Multifunctional Nano-biomaterials For Photoacoustic/Magnetic Resonance Imaging-guided Efficient Photothermal Ablation Of Cancer

PART ? PREPARATION,CHARACTERIZATION ANDBIOSAFETY EVALUATION OF MULTIFUNCTIONALNANO-BIOMATERIALSObjective: 1.To prepare melanin-based nano-liposome-PEG and to test the physical properties.2.To evaluate cytotoxicity and biosafety of Lip-Mel,Lip and Mel.Methods: 1.Lip-Mel and Lip were prepared by a trichloromethane-injection and freeze-drying strategy.The size distribution and zeta-potential of Lip-Mel and Lip were determined.Melanin entrapment content was measured by UV-Vis-NIR spectrophotometer.The particle size of Lip-Mel dispersed in phosphate buffered saline(PBS)was recorded at different time points(0 d,1 d,3 d,7 d and 14 d).2.Lip-Mel dispersed in serum-free DMEM solution was added into each well at different concentrations(0.00,0.40,0.80,1.20,1.60 and 2.00 mg/m L).The toxicities of Lip and free melanin were also tested as the control groups.The concentrations of Lip and melanin corresponded to their content in Lip-Mel.After another 24 h co-incubation,cell viabilities were tested by the typical CCK-8 assay.BALB/c mice(~20 g,4-6 weeks)were randomly divided into thirteen groups as follows: control,1 d Lip-Mel,1 d Lip,1 d melanin,3 d Lip-Mel,3 d Lip,3 d melanin,7 d Lip-Mel,7 d Lip,7 d melanin,14 d Lip-Mel,14 d Lip and 14 d melanin(n = 5 per group),which were then intravenously injected with saline solution(control group),Lip-Mel(100 mg/kg),Lip,and free melanin suspension(groups of 1,3,7,14 days post intravenous injection).The concentrations of Lip and melanin corresponded to their content in Lip-Mel.The behaviors of the mice during the evaluation were monitored.The blood samples and major organs were collected for further analysis.Results: 1.Lip-Mel feature a uniform size/shape and high dispersity,as observed by bright-field optical microscopy.The average hydrodynamic diameters of Lip-Mel and Lip were determined to be 300.5 nm(PDI = 0.187)and 299.3 nm(PDI = 0.104),and the zeta potential were(-42.5 ± 0.935)m V and(-39.4 ± 0.60)m V,respectively.The melanin-entrapment amount in Lip-Mel was calculated by a weight ratio(melanin/Lip-Mel)of 90.9 ?g/mg,which were detected by UV-Vis-NIR absorption spectroscopy.2.The resulting cell viability suggested no obvious cytotoxicity for Lip-Mel and Lip in the analyzed concentration range,even at the high concentration.Comparatively,melanin showed hypotoxicity with the elevated concentration.The blood indexes and physiological analysis showed negligible variation among the different groups.No obvious abnormal behaviors were found among all miceConclusion: 1.Lip-Mel were successfully prepared with high stability.2.The encapsulation of melanin into liposomes can substantially decrease the cytotoxicity of melanin.The histocompatibility of Lip-Mel,Lip and melanin were high.PART ? MULTIFUNCTIONAL NANO-BIOMATERIALS FOR PHOTOACOUSTIC/MAGNETIC RESONANCE IMAGING IN VITRO AND IN VIVOObjective: 1.To observe biodistribution of Lip-Mel in major organs after intravenous injection.2.To evaluate PA and MR performance of Lip-Mel in vitro.3.To evaluate PA and MR enhancement of Lip-Mel on MDA-MB-231 xenograft tumor-bearing nude mice in vivo.Methods: 1.MDA-MB-231 tumor-bearing mice(n = 4)were intravenously injected with Di R-labeled Lip-Mel saline solution(10 mg/m L,200 ?L).Fluorescence images were acquired pre,1 h,3 h,6 h,24 h and 48 h post injection,and the relative fluorescence intensities within the tumor regions were measured.The major organs and tumor of one mouse were harvested for ex vivo fluorescence imaging 24 h post injection.2.Lip-Mel suspension at a concentration of 3 mg/m L was scanned for PAI at different wavelengths ranging from 680 nm to 970 nm(interval = 5 nm)to detect the maximum absorbance for optimized PAI.The quantified PA signal intensities within a region of interest(ROI)of each image were then analyzed by Vevo LAZR software.Different concentrations of Lip-Mel dispersed in PBS ranging from 0 to 5 mg/m L were triggered by the optimal excitation wavelength to acquire the corresponding PA images.In addition,PA images of 3 mg/m L Lip-Mel suspension were obtained at different time points(0 h,6 h,12 h and 24 h)for photostability analysis.The PA intensities of each image were measured.The