Radiation-responsive Polypeptide Nanogels:Preparation And Application In Concurrent Chemo-Radiothreapy

Part1.Preparation and characterization of radiation-responsive polypeptide nanogels.Purpose:Preparation the diselenide-containing radiation-responsive polypeptide nanogels and characterizations of chemical structures and compositions of nanogels.Materials and Methods:m PEG-b-P(LGA-co-CELG)was first synthesized by the ring-opening polymerization of BLG-NCA and CELG-NCA with m PEG-NH2 as macroinitiator,and then deprotection of benzyl group.The 1H NMR and FT-IR spectra,and GPC chromatograms were used for confirming the chemical structures of copolymers.The amphiphilic copolymers spontaneously self-assembled into spherical micellar aggregations in an aqueous environment.The diselenide cross linking nanogel was prepared through substitution reaction between disodium diselenide and the chlorine in the core of methoxy poly(ethylene glycol)-block-poly(L-glutamic acid-co-?-2-chloroethyl-L-glutamate)micelle.DOX was loaded into micelles through a nanoprecipitation technique.The in vitro DOX release profiles of DOX-loaded micelles and nanogels were determined with PBS at p H 7.4 as a medium after 5Gy,10Gy,25Gy,50Gy irradiation.The accumulative DOX release was tested by fluorescence spectroscopy through a standard curve method(kex=480 nm).The morphologies of micelle and nanogel were revealed by transmission electron microscopy(TEM).Results:The diselenide cross linking nanogel was prepared through substitution reaction between disodium diselenide and the chlorine in the core of micelles.1H NMR spectra,FT-IR spectra and GPC confirmed the successful preparation of copolymers.The morphology of micelles and nanogels after X-ray irradiaiton were tested with TEM.The micelles and nanogels displayed spherical morphology with average diameters of 50nm and 40 nm,respectively,which were estimated from TEM micrographs.The DOX loaded nanogels were spherical before irradiation;however,the shape and size changed along with the increase of radiation dose.After 5Gy X-ray irradiation,the spherical nanogels become swollen,with an average diameter increase from 50 to 82 nm.When the radiation dose increased to 25 Gy,apparent shape change and size increase of nanogels were observed.When giving 50 Gy X-ray irradiation,nanogels were completely broken.However,there was no obvious changes in micelles before and after X-radiation.The DLC of micelles and nanogels are 19.47%and 19.89%respectively,and the DLE of micelles and nanogels are 96.71?99.31%respectively.There was no dramatic initial burst release of DOX from the unirradiated nanogel,about 30%for nanogels of the initial loading amount were eventually released in the test duration.When increasing the X-ray dose to 25Gy resulted in the faster and increased release of DOX to more than 60%in 48h,indicating that DOX molecules could be released efficiently from the nanoparticles after X-ray irradiation.As the radiation dose increased from0Gy to 50Gy,the release of DOX increased accordingly.The released percentage of Dox gradually increased with the dose of X-ray and was 81%when a 50 Gy radiation was applied and the maximal release rate of DOX from nanogel was reached at 8 h.This was because there was no obvious structural change to the diselenide block copolymer before irradiation.The loaded Dox can not be easily diffused out without X-ray irradiation.Conclusion:The m PEG-b-P(LG-co-CELG)was synthesized through ROP of CELG-NCA and BLG-NCA,DOX was loaded into nanogels as a model drug.m PEG-b-P(LG-co-Se₂ELG)nanogel was prepared by the crosslink of m PEG-b-P(LG-co-CELG)micelle with Na₂Se₂.The radiation sensitivity of DOX-loaded nanogels was examined after different doses X-radiation.Results indicated the release of incorparated DOX was ascribed to the break of diselenide groups within nanogels induced by X-radiation.The DOX release rate was proportional to the radiation doses.Part2:The in vitro and in vivo anti-tumor effect of diselenide-groups containning polypeptide nanogels.Purpose:To explore the anti-tumor effect of diselenide-groups containning polypeptide nanogels in vitro and vivo.Materials and methods:1)The in vitro cellular proliferation inhibition experiment:A549 cells were divided into radiation groups and non-radiation groups.Radiation groups included X-radiation alone group,Free-DOX+X-radiation group,micelles+X-radiation group and nanogels+X-radiation group;while the non-radiation groups included PBS group,Free-DOX group,micelles group and nanogels group.The A549 cell survival rate were examined after 5Gy X-radiation at 48h.2)Cellular uptake by A549 cells was examined using flow cytometry and confocal laser scanning microscope.3)in vivo experiment:Treatments were started after 2 weeks when