Preparation Of Self-Immolative Polymers And Construction And Application Of Functionalized Nano-Systems

Under the stimulation of specific external signals,responsive polymers can undergo structural changes at the molecular level,resulting in the size and morphology variation of their nano-assemblies.Therefore,tumor microenvironment-responsive polymeric nanocarriers can realize personalized and accurate treatment while making diagnosis for different diseases and patients,which has attracted more and more attention in recent years.Among them,self-immolative polymer(SIP)is a novel material developed in the past decade.Once triggered,irreversible depolymerization occurs from head to tail at the molecular level.To date,through the responsive depolymerization of the backbone,the SIP is transformed into a small molecular quinone methide(QM)or azaquinone methide(AQM),of which application in signal amplification,drug release,in situ fluorescence labeling and microreactor has been reported.However,SIPs containing functional groups on each repeating unit were rarely reported.In this paper,several kinds of imaging functional side groups are grafted to the backbone,which self-assemble into micellar nanoparticles in aqueous solution.Making full use of the structure of polymer chains and the self-immolative feature,we achieve activatable 19F MRI and long-term retention 1H MRI system,high drug loading nanoparticles and ROS amplification system coordinated with QMs and Lapachone based on conversion from nanoparticles to molecular agents.Additionally,in this research paper,two kinds of fluorescent dyes in response to pH and reductive milieu were covalently labeled on the amphiphilic polymer block copolymers to construct dual-ratioetric fluorescent probe nanoparticles,which can provide useful information for understanding the intracellular transport pathway of nanoparticles.The following sections are included in this dissertation.1.A series of SIPs containing allyl,imine structure(nbs),phenylborate(pb),boc-amino(Boc)as triggering moieties,named poly(2-allyl-4-hydroxymethylphenylcarbamate)(Pnbs,PPb,PBoc).Through pegylation and "thiol-ene" click reaction post-modification,19F and 1H MRI functional moieties were introduced into the hydrophobic block side group(DOTA-Gd,hexafluoroisopropanol HFI,bis(trifluoromethyl)phenyl HFB).A series of amphiphilic self-immolative block copolymers triggered by mild acidic pH,ROS and TFA were obtained,BPx-Y(x:triggering moiety,Y:functional group).The amphiphilic block copolymer with pendant-decorated SIP can self-assemble into nanoparticles of about 100 nm in aqueous phase.Fluorine-containing SIPs tend to embedded in the hydrophobic core of nanoparticles during self-assembly,and the 19F NMR/MRI signal is weakened by restricted molecular chain motion.According to the different trigger moieties,in response to the triggering of mild acidic pH and ROS,SIP nanoparticles depolymerize into small molecules,resulting in the dissociation of nanoparticles and the recovery of the movement ability of fluorine-containing groups to a certain extent,which is manifested by the significant enhancement of the 19F NMR/MRI signal intensity.In addition,the nanoparticles co-assembled by BPpb-Gd-HFI containing both DOTA-Gd and HFI functional groups and the BPpb-HFI containing HFI.The 1H MRI signal,which remained basically unchanged before and after ROS triggering was used as an internal reference,and the 19F/1H MRI signal intensity comparison was used to construct an activated dual-mode MRI system.When the content ratio of gadolinium and fluorine is adjusted,the BPpb-Gd-HFI containing gadolinium and fluorine is co-assembled with the BPpb-HFI containing only fluoride.Before and after ROS triggering,the 1H MRI signal enhanced by gadolinium can be used as an unchanged internal parameter,while the 19F signal shows the effect of being activated and greatly enhanced,thus constructing a 19F/1H dual-mode MRI imaging system that can be activated.To further explore the self-immolation process,the model molecule BPBoc-HFB was synthesized and applied to the model reaction of QM in situ capture.In the model reaction,the mechanism of SIP degradation was proved by GPC:the end group needed to expose a group containing lone pair electrons.Once the terminal amino group was protonized,the depolymerization was stopped,while when the amino group was deprotonized,the depolymerization was immediately carried out.The QM generated by SIP degradation produces a competitive response in the system.Once there are thiol captors,multiple elution peaks of large molecules or small molecules become one peak reflected in the GPC curve.NMR,HPLC,ESI-MS have also been used to demonstrate QM-thiol capture.Pendant DOTA-Gd SIP self-assemble into nanoparticles,by injection into a tumor-burdened in tumor mice,using ICP-AES and magnetic resonance imaging(MRI)to keep track of Gd in tumor and long-term MRI imaging,the accumulation of the experimental results show that the QM can realize caused by the degradation of SIP in situ marker for tumor treatment and imaging,long-term imaging function.2.Using ROS responsive SIP obtained from the above synthesis,lapachone(La),camptothecin(CPT)and doxorubicin(DOX),17-AAG,mitomycin C(MMC),indocyanine green(ICG)were packaged in nanoparticles based on SIPs with three different side groups.The correlation between the structure types of these polymers and the drug loading was compared and screened.Due to the large amount of hydrogen bonding on the back bones and side groups of SIPs,it was found that BPpb-HFI NPs had very high drug loading content(DLC)and encapsulation efficiency(DLE).ROS can trigger polymer depolymerization and nanoparticle dissociation,which will release a large amount of lapachone within 24 hours.In the presence of NQO1 and NADH,a large amount of ROS can be amplified by consuming NADH through futile cycle.The ROS produced can release lapachone and generate QM to trigger more SIP degradation,achieving positive feedback.In cell experiments,19F NMR and CLSM were used to prove the intracellular enantiosculation and responsive depolymerization of nanoparticles,further proving that lapachone released in response to ROS and QM generated by SIP degradation can synergistically destroy the antioxidant system of cells,increase the oxidative pressure,and promote the generation of toxicity and eventual apoptosis of cells.3.To understand the cellular internalization mechanism of nanomaterials,dual-ratiometric fluorescence probes in response to pH and reductive milieu were constructed based on double hydrophilic block copolymers(DHBCs)and fluorescence resonance energy transfer(FRET)effects.Co-labeling of rhodamine B(RhB)moieties with disulfide spacer and pH sensitive naphthalimide-based probe(NPI)moieties into stimuli-responsive DHBCs was achieved by one-pot reaction cascade involving the ring-opening of a thiolactone and subsequent disulfide exchanging reaction.The DHBCs can self-assemble into micellar nanoparticles in aqueous solution by slow water addition and adjusting the pH value of the solution.Due to the deprotonation/protonation state transition caused by the neutral to acidic transformation,the fluorescence emission of the NPI on the nanoparticles changes from green to blue,resulting in the corresponding changes in the fluorescence emission properties of the RhB moiety.In addition,in the presence of glutathione(GSH),the removal of RhB caused by disulfide bond rupture can weaken or block the FRET effect between RhB and NPI,thus reducing the fluorescence intensity of RhB moiety.The results of cell experiments showed that the dual-responsive ratiometric fluorescent probe could monitor the change of pH value and GSH concentration in cells,and track the endocytosis process of nanoparticles and their transport routes in cells.