magnetic properties of Lip-Mel and melanin powder were tested by Physical Property Measurement System(PPMS)at 300 K.All MR imaging experiments were conducted on a 7.0 T micro-MRI System(Bruker,Pharmascan)equipped with a small animal-specific coil.Lip(10 mg/m L),and Lip-Mel dispersed in PBS at different concentrations(0-10 mg/m L)were placed in 2 m L Eppendorf tubes for T1-weighted and T1 mapping MRI.The MRI signal intensity(SI)and relaxation time T1 within the region of interest(ROI)were measured.3.For the in vivo PA imaging,MDA-MB-231 tumor-bearing mice(n = 3)were intravenously injected with 200 ?L Lip-Mel at a dose of 10 mg/m L.Then,PA images were collected at different time points(pre,1 h,4 h and 24 h)and average PA intensities in the tumor regions were measured.T1-weighted MR images were collected at different time points(pre,1 h,4 h and 24 h)and pseudo-coloring was applied to each image using Matlab(2016).The method of self-controlled study was used to evaluate the in vivo MRI effect.Average MRI signal intensity(SI)in the tumor region(SItumor)and buttocks muscles(SImuscle)of the same slice were measured.Relative signal intensity(SIr)and the percentage of signal intensity increase(PSII)were calculated.Results: 1.Fluorescence signals within the tumor region behaved in a time-dependent manner.The peak value of fluorescence signals at the tumor site was recorded at 24 h post injection.The ex vivo fluorescence images of the major organs and tumor at 24 h post injection showed there was fluorescence signals within the tumor tissue.2.700 nm was determined to be the maximum absorbance for the potentially optimal PAI.It was found that Lip-Mel showed the concentration-dependent contrast enhancement in PAI and quantitative PA signal intensities increased linearly relative to elevated Lip-Mel concentration while the pristine Lip represented no contrast change even at high concentration.Furthermore,PA images of Lip-Mel suspension(3 mg/m L)were obtained with prolonged time,and the PA signal had no obvious change.The magnetization saturation values of Lip-Mel and melanin are 0.06 and 0.29 emu/g,respectively.It has been found that brighter T1-weighted MR images of Lip-Mel suspension were detected with increased Lip-Mel concentrations.The longitudinal relativity r1 was calculated to be 0.25 m M-1·s-1.3.The PA signal and PSII increased gradually with prolonged observation.Conclusion: 1.Lip-Mel can be effectively accumulated into tumor tissue for effective cancer theranostics.2.Lip-Mel could keep its photostability for potentially continuous PAI.Lip-Mel can enhance PA imaging and T1-weighted MR imaging in vitro and in vivo.PART ? MULTIFUNCTIONAL NANO-BIOMATERIALS FOR EFFICIENTPHOTOTHERMAL ABLATION OF CANCERObjective: 1.To investigate in vitro photothermal property of Lip-Mel and in vitro photothermal ablation against tumor cells and tumor issue.2.To detect in vivo toxicity of PTT assisted by Lip-Mel.Methods: 1.In vitro photothermal property of Lip-Mel was evaluated by laser irradiation of Lip-Mel at different concentrations.808 nm laser was adopted for photothermal evaluation.In addition,different power densities of the 808 nm laser were introduced for irradiation.Water,Lip and melanin were used as controls.The cyclical temperature change of Lip-Mel suspension(melanin concentration: 363.60 ?g/m L,100 ?L)was recorded under 808 nm laser irradiation at a power density of 1.50 W/cm2 and 1.00 W/cm2 for 5 min(laser on),followed by natural cooling to room temperature(laser off)for five laser on/off cycles.Lip and Mel suspension(The concentrations of Lip and melanin corresponded to their content in Lip-Mel.melanin concentration: 363.60 ?g/m L)were exposed to 808 nm laser at the density of 1.50 W/cm2.The temperature changes and infrared radiation(IR)thermal images were recorded by an infrared thermal-imaging camera.2.Lip-Mel-serum-free-DMEM solution was incubated with cells at different concentrations for 12 h of incubation and then the cells were exposed to an 808 nm laser for 5 min at different power densities.The cell viabilities were determined by CCK-8 assay.For confocal observation,the MDA-MB-231 cancer cells were seeded onto four confocal-specific cell-culture dishes(four groups: control,Lip-Mel only,Laser only and Lip-Mel + Laser).The living cells