the targeting can be improved,which has broad application prospects.Cabazitaxel(CTX),a semi-synthetic derivative of natural taxane compounds,has a low affinity for P-glycoproteins that cause paclitaxel and docetaxel resistance.CTX extends survival in castration-resistant metastatic prostate cancer patients who do not respond to docetaxel therapy.However,similar to other taxanes,due to its poor water solubility,the clinical use needs to add Tween 80 and ethanol as a co-solvent,which usually causes strong adverse reactions.In addition,the non-specific distribution of CTX in vivo,lack of targeting,easy to lead to systemic toxicity.Unsaturated fatty acids contain at least one unsaturated double bond in the alkyl chain,α-Linolenic acid(ALA),γ-Linolenic acid(GLA)and docosahexaenoic acid(DHA)are three important polyunsaturated fatty acids.They have certain antitumor effect and synergistic antitumor effect on chemotherapy drugs.Dextran(Dex)is a large molecular polysaccharide drug carrier with good water solubility,biocompatibility,stability and low price.Its structure contains a large number of chemically modified hydroxyl groups,which can be derivated through esterification,etherification,oxidation and other reactions.Using dextran as the carrier,CTX and unsaturated fatty acids were chemically coupled to dextran through a series of chemical reactions,so as to improve the water solubility and targeting of CTX,and achieve the purpose of reducing toxicity and enhancing anti-tumor activity.We first functionalized dextran and connected L-glutamic acid to make it negatively charged after hydrolysis.Lysine containing azide group was also coupled with the functionalized dextran for subsequent click chemical reaction.Then,the linked fragment containing alkyne,lysine and glycine was synthesized,and the three unsaturated fatty acids ALA,GLA and DHA were connected on the linker fragment by amide reaction.And CTX was then connected by esterification reaction to obtain the conjugate of CTX and unsaturated fatty acids(CTX-ALA,CTX-GLA and CTX-DHA),respectively.Finally,CTX-ALA,CTX-GLA and CTX-DHA were connected with functional polysaccharides by specific and efficient click-chemical reaction,and three different conjugates were prepared,which were named Dex-GLA-CTX,Dex-ALA-CTX and Dex-DHA-CTX,respectively.Dex-CTX without fatty acid and Dex-GLA without CTX were synthesized as experimental controls.The CTX drug loading of Dex-GLA-CTX,Dex-ALA-CTX,Dex-DHA-CTX and Dex-CTX were 24.8%,25.4%,21.9%and 21.1%,the GLA drug loading of Dex-GLA was 8.8%.Dex-GLA-CTX,Dex-ALA-CTX and Dex-DHA-CTX contain both hydrophilic and hydrophobic groups,so they can self-assemble into nanoparticles in water.The nanoparticles formed were spherical,uniform in size,without aggregation,and with good dispersion.The particle sizes were all in the range of 61.6-90.0 nm.The Zeta potential were in the range of-49.0-32.1 mV,all of which were electronegative and stable in the systemic circulation.Both the particle size and potential were suitable for tumor treatment.The solubility of Dex-GLA-CTX,Dex-ALA-CTX and Dex-DHA-CTX in water was 33.1,36.3 and 27.4 mg/mL(equivalent to CTX),respectively;however,the solubility of CTX in water is 3-7μg/mL.The results showed that these conjugates could significantly improve the water solubility of CTX.In the rat plasma release experiment,the content of CTX in the plasma of the parent CTX decreased in a time-dependent manner,while the release of CTX in the plasma of all the three conjugates gradually increased with time and then remained stable,indicating that the conjugate substance could release CTX slowly in the plasma and maintain a certain concentration,with obvious slow-release characteristics.In the PBS release experiment,the parent CTX was released quickly,and the cumulative release rate increased to nearly 100%after 12 h,and then remained relatively stable.The cumulative release rate of CTX in the three conjugates increased slowly and remained relatively stable after 48 h.The cumulative release rate of CTX in 72 h