Molecular Beacons in Lipoplex Nanoparticles for Extracellular Vesicles Based Cancer Diagnosis

Abstract Early detection is one of the keys to winning the war on cancerBecause current imaging and tissue biopsy based diagnosis are invasiveexpensiveand in some cases dangerousthey are impractical as an early cancer screenas well as therapeutic and recurrence monitoringLiquid biopsy is an exciting new form of biopsy that can complement cancer detection assays from the analysis of bodily fluids such as blood or urine because it is non invasiveeasiersaferand more cost effectiveWith regards to body fluids based cancer detection and monitoringblood analysis is the most reliable sourcewith three types of analysis emerging as possible approaches for detectioncirculating tumor cellsCTCscell free DNAcfDNAand extracellular vesiclesEVsCTCs have been investigated by many researchers and companies in the pastyearsMany technologies are available for their capture and identificationHoweverCTCs are rareparticularly in early stage cancer patientsandltCTCs inmL bloodmaking quantitative analysis difficult and less reliableMany cancer patients also do not show CTCs in blood because of endothelial to mesenchymal transition in circulationTodayCTC based cancer diagnosis has not been widely used in clinicCell free DNAcfDNAhas gained its success in noninvasive parental testingNIPTBut the content of DNA targets from dead target cells such as early stage cancer is lowwhich requires PCR for amplification and Sequencing for identificationConsequentlythe procedure is costlyandgt $per test in NIPTcfDNA for early stage cancer detection is not provenand many cancers are not initiated by DNA mutationsExtracellular vesiclesEVsare abundant in bloodinmL bloodmaking quantitative analysis easierCodinge gmRNAand non codinge gmicroRNA and lncRNARNAs encapsulated in EVs and membrane proteins on the surface of EVs can serve as good disease biomarkersThey also play important roles in disease metastasis and relapsemaking the capture and characterization of individual EVsnot average information from all EVs in the sample as in the existing methodshighly valuable in disease diagnosis and medicine developmentMany cancer types including pancreatic cancer have very high mortality rates because it is difficult to detect their presence in the early stageCurrent biomarkers for pancreatic cancer such as serum CAprotein face high false positive and negative ratesThe proposed studies seek to develop a robust and user friendly novel molecular beacon in lipoplex nanoparticles for extracellular vesicles based cancer diagnosis and validate its clinic potential by detecting GlypicanmRNA and KRAS mutation in extracellular vesicles from pancreatic cancer patient serum samples for imminent use in the fields of early cancer diagnosis and relapse monitoringThe specific Phase I aims and milestones of this project areSpecific AimDesign and testing of a toehold initiatedTiand catalyzed hairpin DNA circuitCHDCCLN MB which are highly stablespecific and sensitive for GPCand KRAS mutation genesMilestonesiDetection of GPCand KRAS mutation mRNAs in EVs secreted from andltxpancreatic cancer cells inmL blood with andgtrepeatabilityiilyophilized CLN MB nanoparticles for andgtmonths shelf life and andltperformance variationiiiandltvariation inassays using a conventional microscope as an analyzerSpecific AimDual site validation of EV GPCKRAS mRNA biomarker for early PDAC diagnosisMilestonesiUser friendly assay with andlthours assay timeiiandltfalse positive negative prediction from andgtpancreatic cancer patient samples at OSU and MSKCC Project Narrative Current biomarkers for pancreatic cancer such as serum CAprotein face high false positive and negative ratesCirculating extracellular vesiclesEVshave been identified as potential biomarkers in human diseaseWe present a novel technology that enables the direct analysis of biomarkers within individual EVsWe accomplish this without the need for mixing together the contents of all EVswhich results in a faster assay with more sensitive resultsOur novel technique utilizes a tethered lipoplex nanoparticle biochip containing molecular beacons to directly capture individual EVs and detect GPCand mutated KRAS mRNAs within them as biomarkersThis intact EV analysis technique is fasterless expensiveand less prone to error than the current technologiesand could be a valuable tool in the emerging area of liquid biopsy