Design And Activity Study Of Tumor-targeted Cell-penetrating Peptide-drug Conjugates

Tumor continues to be one of the major diseases that threaten human health.Currently,chemotherapy still is a primary means of treating tumor.However,lack of specificity,poorly water soluble,and drug resistance seriously limit the clinical application of conventional chemotherapeutic drugs.Recently,tumor-targeted peptidedrug conjugates become an effective approach for improving the therapeutic index of conventional chemotherapeutic drugs.Despite this,development of ideal tumortargeted peptides remains huge challenge.Intratumoral heterogeneity is one of the important factors that affect the antitumor activity of tumor-targeted drugs.Cell-penetrating peptides(CPPs)are a type of cationic short peptides.Generally,cationic CPPs adsorb readily to a negatively charged membrane of tumor cells via electrostatic interaction,subsequently enter cells by membrane disturbance.Compare with many tumor-targeted peptides,cell membranetargeted CPPs has been received more attentions for delivering conventional antitumor drugs.Despite this,lack of specificity still hampers the clinical application of CPPs.To date,many strategies have been developed for improving the tumor specificity of CPPs.Utilization of acidic tumor microenvironment is an effective strategy to develop tumortargeted CPPs.Histidine,containing an imidazole side-chain,is the only amino acid that can shift from the unprotonated state at neutral p H to the protonated state for positive charge at mildly acidic p H.This feature of histidine can be used to develop efficient acid-activated CPPs: histidine-containing CPPs become positive under acidic conditions,following by absorbing to membranes and entering cells.Melittin,derived from bee venom,is the most classical AMP with multiple functions.Melittin has been widely used for antitumor application due to its extreme membrane-lytic activity.However,lack of specificity seriously limits the antitumor application of melittin.Therefore,many strategies were developed to decrease the toxicity and improve the antitumor activity of melittin.In addition to the membranelytic mechanism,melittin can induce cell death by disrupting organelle membranes and interfering with various signal transductions related to tumor growth after entering cells.Based on the similarity in structure between AMPs and CPPs,we think that melittin is capable of delivering antitumor drugs like CPPs.To improve the antitumor activity of melittin,we synthesized the p H-response melittin analog AAM by replacing the lysines and arginines with histindes.In addition,AAM-1and AAM-2 were synthesized by introduction of cystine at the C-terminus and N-terminus of AAM,respectively.Subsequently,CPT-AAM-1and CPT-AAM-2 were synthesized by conjugation of antitumor drug camptothecin(CPT)to AAM-1and AAM-2 via a disulfide releasable carbonate linker.We expected that these conjugates possess two functions under acidic conditions: at high concentrations,they can kill cancer cells by membrane disruption;at low concentrations,they can kill cancer cells by the released CPT.As expected,FAM-AAM-1 and FAM-AAM-2 could enter cells like CPPs.However,the cellular uptake of FAM-AAM-2 did not show obvious p H-dependent manner.In contrast,FAMAAM-1 showed high cell-penetrating activity under acidic condition compared with FAM-AAM-2.This result demonstrated that that the C-terminus of AAM is more suitable than N-terminus to conjugate FAM.In addition,FAM-AAM-1 showed stronger endosomal escape activity than FAM-AAM-2.This may be because FAM-AAM-1displayed stronger membrane-lytic activity compared with FAM-AAM-2.In vitro experiment demonstrated that CPT-AAM-1 displayed higher antitumor activity than CPT-AAM-2 under acidic conditions.This result further confirmed that the C-terminus of AAM is suitable for conjugation of drugs.The results derived from cell cycle analysis demonstrated that CPT-AAM-1and CPT-AAM-2 killed tumor cells by the released CPT.The results derived from LDH leakage assay and PI uptake assay demonstrated that CPT-AAM-1 was capable of rapidly killing tumor cells via membrane disruption.In B16-F10 bearing mice model,the results demonstrated CPT-AAM-1 exhibited strong tumor-suppressive activity than CPT and AAM-1.Gratifyingly,in two out of eight mice that received CPT-AAM-1 treatment,tumor almost completely