Exploration In Passive Targeting Therapy Of Solid Tumor Based On The EPR Effect

Blood vessels in most solid tumors possess unique characteristics that are not usually observed in normal blood vessels. When tumors reach a size of 2mm, angiogenesis is induced and neovasculature is formed to offer the increasing nutritional and oxygen demands. The blood vessels in tumor are irregular in shape, dilated and defective, and the endothelial cells are poorly aligned with large fenestrations. Literatures report that the vascular pore size of human colon adenocarcinoma could be as wide as 400nm. In normal blood vessels, the smooth muscle layer is essential for the proper response to mediators such as acetylcholine,NO and maintain a constant blood flow volume; solid tumor vessles were found to lack a smooth muscle layer, instead the regulating function its vessels appears to be in a state of permanent dilation to increase the leakage from tumor vessles. Vascular endothelial growth factor (VEGF / VPF), bradykinin (BK), NO, prostaglandin (PGs), MMP (MMPs), and other regulatory factors generate more and strengthen interaction, and tumor vascular permeability was increased. Macromolecular material induced by above-mentioned reasons and could permeate through blood vessels into tumors, and produce passive targeting to tumor tissues. In addition, there is lack and impaired of lymphatic clearance from tumor tissues, hence macromolecules and lipids which are cleared from lymphatic remain in the tumor interstitium for a long time.Macromolecular drugs including liposomes that could not get through blood vessels into the normal tissue greatly increased permeability into tumors and prolonged retention. The phenomenon is known as the Enhanced Permeability and Retention (EPR effect). EPR effect is attribution to anatomical and pathophysiological alteration. The retention of macromolecular drugs prolonged for weeks or even months in tumors. EPR effect can be observed with macromolecules having an apparent molecular size larger than 40KDa and longer plasma half-life.Evans Blue (EB) is a laboratory diagnosis drug, which is widely used for determination of plasma or blood volume and permeability of the blood-brain barrier or capillary. It is a water-soluble dye with four symmetrical sulfonic groups. The chemical reaction of Evans blue occurs between sulfonic acid groups and nucleophilic groups at the protein surface such as the amino groups of lysines or arginines or hydroxy groups from other amino acids to form a sulfonamide or sulfonate ester linkage with protein. Serum albumin plasma protein content is about 60 percent of blood plasma total protein level. Dimensional structure and conformation has given it the ability to combine with other substances, such as the combination of the normal metabolic product of long-chain fatty acids, steroids and foreign non-biological substances like drugs and dyes. EB rapidly and firmly bond with albumin with a non-covalent after into the blood to form a stable complex macromolecules. Reports said that Evans blue was integrated preferentially and only bond with plasma albumin in the blood, when the ratio of Evans Blue and plasma albumin was less than 8.Using a reasonable dose of EB, we applied EB-albumin as a macromolecules model to study its accumulation and retention in tumor tissues, and observed its distribution within tumors was significantly higher than that of other organizations (P <0.01).Cutting off tiny focus of tumor is an important method to prevent recurrence after removing large tumors in surgical operation. It is so difficult to distinguish boundaries between small tumor focuses and normal organizations through the naked eye that completely removing tumors are always not achieved which induce tumor recurrence. Based on the front study of the EB-albumin complex distribution, it is so easily distinguished by the naked eye because EB coloring with the primary tumors and cysts around them were significantly higher than normal tissues, hence we try to develop Evans blue as a tumor coloring agent.EB-serum albumin macromolecules accumulated in tumor through the EPR effect after intravenous injection EB and colored tumors. we observed that EB could even make a small tumor with 1-2 mm sizes to be colored. This offered us the helps to distinguish boundaries between primary tumors and normal tissue in clinical surgery and support cancer-related experiments research. Mice experiments showed that: the best time for the administration of EB is 2-5 days before operation, and the best dose is 0.1-0.2 mgEB / 10g weight.We knew that Evans blue bond with the serum albumin formed a solid macromolecule compounds which had passive tumor targeting through the EPR effect. This prompted us that binding albumin formed macromolecules had an important value in self-assembly and increasing targeting of chemotherapy drugs. So we tried to combine small molecule conventional chemotherapy drug vincristine(VCR) with bovine serum albumin (BSA) in vitro through the weak link, and approached its effects on anti-cancer in the pharmacology.There is no increasing effect in vitro experiments which showed that VCR bonded with BSA was destoried fast in body, then a new balance was formed which was so unstable that could not make full use of EPR effect to achieve tumor passive targeting.LHRHR have been found to be expressed in a number of tumors, which act as an autocrine negative regulator of tumor growth. The tested drug LHRH-PE38 with molecular weight greater than 40KDa is a macromolecular anticancer drug which is tumor-specific integration with the LHRH-oriented elements and toxins mutant protein PE38. Based on the anticancer efficiency and through comparison with PE38 efficacy, we initially apporached the mechanism on tumor targeting distribution of LHRH-PE38.Experiments in vitro showed that, LHRH-PE38 counteracted tumor cells growth which had a large number of LHRHR expressed such as BGC823, MCF-7 and Lovo. Inhibition rate is from 72.6 to 83.3%. When concentration of drug lower was lower and action time was shorter, LHRH-PE38 exerted targeting anti-tumor effects mainly through identification of receptor; With the increased concentration and action time, effect of identifying receptor no longer had obvious advantages, however, tumor cells directly endocytosis drugs played a leading role.In vivo LHRH-PE38 could inhibit the growth of a variety of xenograft. LHRH-PE38 (100μg.kg-1) inhibited the growth of BGC823 xenograft reaching 62.5%, MCF-7 xenograft reaching 51.7%, and Lovo xenograft reaching 34.7%.Experiments on inhibition of Lovo xenograft growth showed that inhibition rate of LHRH-PE38 (100μg.kg-1) was 34.7%, and inhibition rate of PE38 (100μg.kg-1) reached 39.2%. In addition, inhibition rate of LHRH-PE38 (50μg.kg-1) achieved 27.2% and inhibition rate of PE38 (50μg.kg-1) was 20.3%. These results initially indicated that LHRH-PE38 mainly utilized EPR effect through characteristics of macromolecules to realize tumor selective delivery, completing the distribution in pharmacokinetics, moreover, it had coordination effect with identifying LHRHR to further the role of targeting anti-tumor effect. LHRH-PE38 played anti-tumor roles through active targeting identifying LHRHR in coordination with passive targeting EPR effect of macromolecules , and the latter made a leading role in the tumor targeting distribution.In this paper, we preliminarily studied the application of EPR effect in passive targeting therapeutics on solid tumors. EPR effects provided a theoretical and practical basis for chemotherapy of macromolecules, and played a guiding role to reduce the side effects of traditional chemotherapy.