Preparation And In Vivo Integrity Investigation Of Polymer Drug Delivery Systems

Polymeric drug delivery carrier is one of the most important and useful drug delivery systems,which is classified into nanocarrier,microcarrier and macro-carrier.The delivery efficiency of carrier is impacted by many factors,such as size,shape,surface physicochemical properties,integrity,rate of drug release and the form of the drug released,in which integrity is one importantly influential factor.As for the nanocarrier,maintaining their integrity contributes to longer circulation time,more tumor accumulation and higher anticancer efficacy.Maintaining suitable integrity avoids burst drug release from micro-and macro-carriers,which enables their controlled and long-term drug release for better therapy and improves patients' comfort and compliance.Therefore,accurate investigating the integrity of polymeric drug delivery carrier is critically important for optimized drug delivery system.In this dissertation,we developed a method based on the FRET effect of two fluorophores with large Stokes shift for the first time to demonstrate the assemble and dissociation of micelle.Based on this method,we invented an approach for micellar integrity calculation via mathematic derivation and in vitro simulation.We calculated the integrity of this micelle both in vitro and in vivo.In the latter part,we developed two kinds of microparticles with different stability.These microparticles contained different acid responsive linkers and could release small nanoparticles with different release rate.The main content of this dissertation is described as two parts as below:1.To establish an accurate quantification method for measuring micellar integrity,two fluorophores with large Stokes shift NBD-X and MS735 were chosen as FRET pairs.The two fluorophores were conjugated at the end of the hydrophobic segment of poly(ethylene glycol)-poly(caprolactone)(PEG-PCL)block polymer to form PEG-PCL-NBD-X and PEG-PCL-MS735 as donor and acceptor,respectively.PEG-PCL,the obtained donor and acceptor were self-assembled and formed micelles denoted as MicelleD+A.The excitation and emission spectra confirmed the FRET phenomenon between donor and acceptor.The composition ratio of the donor and acceptor in micelleD+A was optimized to ensure strong fluorescence intensity and no obvious self-quenching of fluorophores was detected.We found that the FRET phenomenon of micelleD+A could be used to probe the assemble and dissociation of micelleD+A.Based on the theory of FRET,we developed a quantification formula for micellar calculation via mathematic derivation and in vitro simulation.Finally,we investigated the integrity of micelleD+A both in vitro and in vivo,and found that most of the micelles were intact in the circulation system of mice 72 h after intravenous injection.We provided an accurate method for illuminating micellar integrity.2.To prepare microcarriers that could release small-size poly(amidoamine)(PAMAM)nanoparticles with enhanced permeability of drug into tissues,we developed two methods to prepare two types of microparticles with different structures,assembly principles and release rates.In the first method,we synthesized poly(ethylene glycol)derivatives modified with different end groups(CI-PEG-CI with N-Carbonyl imidazole as end group,CHO-PEG-CHO with benzaldehyde as end group and CDM-PEG-CDM with 2-propionic acid-3-methyl maleic anhydride as end group)as crosslinker.PAMAM and different crosslinkers in aqueous solution were used to form microdroplets in hexane via inversed phase emulsification,and microgels formed in microdroplets via the reaction of the crosslinker and PAMAM.We found that CI-PEG-CI and PAMAM formed very stable microgels;CHO-PEG-CHO and PAMAM formed microgels that would be degraded and would release nanoparticles rapidly in aqueous solution;CDM-PEG-CDM and PAMAM only formed macro-hydrogels because of the rapid reaction between CDM and NH2 and inability of forming microdroplets.To prepare microparticles with more controllable release of small-size nanoparticles,we used droplet-based microfluidic approach to preparemicroparticles with capability continuously release nanoparticles.First,biodegradable amphiphilic polymers PCL-Dlinkm-PAMAMs were first synthesized through the reaction of CDM modified poly(?-caprolactone)(PCL-CDM)and PAMAM via labile amide bonds.By tuning the feed ratio,a series of amphiphilic polymers with different PCL conjugates(PCL-Dlinkm)n-PAMAM(n=1?5)were synthesized.Then,these polymers were assembled into microparticles by the hydrophobic interaction of PCL segments in the droplet-based microfluidic approach.The size of microparticles was well-controlled by adjusting the ratioof the flow rate of continuous phase and dispersed phase.It was found that regularly spherical microparticleswere formed only when more than two PCL segments were conjugated on the amphiphilic polymer.These spherical microparticles were denoted as imCluster and could sustainably release PAMAM nanoparticles after the cleavage of amide bonds in the aqueous solution.