disappeared.Although melittin showed similar tumor-suppressive activity as CPT-AAM-1,melittin displayed obviously in vivo toxicity with a 75% mortality rates.Overall,CPT-AAM-1 with efficient acid-activated cell-penetrating activity and membrane-lytic activity showed a potential in tumor theray.Camptothecin(CPT),a DNA-toxin drug,exerts anticancer activity by inhibiting topoisomerase I.Targeted delivery of CPT into the cancer cell nucleus is important for enhancing its therapeutic efficiency.The nuclear localization sequence(NLS)is the most well-known for mediating transport of multiple carriers into the nucleus.However,low cell-penetrating activity and tumor specificity limit the application of NLS.Therefore,HNLS-2 was constructed by attachment of six histidines to the C-terminus of NLS.Our results demonstrated that the cellular uptake of HNLS-2 was significantly enhanced under acidic conditions.To further improve the cell-penetrating activity of HNLS-2,HNLS-3 was constructed by attaching the hydrophobic peptide sequence PFVYLI to the N-terminus of HNLS-2.Our results indicated that HNLS-3 showed significant p H-dependent cellular uptake efficiency,endosomal escape ability,and nuclear localization activity.More importantly,the HNLS-3-CPT conjugate showed obviously enhanced cytotoxicity and selectivity compared with CPT.Taken together,our findings provide an effective strategy to develop effective CPPs with both cancerand nucleus-targeting properties.Development of efficient nucleus-targeted CPPs can contribute to deliver DNAtoxin drugs.To our knowledge,there has been few reports on this type of CPPs.PFVNLS was constructed by attaching the hydrophobic peptide sequence PFVYLI to the N-terminus of NLS(KKKRKV).In addition,a series of analogs were designed by cyclization and amino acid substitution to obtain efficient CPPs with nuclear translocation activity.First,PFV-MNLS was constructed by introduction of two methionine residues in the sequence of PFV-NLS.Subsequently,PFV-MNLS-1 was synthesized by stapling of PFV-MNLS via methionine bis-alkylation.Surprisingly,we found that introduction of methionine could obviously enhanced the cell-penetrating activity of PFV-MNLS compared with PFV-NLS.We inferred that the increased hydrophobicity can contribute to the improved cell-penetrating activity of PFV-MNLS.Unfortunately,cyclization did not enhance the cell-penetrating activity of PFV-MNLS-1 compared with PFV-MNLS.To further improve the cell-penetrating activity,PFVLNLS was constructed by substitution of methionine for leucine.Although the hydrophobicity of PFV-LNLS was decreased,its cell-penetrating activity was obviously higher than PFV-MNLS.Despite this,both PFV-MNLS and PFV-LNLS displayed efficient cell-penetrating activity as expected.In addition,they displayed nuclear-localization activity.To evaluate the application of PFV-MNLS and PFV-LNLS as drug carriers,Cbl-PFV-MNLS 和 Cbl-PFV-LNLS were constructed by conjugation of two chlorambucil(Cbl)to the N-terminus of PFV-MNLS and PFV-LNLS via an amide bond.Our result demonstrated that Cbl-PFV-MNLS and Cbl-PFV-LNLS showed expected antitumor activity.Cbl-PFV-MNLS and Cbl-PFV-LNLS could kill tumor cells by direct membrane disruption and the conjugated Cbl.Although the in vitro antitumor activity of Cbl-PFV-MNLS and Cbl-PFV-LNLS was significantly higher than Cbl,their tumor-suppressive activities were obviously lower than Cbl.With increasing doses,the tumor-suppressive activity of Cbl-PFV-MNLS was enhanced,while its activity still lower than Cbl.However,the toxicity of Cbl-PFV-MNLS was obviously lower than Cbl.Although the in vivo application of Cbl-PFV-MNLS and Cbl-PFV-LNLS were not ideal,efficient cell-penetrating activity in vitro implies that they have potential for further optimization.In short,we developed efficient tumor-targeted peptides based on CPPs and AMPs,which target cell membranes.This type of tumor-targeted peptides can be used to construct peptide-drug conjugates with cell-penetrating activity and membrane-lytic activity,which can inhibit tumor growth by multiple mechanism of action.We think this type of conjugates can exert their tumor-suppressive activity with low impact by tumor heterogeneity.Although the designed tumor-targeted peptides in this thesis need further optimization,our work provides a strong theoretical support for developing efficient tumor-targeted peptides.