Design And Evaluation Of Targeted Probes And Synthesis And Mechanistic Study Of Targeted Drugs For Hepatocellular Carcinoma

In view of the high harmness of hepatoma carcinoma,the research on diagnosis and therapy was performed in this paper,mainly involving design and evaluation of targeted probes,synthesis and treatment mechanism of targeted drugs.First,all the nine pH probes prompted the spiral structure to open form in rodamine groups through N protonation in acidic pH environment,which resulted inπelectron conjugation to achieve fluorescence turn on.The recognition trend of glucoside<lactoside<galactoside illuminated that the galactose was a targeting group toward HepG2 cells and its recognition was affected by the bulk of targeting group.The HepG2 cells binding trend of RDGal1<RDGal2<RDGal3 supported that RDGal3 had the optimal targeting potential to HepG2 cells due to the stongest affinity of triantennary galactose.The specific recognition mechanism of RDGal3 to HepG2 cells was confirmed.First,compared with other cells,RDGal3 had the strongest fluorescence signal in HepG2 cells,indicating that RDGal3 specifically recognized HepG2 cells.Second,the fluorescence signal colocalization of RDGal3 and Asialoglycoprotein Receptor（ASGPR）antibody,indicating RDGal3 binding to ASGPR.And third,RDGal3 fluorescence intensity decreased after HepG2 cells blocked with galactose.The cell recognition mechanism of RDGal3 depended on the galactose group binding to ASGPR.The fluorescence labeling function of RDGal3 to HepG2 cells was also analyzed.First,The fluorescence based RDGal3 was activated in lysosomal pH range in cancer cells.Second,the fluorescence signal of RDGal3 colocalized with that of lysosomal dye.It indicated that RDGal3 accumulated in lysosome and was activated in lysosomal acidic pH to achieve selective labeling of HepG2 cells.The fluorescence response of RDGal3 in HepG2 cells was a time and concentration dependent manner,and RDGal3 was nontoxic to HepG2 cells,can be used in the early diagnosis of hepatoma carcinoma.Second,the three other metal ion probes can selectively determine Hg²⁺with stable interaction.The Hg²⁺detection was reversible,which was beneficial to probes recycling.The Hg²⁺detection limit using 5 mol/L probes was as low as 10^-8 mol/L,which can be used to detect trace Hg²⁺.The probes binding to Hg²⁺in accordance with the ratio of 1:1,and the mechanism of fluorescence turn on was as follows:Hg²⁺destroyed the spiral structure of the rodamine groups in probes,and the large conjugated structure after ring-opening resulted in Photoinduced Electron Transfer（PET）inhibition to activate fluorescence.The optimal pH range for the fluorescence response of the probes to Hg²⁺was3.5-6.5,which was suitable for the weakly acidic microenvironment of cancer cells.The probes can generate fluorescence via Hg²⁺in organisms,and the fluorescence response was in terms of Gal>Lac>Glu in HepG2 cells,but no significant changes in other cells.The probe Gal produced the strongest fluorescence signal in HepG2 cells.The fluorescence labeling of Gal to HepG2cells mediated via Hg²⁺was a concentration and time dependent manner,and Gal had no cytotoxicity to HepG2 cells,so can be used for safely monitoring Hg²⁺in hepatoma cells.Gal can selectively label HepG2 cells,which provided an idea for targeted research on hepatoma carcinoma.Third,the targeted drugs for hepatoma carcinoma of 5-fluorouracil derivative 5-FUGal and isoglycyrrhizin derivatives ISL1,ISL2 and ISL3 were synthesized.5-FUGal was nontoxic to HepG2 cells,and ISL1 had the highest fatality(IC₅₀ 4.62×10^-5 mol/L).The targeted therapy mechanism of ISL1 was investigated using Isobaric Tags for Relative and Absolute Quantitation（i TRAQ）proteomics.Overall,393 differentially expressed proteins were identified,of which 54proteins were upregulated and 339 proteins were downregulated（Ratio>1.5or<0.67）.The targeted therapy mechanism of ISL1 was revealed as follows:ISL1 induced decreased expression of HK2,and inhibited the glycolysis pathway to induce apoptosis.Down regulation of PEPCK activation inhibited the gluconeogenesis pathway,inhibiting tumor cells growth.Decreased expression levels of Akt1 and IKK caused down regulation of the Akt/IKK/NFκB signaling pathway,suppressing NFκB transcription to inhibit cells growth.Or through suppression of the NO/c GMP/PKG/CREB signaling pathway,CREB activity was regulated negatively to induce apoptosis.Cyt C with increased expression was released from mitochondria to activate Caspase-9,which activated Caspase-6 and Caspase-7 by cleavage cascade,so apoptosis was promoted.ISL1 can inhibit cell proliferation and growth,and activate apoptosis mediated by a mitochondria dependent pathway,can be used as a potential targeted therapeutic agent for hepatoma carcinoma;Functional proteins HK2,Akt1,IKK,PEPCK and Cyt C with anti-hepatocarcinoma activity can be used as candidate proteins.Fourth,porphyrin derivative of Zn TPPGal targeting to hepatocarcinoma was synthesized with excellent fluorescence properties,and metal zinc（Ⅱ）intervention can affect the optical properties of porphyrin.Zn TTPGal can be endocytized by HepG2 cells through selective recognition of tetraantennary galactosyl to ASGPR,which caused the rapid accumulation of the compound in HepG2 cells to stimulate red fluorescence with strong signal,so can be used in drug tracing.Zn TPPGal had a high mortality(IC₅₀ 0.91×10^-5 mol/L)to HepG2 cells,so had a specific targeted therapy effect on hepatoma carcinoma,and the treatment mechanism of Zn TPPGal was investigated via i TRAQ quantitative proteomics.In total,475 differentially distributed proteins were detected,of which 174proteins were upregulated and 301 proteins were downregulated（Ratio>1.5or<0.67）.The targeted treatment mechanism of Zn TPPGal was revealed as follows:Zn TPPGal induced down regulation of EGFR expression,and inhibited the Ras/Raf/MEK/EPK signaling pathway to inhibit cell proliferation,or blocked the PI3K/Akt/m TOR signaling pathway to induce apoptosis.Zn TPPGal triggered endoplasmic reticulum stress to induce increased expression of ASK1,activating the ASK1/MKK7/JNK apoptotic signaling pathway.Up regulation of Cathepsin D expression induced the cleavage activation of Bid,which resulted in cascade activation of Bax and Bak to cause mitochondrial membrane permeability and induce apoptosis.Suppressed expression levels of Rho and JNK blocked stress transduction,inhibiting the Rho/JNK signaling pathway,and leaded to apoptosis.Up regulation of Rag B and Rheb can positively regulate the m TOR signaling pathway activation,which resulted in the inhibition of autophagy pathway.Zn TPPGal can inhibit cell proliferation,growth and survival,and activate apoptosis mainly through endoplasmic reticulum stress pathway and mitochondrial dependent pathway,while do not activate autophagy,can be used as a potential targeted treatment agent for hepatoma carcinoma;Functional proteins EGFR、ASK1、Cathepsin D、Rho、JNK、Rag B and Rheb can be used as candidate proteins for hepatocarcinoma treatment and drugs development.