Experimental Study Of Carrier-free Self-assembled Nanomedicine Based On Pro-apoptotic Peptide Gemcitabine Conjugate For The Treatment Of Pancreatic Cancer

BackgroundPancreatic cancer is a fatal malignant tumor,with a significantly low early diagnosis rate,low resection rate and easy metastasis and recurrence.Chemotherapy is the mainstay of pancreatic cancer treatment,with gemcitabine(Gem)remainning the first-line regimen for pancreatic cancer chemotherapy.Gem-induced DNA damage is an mitochondria involving endogenous regulatory pathway,although increased expression of apoptosis inhibitory proteins upon Gem treatment makes tumor cells apoptosis-resistant,resulting in Gem resistance.The rapid development of nanomedicine and nanodrug delivery technology has also promoted the application of tumor drug therapy.Most of the nanomedicines need to use inactive carriers,which not only have low drug loading capacity,but also have toxic side effects of carrier materials.Therefore,how to design a safe and non-toxic nanomedicine for gemcitabine resistance pancreatic cancer,is an urgent clinical problem,while the developed nanomedicine can be used to improve the efficacy of pancreatic cancer treatment.Carrier-free nanodrugs prepared by self-assembly or co-assembly of pure drug molecules have been widely studied due to their high drug loading capacity and low toxic side effects properties.Photodynamic therapy has been approved for many tumor treatments in many countries around the world due to its advantages of high efficiency,low toxicity,spatial and temporal control,and has also made some progress in clinical research for pancreatic cancer.The design of light-activated reactive oxygen species-responsive photodynamic prodrugs for pancreatic cancer treatment,can improve the spatio-temporal controllability of drugs.In addition,peptide drug conjugate(PDC),which are compounds obtained by coupling antitumor drugs with small molecule peptides through a linker arm,have become one of the new avenues for tumor therapy.Based on the above mentioned background,this work has been conducted aiming for the design and synthesis of a stimuli-responsive PDC prodrugs based on gemcitabine,a first-line therapeutic drug for pancreatic cancer.The pro-drug and photosensitizer were then generated as a simple and green pure drug carrier-free nanoparticles.Combining the spatio-temporally controllable nanomedicine concept with the combined drug delivery format,new ideas of novel prodrugs carrier-free nanoparticles in the treatment of pancreatic cancer were explored.ObjectIn this study,we developed a simple green drug carrier-free self-assembled nanoparticles for the treatment of pancreatic cancer.We explored the self-assembly mechanism and anti-tumor mechanism of nanomedicine,and evaluate its anti-tumor efficacy in vitro and in vivo.MethodsThe pro-apoptotic peptide Smac N7 was first synthesized by solid-phase peptide synthesis,and then Smac N7 was covalently coupled with Gem via an ROS-sensitive linker(vinyldithioether)to form the pro-apoptotic peptide gemcitabine conjugate(Gem-vinyldithioether-Smac N7,GVS).The prodrugs and the photosensitizer chlorin e6(Ce6)were then underwent a one-step precipitation method to form a simple and green pure drug-free carrier self-assembled nanomedicine.The basic properties of nanodrugs including morphology,particle size and surface potential were investigated by detailed characterizations;the mechanism of nanodrug self-assembly formation was probed by molecular dynamics simulations.At the cellular level,the uptake of carrier-free self-assembled nanodrugs by pancreatic cancer cell PANC-1 was investigated and the in vitro anti-tumor mechanism of nanodrugs was verified.Finally,a xenograft tumor mouse model of human pancreatic cancer PANC-1 cells was developed to verify the antitumor effects of nanodrugs in vivo as well as their biosafety.ResultThe pro-apoptotic peptide gemcitabine conjugate was successfully synthesized and characterized by nuclear magnetic resonance and high resolution mass spectrometry.Its photolysis effect was then confirmed by high performance liquid chromatography.Under the intermolecular force of non-covalent interaction,Ce6 and GVS self-assembled to form quasi-round spherical particles Ce6-GVS NPs.The hydrated particle size is about 81.89 nm and the potential is-34.40 mV.Western blot experiments showed that nanoparticles could bind to and reduce the expression of inhibitor of apoptosis protein.The characterization of mitochondrial membrane potential damage and DNA nuclear damage also showed the tumor inhibition effect in cells.In the animal experiments,tumor growth was significantly inhibited in the presence of nanomedicine and light illumination,while the biosafety assays confirmed the good compatibility of the nanomedicine.ConclusionWe successfully synthesized a photoactivated ROS responsive pro-apoptotic peptide gemcitabine conjugate and constructed a pure drug carrier-free self-assembled nanoparticles with good drug loading and safety.The combination of apoptosis,chemotherapy and photodynamic therapy can effectively inhibit tumor growth.Therefore,carrier-free self-assembly nanoparticles Ce6-GVS NPs is a promising strategy for the treatment of pancreatic cancer.