Design And Synthesis Of Polymersomes For Biomedical Applications

Polymersomes are hollow nanomaterials with a typical structure consisting of a lumen,a membrane,and coronas,which are prepared by macromolecular self-assembly.They have shown great potentials in diverse biomedical applications,but still face a variety of challenges in design principles,preparation methodology,biocompatibility,biodegradability,structural stability,loading and delivery efficiency,and so forth.Especially,the following three scientific problems are yet to be solved when polymersomes are used as drug delivery vehicles:（1）How to prepare polymersome vectors that can achieve both stable delivery and efficient release?（2）How to develop polymersome vectors for safe and highly efficient loading and delivery of biomacromolecules,and to propose the design principles accordingly?（3）How to design polymersome vectors for programmed release of small molecule drugs,and to predict and achieve precise control on the drug release?To solve the above problems,in this thesis,on the one hand,we designed and prepared a polymersome with balanced electrosteric stability and biodegradability（Chapter 2）;on the basis of such platform,safe and efficient loading of biomacromolecules such as protein and nucleic acids,as well as efficient delivery of plasmid were achieved.The principles for designing polymersome vectors with ultrahigh biomacromolecular loading efficiencies were proposed accordingly（Chapter3）,and a further justification by computer simulation was carried out（Chapter 4）,offering new ideas in the design and development of biomacromolecular nanovectors.On the other hand,by the design and preparation of polymersomes with inhomogeneous membranes,in combination of the analysis on the phase structures on the membrane of polymersomes,the prediction of programmed release kinetics of small molecule drugs was achieved,providing a reliable approach for the design of delivery vectors for small molecules（Chapter 5）.In detail,this thesis focuses on the following four aspects:1.To meet the challenge of balancing electrosteric stability in solution during drug delivery and biodegradability during drug release,we designed and synthesized a photo-cross-linkable yet biodegradable polymersome with a membrane rich of ester bonds.First,we designed and synthesized a novel amphiphilic diblock copolymer,poly（ethylene oxide）₄₅-b-poly（（α-（cinnamoyloxymethyl）-1,2,3-triazol）caprolactone）₉₀(PEO₄₅-b-PCTCL₉₀).Such copolymer can self-assemble into polymersomes.Due to the disruption of crystallinity of the polycaprolactone（PCL）chain on the membrane by cinnamate side groups,the ester bonds in the polymersome membrane degrade faster.Besides,the cinnamate groups undergo cross-linking upon UV irradiation,which not only stabilizes the polymersome,but also tunes the permeability and biodegradability of the polymersome membrane.According to the biodegradation test results,cross-linked polymersomes can be degraded under the catalysis of either lipase or acid.When the degradation is catalyzed by lipase,ester bonds in both backbones and side chains degrade simultaneously.When the degradation is catalyzed by acid,the degradation process starts mainly on the side chains,and then gradually shifts to the backbones.With the aid of transmission electron microscopy（TEM）analysis,membrane perforation of cross-linked polymersomes during degradation can be observed.In addition,homopolymer PCTCL₁₃₃ and statistical copolymer P(CL₁₅₆-stat-CTCL₂₈)were synthesized as control to further confirm the amorphous character of PCTCL and its degradability after cross-linking.With a further cytotoxicity test and a drug release test,the polymersomes exhibit good potential in biomedical applications,especially for drug delivery uses.2.To meet the challenge of low biomacromolecular loading efficiencies for safe delivery by non-cationic polymersomes,we proposed two principles for highly efficient loading of proteins and nucleic acids:acid-induced adsorption（AIA）and affinity-enhanced attraction（AEA）.PEO₄₅-b-PCTCL₉₀ was also designed according to these principles.The principle of AIA depicts:The triazole groups within the side chains of PCTCL are able to facilitate transportation of protons by their lone pairs,which subsequently catalyzes nearby ester bonds to hydrolyze,thus exposing carboxylic and hydroxyl groups for adsorption of proteins by electrostatic interactions or by formation of hydrogen bonds.The principle of AEA depicts:The triazole groups attract electron-rich and negatively-charged nucleic acids by affinity interactions including anion–πinteractions andπ–πstacking,which can be further boosted by theπ-electron-rich cinnamate groups.With the help of these intermolecular interactions,PEO₄₅-b-PCTCL₉₀ can efficiently load hemoglobin,plasmid,and RNA during self-assembly via powder rehydration method,achieving loading efficiencies of 79%,75%and 85%,respectively.These values exceed the maximum loading efficiency of densely-packed hollow spheres by passive loading（driven by passive diffusion）,which is 74%,confirming the existence of active loading mechanism.By further evaluating the significantly lower loading efficiencies by non-cationic control polymers,PMPC₆₀-b-PCL₂₅ and PEO₄₃-b-P（αCl CL）₁₁₁,via the same loading procedures,we further confirmed the importance of the side chains of PCTCL in the loading process.Besides,we did a detailed analysis on the mode of polymersomes loaded with biomacromolecular cargoes and evaluated the influence of loading methodologies.In addition,we performed in vitro transfection tests by green fluorescent protein（GFP）-encoded plasmid-loaded