Liposome composed of a lipid bilayer comprising phospholipids (PL) and sterols such as cholesterol (Chol) hasbeen extensively used for packaging and delivery of therapeutic agents due to its intrinsic biocompatibility andbiodegradability. While most approved liposomal nanotherapeutics can improve pharmacokinetics (PK) andreduce systemic toxicities improvements in therapeutic efficacy and overall survival are disappointingunderscoring the urgent need for enhanced therapeutic delivery. Chol plays a critical role in fortifying membranepacking and reducing bilayer fluidity and permeability by promoting the liquid condensed state in lipidmembranes enhancing bilayer rigidity and strength. Lipid bilayers with high levels of Chol are generally morestable than those without or with less Chol. However under the physiological environment Chol is rapidlyextracted from the bilayer by biomembranes and serum proteins which jeopardizes bilayer stability and resultsin premature content leakage fast blood clearance and unwanted adverse effects leading to suboptimal clinicefficacy. In addition although enhanced permeability and retention effect allows nanotherapeutic accumulationto the periphery of diseased tissues intracellular internalization and tissue penetration remain inefficient due tothe tenacious resistance imposed by high interstitial fluid pressure and dense extracellular matrix compromisingthe therapeutic outcome. These phenomena present formidable barriers for lipid bilayer-based therapeuticdelivery. To tackle these key challenges the overall vision of my research program is to establish a stabilizedlipid bilayer with improved physicochemical properties that can further improve drug delivery and selectivelyfortify intracellular uptake and infiltration at target sites. We have established a Chol-derived PL via covalentlyattaching Chol to a PL with varied stimuli-responsive linkages. Via systemic structure activity relationship studieswe demonstrated that Chol-derived PL blocked Chol transfer prevented payload leakage prolonged circulationtime and augmented efficacy in treating lung inflammation Alzheimers disease lymphoma pancreatic andtriple negative breast cancer models which were linker chemistry dependent. For the next five years the goalsof this proposal are to 1) unravel the underlying mechanisms and principles on how the structural alterations ofa sterol-modified PL bilayer that forms liposome but cannot shuttle between biomembranes will affect drug andgene delivery via substituting Chol with other membrane sterols; and 2) establish a universal ultra pH-sensitivecharge-reversal delivery platform to boost the cellular uptake and tissue penetration efficiency via incorporatingan intelligent build-in cationization mechanism that selectively triggers effective adsorption-mediated endocytosisand transcytosis at diseased tissues. Completing these studies will provide fundamental and functionalcorrelations of bilayer properties with therapeutic delivery enable us to establish a set of design rules governingthe optimal interactions between lipid bilayer and encased drugs and provide a paradigm-shifting toolbox toadvance the drug delivery technologies facilitating clinical translation of treating human diseases.