the tumor volume of the nude mice reached approximate 80 mm³.The tail-vein injections of 0.1 ml of PBS alone,free DOX,DOX-loaded nanogels or micelles with an equivalent DOX dosage(5.0 mg DOX per kg body weight)in PBS were performed every 4 days during the treatment time.The irradiation was performed approximately 10 h after the DOX solution injection.There were totally five times of durg solutions injection and raidiation during the treatment.The data of tumor volume and body weight of mice were collected every other day and analysed at last.The tumor lesion of mice were exposed to a single dose of 5 Gy irradiation after injection.The anti-tumor effect DOX loaded nanogels were assessed by analysing the results of tumor volume,HE stain,IHC and TUNEL.Mice serum were also collected for evaluating the side effect to liver,kidney and heart.Heart,liver,spleen,lung and kidney of mice were also fixed,slided and stained with HE.4)To investigate the anti-tumor efficacy of DOX loaded nanoparticles in vivo,female BALB/c nude mice bearing with A549 cells xenograft model were treated with DOX loaded micelles and DOX loaded nanogels.All the mice were divided into eight groups according to the presence of 5Gy X-ray irradiation:PBS(a),free DOX group(b),DOX loaded micelles group(c),DOX loaded nanogels group(d),radiation group(e),free DOX+irradiation group(f),DOX loaded micelles+irradiation group(g),DOX loaded nanogels+irradiation group(h).PBS treated groups was considered as negative control.Results:The in vitro proliferation inhibition experiment showed that the survival rate of A549 cells decreased with the concentration of DOX increased.Raidiation groups gained better inhibition capacity compared with non-raidiation groups under same DOX concentrations.A549 cells incubated with DOX-loaded nanogels combined with 5Gy irradiation exhibited lowest A549 cell survial rate compared with the other groups.After 2 h incubation with free DOX and DOX loaded nanoparticles,red fluorescence can be seen in the treated cells.A549 cells treated with DOX loaded nanogels combined with 5 Gy X-radiation exhibited strongest fluorescence though similar intensity of fluorescence were observed before radiation.Results from flow cytometry were consistent with the CLSM.The in vivo anti-tumor effect of DOX loaded micelles and nanogels were evaluated using tumor bearing nude mice.Data of tumor volumes throughout the treatment process were collected and analyzed.Tumor growth in the PBS treated group presented the most rapid growth.For the irradiation groups,their antitumor efficacies were better than the rest non-irradiation groups.The combination of free DOX or micells and radiation displayed enhanced therapeutic efficacy than radiation alone,but worse than nanogels combined with radiation.In other words,the best treatment effiacacy occurred in nanogel+radiation group.The tumor volume after treatment were also analysed,and the results were consistant with the in vivo tumor volumes.For HE staining,necrotic area of each slide were calculated to evaluate the antitumor effect of all the agents.The tumor tissue in the PBS group contained dense tumor cells with clear cellular morphologies and almost no necrotic area.The pathology of the all DOX prescriptions treated groups showed cancer cells death and deterioration in different degrees,with irregular necrotic area.Apparently,the necrotic area in nanogels+radiation group was the biggest among all eight groups.In TUNEL assays,less apoptotic cells were observed in tumor tissues treated with PBS,while different degrees of cellular apoptosis were detected in all DOX formulation treated groups.DOX loaded nanogels combined with X-radiation group showed the most serious cellular apoptosis compared with DOX-loaded micelles and free DOX,which is consistent with the disciplines of antitumor efficacies in vivo and H&E analyses.The pathological changes of the Vital organs and serum index were also evaluated to assess the security of different DOX formulations in vivo.DOX-loaded micelles and nanogels groups just exhibited better therapeutic effect and mild side effect.Conclusion:In vitro and vivo experiment were conducted to evaluate the radiation sensitivity of diselenide-containing polypeptide nanogels.Confocal fluorescence microscopy and flow cytometry demonstrated that DOX-loaded nanogels could deliver DOX into A549 cells more efficiently than the parent DOX-loaded micelles and free DOX after irradiated by 5Gy X-ray.What'more,the DOX-loaded nanogels showed efficient proliferation inhibition of tumor in vitro and in vivo combined with concurrent external irradiation.The properties endowed the great potential for smart drug delivery during concurrent CRT to tumors with enhanced therapeutic efficacy and reduced toxicity.