| The chemotherapy is one of the most common strategies against tumor.The low antitumor efficacy and serious toxicity,however,have limited the clinical application of the chemotherapy.At present,developing nano-drug delivery systems(nano-DDSs)for anticancer drugs have long been a focus for cancer therapy.Nevertheless,some drawbacks of nano-DDSs have significantly limited the clinical application,for example poor biocompatibility,poor stability,complex carrier construction,and carrier-mediated serious toxicity.The well-designed carrier construction and the high-efficiency nano-DDSs development still remain challenges.Short peptides(less than five amino acids)have attracted great attention as a new star in nanomaterials owing to excellent biocompatibility,low immunogenicity and easy synthesis.Various types of short peptide-based nanostructures ranged from the nanomicelles to cylindrical nanofilaments,twisted nanoribbons.Among them,the phenylalanine dipeptide(NH2-Phe-Phe-COOH,FF)is one of the well-known short peptides,which has a great capability to form an orderly nanostructure via π-π stacking.The orderly nanostructure has a distinct hydrophobic core with a promising prospect as a carrier for drug delivery.The filamentous nanostructures under physiological conditions make them unsuitable as intravenous formulations due to the entanglement of long fibers and the risk of thrombus.Therefore,in response to the disadvantages of the conventional FF formulations,we customized a dipeptide-based rhein derivative.A structure-based virtual screening of a library of small molecules was conducted to identify the high-affinity molecule(camptothecin,CPT).The influence of CPT-encapsulated nanoassemblies on the intermolecular interactions between CPT and dipeptide-based derivative,cellular uptake,cytotoxicity and antitumor efficiency are systematically investigated.Given the above studies,we widen the short peptide-based research scope.The“space-matching”co-nanoassembly was constructed by assembling a dipeptide-based photosensitive(pheophorbide a-diphenylalanine peptide,PPA-DA)with a hypoxia-activated camptothecin(CPT)prodrug((4-nitrophenyl)formate camptothecin,N-CPT).The influence of co-nanoassemblies on the formation mechanism,cytotoxicity,biodistribution,pharmacokinetics and antitumor efficiency were also systematically evaluated.Firstly,we successfully synthesized Rhein-FF via acylchloride esterifiication,and the final product was characterized by 1H-NMR.The dissipative particle dynamics(DPD)simulation was applied to study the macroscopic morphology and structure.The high throughput virtual screening of small molecules from the Traditional Chinese Medicine(TCM)database based on molecular docking was used to find the optimal cargos that matched the synthesized dipeptide derivative.According to the screening results,there was significant binding energy between camptothecin(CPT)and this derivative,accompanied by a high affinity.The low derivative-affinity norcantharidin(NCTD)was selected as a negative control.Differences in intermolecular interactions between these two drugs and the derivative were investigated by molecular dynamic(MD)simulations.CPT and the derivative could co-exist together in the CPT-encapsulated assembly box,resulting from strong interactions between CPT and the derivative.However,dispersed NCTD pould exist on the surface of the assembly.The CPT-encapsulated nanoassemblies showed spherically shaped nanostructures with a uniform size around 150 nm by dynamic light scattering(DLS).On the other hand,the particle size of NCTD-loaded nanoassemblies displayed two distinct peaks.Transmission electron microscopy(TEM)demonstrated that NCTD-loaded nanoassemblies were inhomogeneous,probably due to the incompatibility between NCTD and the derivative.The cellular uptake of dipeptide-based nanoassemblies was evaluated in mouse breast cancer cells(4T1).Free coumarin-6 or coumarin-6-labeled dipeptide-based nanoassemblies were incubated for 0.5 or 1.5 h.The result showed that the intracellular fluorescence intensity of nanoassembly-treated cells was significantly higher compared to that of free coumarin-6-treated cells.We investigated the cytotoxicity of free CPT and CPT-encapsulated nanoassemblies against 4T1 cells.CPT-nanoassemblies could effectively inhibit 4T1 cells growth due to increased cellular uptake of CPT.The in vivo anticancer efficacy was investigated in 4T1 xenograft tumor-bearing mice.The PBS group showed a rapid increase in the tumor volume.Within the treatment