polymersomes.The results indicate a transfection efficiency higher than 95%within 72 hours in fetal bovine serum（FBS）-containing culture medium and a fluorescence intensity two times higher than groups treated with free plasmid regardless of the plasmid size.This further confirmed that the polymersome vectors achieved successful intracellular delivery of biomacromolecules without affecting their expression,showing the potency of this polymersome in the biomedical applications such as gene therapy.3.To further justify and analyze the above-mentioned principle of AEA,we carried out molecular dynamics（MD）simulations.The software of Materials Studio was used.First,model polymers corresponding to the loading tests were created and optimized,which are PEO₁₀,PMPC₁₀,PCTCL₁₀,and PCL₁₀.Models for protein and nucleic acids were also created and optimized.Then,the models for each interactive system of polymer and model biomacromolecule were constrained into a virtual repeating unit cell（“amorphous cell”）.After applying molecular dynamics simulations on these cells,the interactions between each polymer and model biomacromolecule were simulated.Finally,by further analyzing the evolutions of energies and molecular configurations,the AEA principle can be justified.Four different aspects of the simulations were analyzed to unravel the interactions between polymers and biomacromolecules:（1）The dynamics simulation procedure.The initial state and output state,as well as the energy evolution curves were acquired for each interactive system.With these data,the corresponding interactions between each polymer and macromolecule can be analyzed and concluded:affinity interactions correspond to elevated total energy,and repulsive interactions correspond to declined total energy;（2）The effect of temperature on AEA.By analyzing the dynamics simulations at raised temperature（from 298 K to 335 K）,we confirm that AEA effect can be promoted,but the nature of interactions was not altered;（3）The origin of AEA from PCTCL.We designed two polymer models,poly（α-triazol caprolactone）(PTCL₁₀)with only triazole groups as side chains,and poly（α-cinnamoyloxycaprolactone）(PCCL₁₀)with only cinnamate groups.The dynamics data of these polymers were acquired and compared to those of PCTCL and PCL.The results indicated that both triazole groups and cinnamate groups contribute to the AEA effect;（4）The intermolecularπ–πstacking between polymers and biomacromolecules.With the quench method of molecular dynamics,π–πstacking between model biomacromolecules and both triazole and cinnamate groups was confirmed.The results of these analyses show good consistency with the actual loading experiments,indicating that strong AEA effects significantly enhanced loading of nucleic acids during the actual loading experiment by using PEO₄₅-b-PCTCL₉₀ polymersomes.4.To meet the challenge for the prediction and precise control over the design of polymersomes for programmed release of small molecule drugs,on the basis of the PEO-b-PCTCL polymersome,we introduced crystallizable PCL to induce phase separation on the membrane and predicted the phase morphology by Meso Dyn simulations to achieve the orthogonal tuning of membrane permeability with the assistance of photo-cross-linking.We synthesized PEO₄₃-b-P(CL₄₅-stat-CTCL₂₅),PEO₄₃-b-P(CL₁₀₈-stat-CTCL₁₆)and PEO₄₃-b-PCTCL₄-b-PCL₇₉.Due to the fact that PCL and PCTCL are not fully compatible with each other,these copolymers can self-assemble into polymersomes with inhomogeneous membranes via both solvent-switch method and powder rehydration method.To further reveal the phase separation behavior on the polymersome membrane,the module of Meso Dyn from the software of Materials Studio was used.By incorporating different amount of PCL and PCTCL,the phase structures and thus a phase diagram can be readily afforded.By further adding a shear,the phase behavior under cross-linking can be simulated and analyzed.Finally,by the loading and release of doxorubicin with the polymersomes,the membrane permeability by controlling phase separation and by cross-linking the membrane was evaluated and confirmed,indicating the feasibility of this permeability-tuning strategy in the design of polymersomes for programmed drug release.In summary,to deal with the critical issues in the science of polymersomes,this thesis first proposes the design strategy and preparation method for the construction of cross-linkable yet biodergradable polymersomes,solving the contradiction between safe delivery of drugs and biodegradability of the polymersomes;then two novel principles of AIA and AEA for highly efficient loading of biomacromolecules are proposed,overcoming the bottleneck of low biomacromolecular loading efficiency and lack of rationale for high loading efficiency in the field of polymersomes,which may be further extended to other drug vectors,functional biointerfaces,and so forth.Furthermore,the proposed principles have been confirmed by a combination of loading experiments,computer simulations,and theoretical analysis.With in vitro transfection experiments,the capability of the polymersomes for intracellular macromolecular delivery was confirmed,coping with the difficulty for polymersomes to perform highly efficient intracellular delivery of macromolecules.Besides,in this thesis we propose an approach for orthogonal control of drug release rate,by design the phase separation structure within the polymersome membrane in combination with membrane cross-linking.This approach is analyzed theoretically by using computer simulations,providing a novel exemple for the design of programmed drug release systems.