and dead cells were co-stained with calcein-AM and PI solution for confocal observation.3.The tumor-bearing mice were divided into four groups(6 mice per group)randomly.The first group was set as the control group,which was intravenously injected with saline solution(200 ?L).The second group was Lip-Mel only group intravenously receiving Lip-Mel suspension(200 ?L)at a concentration of 10 mg/m L.The third group was the Laser only group,which was intravenously injected with saline solution(200 ?L)followed by 808 nm laser exposure for 10 min at a power density of 1.50 W/cm2.The fourth group was Lip-Mel + Laser group,which was intravenously injected with Lip-Mel suspension(10 mg/m L,200 ?L)followed by 808 nm laser exposure for 10 min at a power density of 1.50 W/cm2.The temperature and IR thermal images were recorded by an infrared thermal-imaging camera.The tumor-volume change of four groups was monitored with a digital camera and the weight of each mouse was recorded every other day after PTT.The tumor-volume changes were normalized using the relative tumor volumes V/V0(V0: the initial tumor volume before the treatment).One mouse of each group was sacrificed 24 h post treatment,and the main organs(heart,liver,spleen,lung and kidney)and the tumor issues were collected and fixed in a 4% paraformaldehyde solution.Finally,the main organs were stained with H&E,and the tumor tissues were stained with H&E,Td T-mediated d UTP Nick-End Labeling(TUNEL)and Ki-67 for histopathology analysis.Results: 1.The temperature increased significantly with the elevation of melanin concentration and NIR irradiation power.The photothermal performance of Lip-Mel did not show any obvious decay during the irradiation cycling.It was found that Lip-Mel triggered a remarkable temperature increase(?T = 51.5 o C,which was higher than that of free melanin(?T = 42.7 o C)and Lip(?T = 2.4 o C).2.It was found that the cell viability decreased with elevated melanin concentrations and laserdensity.In vitro photothermal ablation of MDA-MB-231 cells was further detected by CLSM.No dead cells were found in the control group,showing the green fluorescence of Calcein AM staining.Negligible dead cells were found in the Laser only and Lip-Mel only groups,as indicated by the strong green fluorescence and very weak red fluorescence of PI staining.In contrast,in the Lip-Mel + Laser group,all the cells showed a strong red fluorescence,indicating the presence of large numbers of dead cells.3.The surface temperature of tumors in the Lip-Mel + Laser group increased from 23.8 ± 1.2 °C to 51.1 ± 2.5 °C under irradiation for 10 min.Comparatively,the temperature at the tumor region of mice in the Laser only group showed only a slight temperature change.Two days after PTT,the tumor tissues in the Lip-Mel + Laser group were necrotic,leaving black scars in the initial tumor regions.After twenty days,the black scar disappeared,leaving the complete eradication of tumor.In contrast,tumors of the other three groups grew significantly.As shown in H&E-stained tumor sections,there were only a small portion of purple blue(normal nucleus)and many more deformed nuclei in the Lip-Mel + Laser group,indicating the high apoptosis and coagulative necrosis of the cancer cells.From the TUNEL assay result,representative apoptosis-positive cells were indicated by dark-brown nuclei in the Lip-Mel + Laser group.Immunochemical staining of Ki-67 on tumor sections showed the in vivo proliferative activities of the four groups,where proliferative cells were stained brown.The Lip-Mel + Laser group exhibited a significant suppression effect(the least brown nuclei)on tumor cell proliferation while the other three groups showed almost no killing effects on the proliferative activity of tumor cells.H&E staining of the main organs showed no obvious histopathological lesion.Conclusion: 1.Lip-Mel exhibits good photothermal performance.The photothermal stability of Lip-Mel is goog and Lip-Mel can as a reusable photothermal nanoagent.Lip-Mel exhibited higher photothermal-conversion efficiency over free melanin.2.Lip-Mel can be used as the photothermal agent for in vitro ablation against cancer cells.3.PTT assisted by Lip-Mel is effective to tumor ablation.4.PTT has few adverse effects of PTT on the health and normal organs of the mice.