Innovation And Functional Exploration Of Precise Encoded Polymeric Amphiphiles With Direct Sequence Readout

Polymeric nanoparticles(NPs)are widely used as drug carriers in diagnostic and therapeutic areas.Currently,an increasing number of NPs with different chemical,physical and biological properties are being fabricated with the aim of controlling their biological fate and addressing multiple in vivo delivery barriers.However,in vivo characterization of polymeric nanoparticles using traditional fluorescent probe labeling techniques provides only qualitative results.The spatiotemporal quantification of nanoparticles at the organ and tissue level after systemic(i.e.,intravenous)administration is critical to understand the relevance of nanoparticle properties to trafficking pathways and biodistribution.Therefore,quantification of the absolute biodistribution of polymeric NPs remains a formidable challenge to further advance nanomedicine design and translational assessment.Based on the above scientific questions,this dissertation focuses on modular synthesis,sequencing of sequencedefined digital polymers,and quantification of digital NPs in complex biological environments.The dissertation consists of the following three main parts:1.The field of controlled synthesis of monodisperse sequence-defined polymers has developed rapidly in the last decade,with the emergence of a variety of novel precision polymers and a clearer articulation of polymer structure-property relationships.Through a modular iterative synthesis strategy,we have developed"forward" and "reverse" synthesis methods to obtain a series of degradable digital polymers.A series of amphiphilic digital polymers were obtained by forward synthesis based on aryl azides that undergo Curtius rearrangement into aryl isocyanates and react effectively with terminal hydroxyl groups.These amphiphilic digital polymers consist of UV-reactive modules,precision poly-Nphenylcarbamate modules with controlled sequences and cascade depolymerization,and hydrophilic PEG dendrimer modules.The coding of the precision polymers is achieved by changing the N-(phenyl)carbamate substituent.We also verified the reaction orthogonality of the Curtius rearrangement and the Hofmann rearrangement.We then synthesized both monomers and dimers containing both hydroxyl and acyl azides or phenyl amides.The inverse synthesis by orthogonal chemistry led to poly-N-phenylcarbamate blocks with controlled precise sequences,and finally photo-reactive groups were installed at the end of the polymer chains to obtain completely degradable digital polymers.2.The regulation of monomeric sequences in biological macromolecules(e.g.,proteins,DNA)is a central concept in biology that has been intensively studied by the biochemical and biophysical communities for decades.Biopolymers have developed sophisticated sequencing methods that have driven the boom in structural biology.However,sequencing of synthetic polymers remains a great challenge,which is largely constrained by the inherent imprecision and polydispersity of traditional polymers.In recent years,sequencing of monodisperse synthetic polymers has been greatly developed with the introduction of the concept of precision polymers and corresponding synthesis strategies.Based on the successful synthesis of digital polymers,we further explored the determination of their sequences.The amphiphilic digital polymers were dispersed in aqueous solution and triggered by UV irradiation.Samples were taken at different incubation times for matrix-assisted laser desorption ionization time-of-flight mass spectrometry(MALDI-TOF MS)analysis and degradation intermediates were monitored to determine the sequence of the amphiphilic digital polymers.Direct sequence readout was accomplished by MALDI tandem mass spectrometry,thanks to the high-affinity binding of alkali metal ions to PEG dendrimers and the selective cleavage of benzyl-carbamate linkages.3.We furthcr found that precise amphiphilic polymers can assemble into digital micelles tightly controlled by oligourethane sequences.The mixture of four types of digital micelles could be identified,sequence-decoded,and quantified by MALDI and MALDI imaging at cellular,organ,and tissue slice levels upon in vivo administration,rendering direct comparison of corresponding biological behaviors on the same animal entity in a label-free manner.By sorting immune cells and stromal cells in the spleen,heated electrospray ionization(HESI)-FTMS(Thermo Scientific Orbitrap Eclipse Tribrid module)was used to study the content of digital macromolecules in different types of cells in the spleen.To explore the design generality for digital micelles self-assembled from encoded amphiphiles,we further propose the concept of digital lipids by incorporating encoded oligourethane segment into conventional double-chain lipids for the construction of digital lipid nanoparticles and their application in the delivery and quantification of siRNA in living cells.Microfluidic co-assembly of amphiphilic digital polymers and traditional amphiphilic block copolymers was used to obtain barcoded micelles,which were used to investigate the size effect of the biological behavior of PEGylated nanoparticles.