Cypate-loaded Polymeric Micelles For Simultaneous Cancer Imaging And Efficient Photothermal Therapy

Objective: In this work, the preparation and application of cypate-loaded micelleswere explored. We demonstrate a theranostic micelle system for cancer NIRF imaging withenhanced signal to noise ratio (SNR) and superior photothermal therapy. The copolymersconsisting of monomethoxy poly(ethylene glycol) and alkylamine-grafted poly(L-asparticacid) are assembled with carbocyanine dye (cypate) into the theranostic micelles, whichexhibit small size, high loading capacity, good stability, sustained release behavior, andnegligible cytotoxicity. Moreover, we further observe that the micelles exhibit superiorhigh imaging contrast and photothermal effect on cancer cells, causing efficientphotothermal tumor regress upon photo-irradiation.Methods:(1) Alkyl amines (decylamine) were conjugated to the aspartate sidechains of the block copolymer (poly (ethylene glycol)-poly (aspartate) block copolymers)by amide bond, all the polymers were characterized by IR,1H-NMR.(2) The polymericmicelles containing cypate were prepared by the dialysis and film-forming method,respectively. The Preparation procedures were optimized with the particle size,polydispersity index（PDI）, entrapment efficiency, drug-loading. The morphology and sizeof micelles were measured using scanning electron microscopy (SEM) and dynamic lightscattering (DLS), The drug loading and encapsulation efficiency were measured usingUV-VIS spectrophotometer, The pyrene fluorescence probe was used to estimate thecritical micelle concentration(CMC) of micelles.(3) MTT Assay was used to estimate thein vitro cytotoxicity of copolymers on A549cells, Furermore, MTT assay was also used todetermine the photothermal cytotoxicity of the micelles against A549celles under785nmphotoirradiation at1W/cm2.(4) In vitro cell uptake of micelles was measured using aUV-VIS spectrometer.(5) We monitored the generation of singlet oxygen from cypateusing1,3-diphenylisobenzofuran (DPBF) as a probe which can specifically be quenchedin the presence of singlet oxygen.(6) To monitor the levels of reactive oxygen species onA549cells, dihydroethidium was utilized as a probe to moniter the generation of ROSupon photoirradiation.(7) To elucidate the mechanism of photoirradiation-triggeredsynergy, acridine orange (AO) was used as a marker to monitor the integrity of lysosomal membranes upon photoirradiation.(8) To validate the cell death triggered by photothermaleffect, we used trypan blue staining to validate cell injury, Trypan blue dye molecules caneasily penetrate plasma membranes of dead cells to stain the nuclei, but viable cells willnot be stained.(9) IVIS Lumina II imaging system was used to evaluate the cancer imagingand tissue distribution of micelles on mice bearing A549tumor.(10) The in vivophotothermal anti-tumor efficacy of the micelles was evaluated in mice bearing A549andmurine heptic H22tumor with neoplasm volume.Results: Decylamine (DA) was grafted into the backbones of copolymers viaaminolysis for the synthesis of mPEG-b-PAsp(DA). The polymer exhibited the low criticalmicelle concentration values (2.36μg/ml), and the polymer was subsequently employed toencapsulate carbocyanine dye cypate. The cypate-loaded micelles had the average diameterof42.3nm with the polydispersity index of0.09, and displayed a spherical morphology,high drug loading of50%and encapsulation efficiency of95%, The copolymer in therange of5~180μg/ml had no significant change in cell viability against A549cells after6h and24h incubation, indicating that the copolymer has no obvious cytotoxicity, However,cypate-loaded micelles effectively caused cell death under785nm laser photoirradiation,The in vivo anti-tumor efficacy showed cypate-loaded micelles without photoirradiationresulted in a similar growth behavior to that of control group, indicating that the micellesare nontoxic to tumor and exhibit no therapeutic effect without photoirradiation. However,cypate-loaded micelles displayed strong tumor necrosis and regression uponphotoirradiation, and finally resulted in the superior anti-tumor efficacy.Conclusions: We demonstrate a theranostic micelle system encapsulatingcarbocyanine dye cypate with small average diameter, good stability, negligiblecytotoxicity, polymeric micelles can facilitate preferable accumulation and long-termretention of cypate at tumor. Simultaneously, the micelles can trigger significantphotothermal damage on cancer cells, leading to the successful tumor regress uponphotoirradiation. We believe that the theranostic micelles are a promising vehicle forefficient cancer imaging and therapy. The results also provide a theoretical andexperimental basis of carbocyanine dye in the field of cancer imaging and photothermaltherapy.