was 38.5%-52.1%,indicating that the three conjugates were relatively stable in PBS.The antitumor activity of the conjugates in vitro was evaluated using human prostate cancer cells DU 145 and PC-3.The content of intracellular free CTX released by conjugates was not significantly different from that of the parent CTX,but the total intracellular CTX content of conjugates was significantly higher than that of the parent CTX,indicating that more conjugates entered the cell than the parent CTX.In the cell proliferation inhibition experiment,the three conjugates inhibited cancer cells growth in a dose-dependent manner and there was no significant difference in growth inhibition among the conjugates and parent CTX.The results indicated that the conjugates and the parent CTX had similar inhibitorty effects aganist cell proliferation.In the cell apoptosis experiment,there was no significant difference among the three conjugates and the parent CTX,indicating that the conjugates and the parent CTX had similar ability to induce apoptosis.The pharmacokinetics of Dex-GLA-CTX in BALB/c mice were studied.Pharmacokinetic results showed that the area AUC(0-∞)of total CTX,free CTX and the maximum blood concentration(Cmax)of the conjugate Dex-GLA-CTX were significantly higher than those of the parent CTX.The AUC(0-∞)of total CTX of Dex-GLA-CTX was 16.9 times that of the parent CTX.The clearance rate(CL)of total CTX and free CTX of Dex-GLA-CTX in plasma was significantly lower than that of the parent CTX.Above results indicated that the conjugate Dex-GLA-CTX had longer circulating time than the parent CTX in vivo.The tissue distribution of Dex-GLA-CTX in DU 145 tumor-bearing BALB/C-nu nude mice were studied.The results of tissue distribution experiment showed that the content of free CTX in tumor treated with the parent CTX decreased gradually with time,while the content of free CTX and total CTX in tumor treated with the conjugate Dex-GLA-CTX first increased and then decreased with time.The concentration of free CTX in tumor in CTX group was higher than that in Dex-GLA-CTX group at 1 h,and lower than that in Dex-GLA-CTX group at other time points.At 12,24 and 96 h,the concentration of free CTX in the Dex-GLA-CTX group was 10.7,17.7 and 20.4 times that of the parent CTX.The amount of total CTX in the Dex-GLA-CTX group was significantly higher than that of free CTX.The content of free CTX of the conjugate Dex-GLA-CTX in normal tissues decreased with time,and the content of free CTX of the conjugate in tumor was higher than that in normal tissues after 24 h.The results showed that the conjugate Dex-GLA-CTX could accumulate and continuously release free CTX at the tumor site,showing tumor targeting,tissue selectivity and sustained release.In vivo tumor inhibition experiment was carried out using human prostate cancer cell DU 145 and PC-3 tumor bearing nude mice administrated with two doses of 4 mg/kg and 6 mg/kg through vein tail.The tumor volume and weight of nude mice were measured twice a week.The experimental results showed that the tumor inhibitory activity of the three conjugates was higher than that of the parent CTX.The survival of nude mice in Dex-GLA-CTX group was the longest among all groups.The order of antitumor activity of the three conjugates was Dex-GLA-CTX>Dex-ALA-CTX>Dex-DHA-CTX.The results also showed that the order of the synergistic effect of unsaturated fatty acids on CTX was GLA>ALA>DHA.The results of DU 145 tumor bearing nude mice also confirmed that the antitumor activity of Dex-GLA-CTX was higher than that of Dex-GLA,Dex-CTX and CTX,and the body weight of Dex-GLA-CTX group was higher than that of CTX group,indicating that GLA could improve the antitumor effect of the conjugate.In summary,the three synthesized conjugates had many advantages,such as high drug loading,good water solubility,suitable in size,sustained release,tumor targeting,high efficacy and low toxicity.Among them,the conjugates Dex-GLA-CTX had the strongest antitumor activity,which is of value for further research and development.