Preparation Of Pinoditerpene Derivative-Melanin Nanoparticles And Their Mechanisms Of Inducing Apoptosis In HCC Cells

Hepatocellular carcinoma(HCC)as the main histological subtype of liver cancer,is characterized by rapid disease progression,high metastasis rate,poor efficacy and prognosis,and high mortality.Currently,chemotherapy is one of the main therapies for HCC.However,some drawbacks including high side effects,high price and low bioavailability in vivo have limited the application of most chemotherapeutic agents.Therefore,it is urgent to develop cheap anticancer drugs and explore ways to increase their effectiveness and reduce their toxicity.To achieve the above objectives,two measures can be adopted:screening novel highly-efficient and low-toxic anti-cancer compounds/lead compounds from plants;enhancing of the tumor targeting and activity of anti-cancer compounds/lead compounds by chemical modification or designing novel drug delivery systems.Herein,screening of compounds with anti-HCC activity from 60% ethanol extract of Pinus koraiensis pinecones were conducted through various purification technologies.Based on the lead compound dehydroabietic acid,a pinoditerpene derivative(DD2)with superior activity was synthesized through a two-step chemical reaction,and its anti-tumor mechanisms in Hep G2 cells and BALB/c tumor-bearing nude mice were explored,and its safety in vivo was evaluated.In addition,a chitooligosaccharide-melanin complex(CMC)suitable for carrying DD2 was prepared,and its chemical structure and the mechanisms of affinity for Hep G2 cell membrane/nucleus were investigated.Finally,nanoparticles(CMC-DD2)with tumor chemotherapy(CT)and chemodynamic therapy(CDT)effects were constructed and mechanisms for synergistic effect of CT and CDT in vitro and in vivo have been thoroughly explored.Results showed that compounds composed of 15-hydroxydehydroabietic acid,dehydroabietic acid,15-hydroxy-7-oxodehydroabietic acid,7α-hydroxydehydroabietic acid and inonotusic acid were the main anti-tumor efficacy material basis of 60% ethanol extract from Pinus koraiensis pinecones.In addition,the anti-HCC activity of DD2 was much higher than that of dehydroabietic acid and paclitaxel(PTX),suggesting that the polymerization of multiple tricyclic skeletons could effectively enhance the toxicity of dehydroabietic acid to Hep G2 cells.The cell phenotype assay and western blot results showed DD2 entering Hep G2 cells could elevate ROS and cytoplasmic calcium levels,induce DNA damage,promote cell cycle arrest in G2/M phase,trigger mitochondrial dysfunction,and initiate mitochondrial apoptosis containing P53-Puma-Bcl-2/Bax axis through caspase enzyme cascade reaction.A Hep G2 cell xenograft tumor model was established using BALB/c nude mice.After intratumoral administration,DD2 could induce severe DNA damage i n tumor tissues and activate mitochondrial apoptosis including the P53-Bcl-2/Bax axis to suppress tumor growth,whose anti-HCC activity in vivo was significantly stronger than that of PTX.Liver and kidney function tests and H& E staining of major organs confirmed the reliable safety of DD2 in vivo.Structural investigations showed CMC exhibited a graphite-like layered structure,where chitooligosaccharide derivatives,5,6-dihydroxyindole-2-carboxylic acid(DHICA),5,6-dihydroxyindole(DHI),and dopa derivatives were interconnected by covalent bonds to form the layer struc ture of CMC(primary structure).CMC exhibited strong affinity for Hep G2 cell membranes by binding phosphatidylserine,glycoproteins,glycolipids,and glycosaminoglycans that accumulated on the cell surface,and interacted with DNA and/or RNA binding proteins located in/around the cell nucleus to target the nucleus and disrupt cell proliferation/apoptosis processes.Nanoparticles(CMC-DD2)of CMC and DD2 were constructed using a nanoprecipitation method.Especially,the hydrophobic interaction,π-π stacking force and hydrogen bonds between CMC and DD2 jointly drived the selfassembly and maintained the stability of the nanoparticles.CM C-DD2 was stable under physiological conditions,but it could be dissoci ated to release DD2 in response to weak acid,especially ROS stimulations.In addition,the surface of CMC-DD2 was positively charged in the weak acid environment,but became negatively charged in neutral solution,which predisposed CMC-DD2 to enter Hep G2 cells,escape capture by lysosomes and target the nucleus.After entering cell nucleus,CMC-DD2 altered the DNA conformation by binding bases of DNA,and dissociated in response to intracellular ROS and release d nitrate ions,· OH and DD2 to initiate DNA damage-induced mitochondrial apoptosis.After entering tumor-bearing nude mice via the tail vein route,CMCDD2 could enrich in the tumor and promote tumor ablation by elevating ROS levels,disrupting DNA structure and activating mitochondrial apoptosis.The safety assessment showed that CMC-DD2 caused no significant weight loss,liver and kidney damage and major organ lesions in tumor-bearing mice,and its in vivo safety was excellent.In the present study,the main anti-HCC compounds in the 60% ethanol extract from Pinus koraiensis pinecones have been identified,and DD2 with significant anti-cancer activity has been synthesized based on the isolated compound,with the investigation of its anti-tumor mechanisms.A drug carrier CMC suitable for delivering DD2 was prepared and its chemical structure and mechanisms of affinity for Hep G2 cell membrane/nucleus were explored.The charge-reversal,nucleus-targeted,intracellularly degradable,DNA-affinity CMC-DD2 composed of CMC and DD2 was constructed,dem onstrating that nanoparticles could promote tumor ablation by producing synergistic CT and CDT effects on tumors.This study is of practical significance for the discovery of novel chemotherapeutic drugs and the design of mul tifunctional nanoparticles,and contribute a reference for tumor targeting and precision therapy.