Construction And Application Of Azoreductase-Responsive Near-Infrared Fluorescent Polymer Nanoprobe

Fluorescent probes have attracted tremendous attention as a versatile tool for analytical sensing and optical imaging because of their high sensitivity,ultra specificity,rapid response time and strong visibility.In addition to unique light-responsive properties,azobenzenes also own another important feature called reduction response indicating that azobenzenes can be destroyed by azoreductase or some reducing agents including Na2S2O4 and SnCl2.The fluorescence intensity of fluorophores is always quenched by azobenzenes as a result of ultrafast isomerization of azo bonds,whereas it will be enhanced once the azo bonds are cleaved efficiently.Therefore,azobenzenes are often designed as switches of fluorescence in the scaffold of probe structures.Azoreductase mainly exhists in colon and there are a variety of microfloras which can produce many types of reductases including azoreductases.During the past decades,azoreductase-responsive fluorescent probes have been significantly demonstrated in the application of target imaging in colon.In recent years,nanoparticles prepared by diblock polymers have been extensively focused on for their potential in drug delivery,diagnosis and imaging,tissue engineering,biosensor and bio separation.Polymerization-induced self-assembly(PISA)is an efficient,controllable,one-pot and facile approach for the preparation of block polymeric particles with tunable shape and size via controlled/living radical polymerization(CRP)at relatively high solids(10-50%w/w).Compared to traditional solution self-assembly,PISA strategy exhibits great excellence to prepare high concentration drug-loaded nanoparticles in the delivery and release of drug for its convenience and simplicity without any further postpolymerization procedure.Furthermore,the introduction of azo-containing fluorescent probe to the polymer chain to prepare nanoprobe offers the chance of increasing treatment efficacy and bioimaging in colon.As is well known,near-infrared(NIR)light,compared to ultraviolet visible(UV-vis)light,exhibits better tissue penetration,lower autofluorescence and less damage to tissue in bioimaging.Hence,azo-containing polymer nanoprobes are feasible in colon-targeted drug delivery monitored by NIR fluorescence and at the same time also show good biocompatibility,which provide an excellent technique for colon-specific drug release and imaging.In this work,two types of Aza-BODIPY containing polymer probes were designed and synthesized based on the reduction response performance of azobenzene.The NIR fluorophores Aza-BODIPY with high molar extinction coefficients and large fluorescence quantum yields were connected to the polymer segment to construct two types of azoreductase-responsive NIR nanoprobes,which can be employed to colon target-oriented probe for the application of drug release monitored by enhanced fluorescence.The specific content is as follows:1.The azo-containg NIR probe ADP-Azo was designed and synthesized,which consisted of azo moieties and NIR fluorophores Aza-BODIPY.The product ADP-Azo was synthesized successfully and characterized correctly by 1H NMR and matrix-assisted laser desorption ionization time-of-flight mass spectrometry(MALDI-TOF MS).In the presence of azoreductases,azo bonds were cleaved efficiently and subquenebbtly the NIR fluorescence was turned from "off" to "on",which was confirmed by UV-vis and FL.The polymer mPEG2k-ADP-Azo were also synthesized by CuAAC(Cu-catalyzed azide/alkyne cycloaddition)"Click" reaction between mPEG2k-N3 and alkynes of ADP-Azo,which were also well characterized by 1H NMR and GPC.The polymer probes self-assembled into spherical micelles with about 38 nm observed by TEM and DLS,whose reduction-responsive behaviors were studied by adding Na2S2O4.DOX-loaded micelle nanoprobes were also prepared by same procedure except the presence of Doxorubicin(DOX)during the process of self-assembly.In the environment of mimicking colon(hypoxic,37?),DOX-loaded micelles were incubated in the presence of Na2S2O4 and azoreductase respectively to investigate the behaviors of drug release.As a result,it was demonstrated that the DOX-loaded NIR nanoprobe exhibited excellent performance in drug release with the real-time enhancement of NIR fluorescence of Aza-BODIPY,which was characterized by UV-vis,FL,TEM and DLS correspondingly to assess the morphologies and sizes of DOX-loaded micelles before and after reduction,changes of the absorbance of azo bonds and variation on NIR fluorescence intensity.2.The other type of NIR nanoprobe was prepared by PISA approach in situ.First of all,the unique functional macromolecular RAFT agent was synthesized with the Al-ADP-Azo-CDPA and hydrophilic block PPEGMA using CuAAC "Click" reaction.1H NMR and GPC were utilized to confirm the structure accurately.The azoreductase-responsive NIR polymer nanoparticles PPEGMA-ADP-Azo-PBzMAx with controllable morphologies and sizes were fabricated in the presence of the typical solventphobic monomer BzMA in alcohol by PISA approach in situ.Subsequently,the reductant-responsive behaviors of PPEGMA-ADP-Azo-PBzMAx were studied by UV-vis,FL,TEM and DLS in simulated colon environment.It must be pointed out the experiment condition of different nanoparticles were identical completely.As shown in this work,the micelle PPEGMA-ADP-Azo-PBzMAs with smaller diameter around 33 nm exhibited more obvious enhancement of NIR fluorescence intensity.The aggregation of NIR fluorophore Aza-BODIPY in the core of assemblies aroused ACQ effect.Under the action of reduction reaction,the degradation of azo bonds resulted in the cleavage of polymer and disassembly of micelles,which caused NIR fluorophore connected to hydrophilic polymer to eliminate the ACQ effect.Consequently,it can be observed that NIR fluorescence enhanced gradually with the increasement of reduction time.DOX,a model drug,was encapsulated into nanoparticles prepared by PISA strategy in situ for the fabrication of azoreductase-responsive NIR DOX-loaded nanoprobe PPEGMA-ADP-Azo-PBzMA5@DOX.The behaviors of the nanoprobe PPEGMA-ADP-Azo-PPEGMA5@DOX on NIR fluorescence reporting drug release were subsequently investigated with the addition of azoreductase in the mimicking colon environment(hypoxic,37?).