Metabolomic Study Of Coronary Artery Disease/Preparation Of A Novel Biomimetic Self-Delivering Nano-drug And Their Application In Cancer Therapy

The whole thesis was divided to two parts.Part Ⅰ was based on the work that Ⅰcompleted in China about metabolomics on coronary artery disease(CAD).Problem:CAD is the leading cause of morbidity and mortality worldwide.Therefore,it is particularly important for predicting and controlling CAD.Traditional risk factors cannot promote prediction capacity for the patients with coronary artery disease(CAD),who usually do not show apparent symptoms until they develop acute myocardial infarction(AMI).As such,novel predictive diagnostic strategies are essential to accurately define patients at risk of acute coronary syndrome.Contents:in the Part Ⅰof this thesis,the serum of CAD-related patients was collected and a metabolomics method based on UPLC-QTOF-MS was established.Then a multivariate statistical model was established to explore novel biomarkers and used to distinguish different stages of CAD development.Part Ⅱ was based on the nano-platform made from bottom-up approach that contains 100%active pharmaceutical and photosensitizer ingredients of Pheophorbide A and Irinotecan conjugates(named PI)which was made in the lab of University of California Davis.Chemotherapy has been one of the major clinic treatment approaches for cancers in the past few decades.However,the unbearable toxicity of chemotherapeutic agents greatly limited their clinical applications.Drug self-delivery systems(DSDSs)have advantages of unprecedented drug loading capacity,minimized carrier-related toxicity and ease of preparation,which represent one of the promising approaches for addressing these issues.Problem:the colloidal stability and blood circulation time of PI still need to be improved.Contents:in the Part Ⅱ of this thesis,we report on the development of a novel biomimicry drug self-delivery system by the integration of a top-down cell membrane complexing technique into our self-delivery multifunctional nano-platform.In this thesis,the main contents and innovative work are as following:(1)Collecting samples of CAD patients and establishing a stable and reliable analytical method based on ultra-high performance liquid chromatography with quadrupole time-of-flight mass spectrometry.A new generation of high-resolution mass spectrometry technologies such as quadrupole time of flight(Q-TOF)are more advantageous for the analysis of unknown compounds,they can obtain more accurate mass spectrometry(MS/MS)through tandem mass spectrometry.Therefore,in the metabolomics application,whether targeted or untargeted,has unparalleled advantages in describing biological processes or interactions between organisms and the external environment.This study helps us to understand the mechanism of the disease more in-depth.After completed several parameter optimizations,a UPLC-QTOF-MS method has been established for the continuous analysis of a total of 143 samples and QC samples.Based on the QC samples,a PCA score plot and the RT and peak areas representing metabolites were used.And finally confirmed that the reproducibility of the analytical method and the stability of the instrument can meet the requirements of the metabolomics study which can help us to perform the data mining analysis in next step.In this process,for the first time,specific information on the diet and drug use of the Chinese population was used to pre-screen specific serum metabolite information to avoid interference with follow-up CAD disease-related biomarker analysis.(2)Based on the collected mass spectral data,a multivariate model was established using metabolomics to discover novel biomarkers.In this study,non-targeted metabolonic profiling using ultra-perfornance liquid chromatography coupled to time of flight mass spectrometry(UPLC-QTOF/MS)was performed in combination with multivariate statistical model to analyze the serum samples of patients with stable angina(n＝38),acute myocardial infarction(AMI)(n=34)and healthy age-and gender-matched controls(n=71).Results showed a clear distinction in metabolomic profiles between stable angina and AMI when using OPLS-DA with both positive and negative models.Internal cross-validation methods were used to confirm model validity with an area under the curve(AUROC)=0.983.We identified various classes of altered metabolites including phospholipids,fatty acids,sphingolipids,glycerolipids and steroids.We then demonstrated the differential roles of these metabolites using multivariate statistical model.Phospholipids previously associated with CAD were shown to have lower predictive capacity to discriminate AMI patients from stable angina patients.Interestingly,ceramldes,bile acid and steroids hormone such as Cer(tls:0/16:0),Cer(d18:0/12:0),dehydroepiandrosterone sulfate(VIP scores of 1.99,1.97,1.64,respectively),were found to be associated with the progression of CAD.These results suggest that metabolomic approaches may facilitate the development of more stringent and predictive patient criteria in the diagnosis and treatment of CAD.(3)A biomimetic nano drug self-delivery system with polylactide-co-glycolic acid(PLGA)as the core was established.Based on the PI molecule,this research successfully applied the engineering teehnology and nanotechnology to prepare a new type of nanopartiele self-assembled by PLGA and PI,and coated it by co-extruding PLGA-PI with red blood cell membrane.The resulting nanoparticles were then eharacterized to determine the suceess of the coating.The in vitro simulated stability experiments showed that PLGA-PI and RBC-PLGA-PI have very good stability within 30 days.We use PLGA and red cell membranes to modify PI nanoparticles to improve their stability and do not want to change the properties of PI.In vitro laser trigger experiments,we have successfully conflrmed that the newly prepared nanoparticles still have photodynamic therapeutic(PDT)and a photothermal therapeutic(PTT)and property of drug release by laser trigger.Finally,we confirmed the therapeutic effect of the resulting nanoparticles on two eancer cell lines which approved by the FDA for photothermotherapy.In this study,PLGA and PI molecules were self-assembled by co-precipitation for the first time,and then erythrocyte membrane vesicles were coated on them to prepare novel biomimetic nanomedicines with PLGA as the core.(4)A biomimetic polymer-free nano-drug self-delivery system based on PI molecules was established.In this study,in order to increase the drug loading capacity,the polymer-based system was discarded,and red blood cell membrane was successfully prepared by integrating top-down into our bottom-up multi-functional nano-platform.Compared to conventional cell membrane coated nanoparticles with polymer framework as core and relatively low drug loading,this system consisting of red blood cell membrane vesicles complexed PI(RBC-PI)is polymer-free with up to 50%API loading.RBC-PI exhibited 2 times longer half-time and 10 times higher area under curve in pharmacokinetic study and much lower macrophage uptake compared with the parent PI nanoparticles.This research innovatively solved the problem of strong co-precipitation caused by the attraction of strong electrostatic force.It successfully used erythrocyte membrane vesicles as carriers and PI molecules to self-assemble into a novel polymer－free core drug-biomolecule self-delivery system,and the use of cryo-transmission electron microscopy to characterize RBC-PI,morphologically explains the nano-self-assembly process.Later,it was confirmed through experiments that this nano-drug system has both synthetic drug functions and biological functions.RBC-PI retained the excellent chemophototherapeutic effects of the PI nanoparticles,but possessed superior anti-cancer efficacy with prolonged blood circulation,improved tumor delivery,and enhanced photothermal effects in animal models.This biomimicry DSDS takes advantages of the best features from both systems to make up for each other’s shortcomings and posed all the critical features for an ideal drug delivery system.