Application Of A Poly(L-glutamic Acid)-Combretastatin A4 Conjugates(PLG-CA4) Nanoparticles For The Treatment Of Colon Cancer

Malignant tumor is the first killer to threaten human health.Colorectal cancer morbidity and mortality is in the top three both in men and women.In the treatment of colon cancer,only the early staged tumor surgery can achieve better results.For the advanced cancer patients,the chemical treatment occupies an important position,but traditional chemotherapy drugs do not bring the satisfaction of the anti-tumor effect.Tumor growth and metastasis are mainly dependent on the formation and maintenance of blood vessels.The tumor cells have the growth limitation without vascular support.Small molecule vascular disrupting agent(VDA)is different from traditional chemotherapy drugs,which eliminates tumor tissue by blocking tumor neovascularization and lack of feeding.Combretastatin A4(CA4)is a leading agent in vascular disrupting strategies for tumor therapy.Although many small-molecule prodrugs of CA4 have been developed to improve its solubility,the overall therapeutic efficiency is moderate.A key reason for this is the reversible effects of CA4 on tubulin as well as the rapid clearance from plasma and tissues.Since the last century 90's,nano-drugs are studied by large-scale,which have accumulated a lot of experience in improving the characteristics of small molecules.The preparation of nano-carrier can improve the circulating time of small molecules in the body,while solving the drug biocompatibility.At the same time,the EPR effect can improve the passive target of the tumor,prolong inhibition of tumor growth,to achieve a good anti-tumor effect.In this study,we proposed a poly(L-glutamic acid)-combretastatin A4 conjugates(PLG-CA4)to fulfill the requirements for fully liberating the potential of CA4 on tumor therapy: enhanced accumulation and retention of CA4 in the tumor tissue,main distribution and gradual release around tumor blood vessels,resulted in prolonged vascular disrupting and markedly enhanced tumor therapeutic efficiency.To clarify this,we examined the therapeutic efficiency of PLG-CA4 on murine colon C26 tumor model and compared with commercial combretastatin A4 phosphate(CA4P).PLG-CA4 showed significantly prolonged retention in plasma and tumor tissue,and was mainly distributed around the tumor vessels.In details:1)Poly(L-glutamic acid)(PLG)-based polymeric-drug conjugates have consistently been used for drug development.PLG is biocompatible,biodegradable,and can be easily modified.CA4 was conjugated to PLG-g-mPEG.The final PLG-CA4 conjugates had a CA4 drug loading content(DLC %)of 33.7 % and its molecular weight 5.7×104 Da,Zeta potential-7.9±0.8 mV.We tested the diameter of 28.0 nm,formed spheres in PBS by TEM and Dh of 36.4 nm by DLS.We tested the in vitro release of CA4 from the conjugates in distilled water,PBS pH 7.4 and pH 5.5,we also tested in BSA solution which similar to the plasmas.The PLG-CA4 is stable in the presence of plasma and released slowly.PLG-CA4 significantly had the characteristics of long plasma retention time by comparing with the pharmacokinetic data of PLG-CA4 and CA4 P.The biodistribution measured the contents of PLG-CA4 and CA4 P in different tissues by HPLC and Ex vivo.The side effect of PLG-CA4 in the main organs was evaluated by the normal organ H&E stain.2)We demonstrated that PLG-CA4 had tumor vascular targeting which can disrupt tumor blood vessels for a long time and play a role in inhibiting tumor growth through MSOT test.In the immunofluorescence imaging test,PLG-CA4 was labeled Rho B and blood vessles were stained by CD31,it was proved that PLG-CA4 was mainly accumulated in the area full of tumor blood vessels and had the ability to targert the blood vessles.We stained the blood vessles by CD31 after adminastration and observed the number of blood vessels in PLG-CA4 group was less than that in CA4 P group,and the number of tumor blood vessels in CA4 P group was significantly decreased after 1 h of administration,and rebounded after 4 h,indicated that PLG-CA4 had a long retention in the tumor blood vessels and played anti-tumor effect.We also built the tumor tissue perfusion model by Hochest33342 stain technique.3)The PLG-CA4 and CA4 P could inhibit the growth of C26 colon cancer cells by MTT and CCK-8 test.CA4 P and PLG-CA4 were analyzed after administration at different time points by H&E stained,we can observe that the tumor relapse was rapid in CA4 P group,especially the area of tumor necrosis was reduced to 10% at 24 h,while the PLG-CA4 group showed sustained tumor inhibition effect,which can reach 78.9% of the necrosis rate at 72 h.The tumor suppression rate in the two groups was 73.65% in the PLG-CA4 group and 24% in the CA4 P group after treament on day 17 by testing the tumor volume and the difference was statistically significant.We also observed the nano-drug had no obvious side effect by analyzing the weight change.4)PLG-CA4 combined with platinum-based drugs had obvious inhibitory effect,but the decrease of body weight indicated that side effect was obvious.PLG-CA4 combined with PLG-CDDP can maintain advanced antitumor effect while reducing the side effect of drugs.