period,CPT-encapsulated nanoassemblies and free CPT could significantly inhibit the tumor growth compared with the PBS group.However,CPT nanoassemblies showed greater antitumor activity than free CPT.There was no significant change in the body weight in all groups.On the basis of study above,a novel FF-based photosensitive derivative(PPA-DA)was designed and synthesized by conjugating FF to pheophorbide.Additionally,a high-affinity hypoxia-activated camptothecin prodrug(N-CPT)was synthesized by coupling(4-nitrophenyl)formate to the C20’-oxygen of camptothecin(CPT).Both PPA-DA and N-CPT spontaneously co-assembled into one nanoassembly with a mean diameter of-60 nm.The nanoassembly was subjected to PEGylation(DSPE-PEG2K)to improve the stability and increase the blood circulation time of the nanomedicines.A spherical shape of the PEGylated co-nanoassemblies exhibited diameters of～70 nm.High colloidal stability of PEGylated co-nanoassemblies was suitable for further study.The interactions between PPA-DA and N-CPT were analyzed using molecular docking to evaluate the co-assembly mechanism.The PPA-DA&N-CPT complex with a low energy represented a favorable stable system compared to the PPA-DA&CPT complex due to the addition of benzene in N-CPT.Based on the aggregation-caused quenching(ACQ)of intrinsic fluorescence by photosensitizers(PSs),we further attempted to understand the mechanisms underlying the interactions of N-CPT or CPT with PPA-DA using an experimental tool.The insertion of the N-CPT or CPT molecules into PPA-DA mono-assembly disturbed the aggregation of PSs and partially restored the ACQ effect of PSs.The N-CPT&PPA-DA complex exhibited lower fluorescence signals than the CPT&PPA-DA complex,suggesting that N-CPT matched better with PPA-DA.We also calculated the bond angles between(4-nitrophenyl)formate and CPT using molecular dynamic(MD)simulations.The bond angle of-O-C20-in N-CPT rotated continually at angles ranging from 110～130°during the MD process.Compared to the rigid and planar structure of CPT,the conformation flexibility of N-CPT favored the dynamic co-assembly with PPA-DA.The DPD simulations were conducted to reveal the co-assembly morphology.The co-assembly did not form the classical core-shell type of structure,but consisted of many small microphase regions.The hypoxia-activated cytotoxicity of both CPT and N-CPT was evaluated under normoxic/hypoxic conditions using the standard cell viability(MTT)assay.CPT exerted stronger antitumor effect than N-CPT in the normoxic environment.The cytotoxicity of both CPT and N-CPT under the the hypoxia inducer(CoCl2)-induced hypoxic condition was obviously decreased under the low oxygen condition compared to the normoxic condition.The O2 concentration was further decreased after irradiation.Upon irradiation with 660 nm light,the PPA-DA mono-nanoassembly efficiently killed cancer cells.The balb/c mice bearing 4T1 tumor xenografts were established to evaluate the biodistribution of the Dir-labeled co-nanoassemblies.The level of the Dir-labeled co-nanoassemblies that accumulated in the tumor was higher than free Dir.The effect of the co-nanoassemblies on the pharmacokinetics was studied by intravenously injecting the co-nanoassemblies and free N-CPT.The N-CPT from co-nanoassemblies showed a notably prolonged circulation time compared to free N-CPT.A much higher concentration of CPT derived from co-nanoassemblies was determined in tumors than the group treated with N-CPT solution using the HPLC method,mainly due to the favorable pharmacokinetic behavior of co-nanoassemblies.Upon exposure to a 660 nm laser,a high level of PPA-DA-activated hypoxia was produced.Subsequently,a greater number of hypoxia-activated N-CPT molecules were bio-converted to CPT molecules.The in vivo antitumor activity of PEGylated co-nanoassemblies was studied in nude mice bearing 4T1 xenograft tumors.Compared to the PBS group,the free CPT and free N-CPT groups displayed a moderate tumor inhibitory capability.Additionally.PPA-DA mono-nanoassemblies and co-nanoassemblies exhibited better tumor inhibitory effects than the other groups.The growth of tumors in the co-nanoassemblies group was reduced compared with the PPA-DA mono-nanoassemblies group.A notable change in body weight was not observed among all groups.The H&E staining of major organ sections did not reveal noticeable histological deficiencies.These results indicated that PEGylated co-nanoassemblies showed potent antitumor efficacy and safety,promising in the clinical application. |
