| Objective:Rapid tumor growth would create hypoxic environment in the center during tumor progression,and necrosis will be happened.On the other hand,various treatment strategies will kill the tumor cells to eliminate tumor,and complicated by necrosis generation.Thus,Necrosis in tumor can be applied to detect and evaluate tumor biology and treatment responses.The necrosis in the center of tumor also can be a targeting site to develop the targeting therapy to variable tumor cells surrounding necrotic zone.In addition,huge cancer stem cells have been proved in the hypoxic zone surrounding necrotic area in tumor,which are responsible for tumor recurrence,metastasis and treatment resistance.The therapeutic drugs delivery on the basis of necrosis targeting also play a scientific and clinical values to treat cancer stem cells.However,the relatively poor blood supply and high interstitial pressure in necrotic tissue impede the drug delivery.Hypericin,extraction from hypericum perforatum,is a small necrosis targeting molecule with satisfactory necrosis affinity.However,hypericin is hydrophobic and easy self-aggregation in saline,its bioavailability will be greatly decreased without appropriate delivery medium in animal body.Previous studies use organic solvents as hypericin delivery medium.However,new negative problems will occur during hypericin application.Organic solvents are toxic to animal bodies including hemolysis and cell membrane rupture.Moreover,the organic solvents will be diluted by blood after injection,and hypericin will aggregate and turn into big particles,and then be engulfed by mononuclei phagocyte system in liver and spleen.How to safely and effectively deliver hypericin to animal body is the key step for hypericin clinical translation.Nanoparticles demonstrate a special tumor accumulation as a result of the special sizes and large surfaces,and are capable of modify the encapsulated drug properties.Nanomaterials have been used in drug delivery as the vector for a long time.Organic nanoparticles show a higher safety for biodegradable property.Polyethylene glycol(PEG)and polycaprolactone(PCL)are very common in organic nanoparticles synthesis,they are intoxic and easily degraded in animal body.Gold nanoparticles,capable of X-ray imaging and of high electron density,have been used for drug and vector detection in previous studies.Thus,we propose the hypothesis:Nanoparticles using PEG-PCL as the shell encapsulate hypericin and gold in the core,the nanoparticle enables fluorescence and necrosis affinity from hypericin,and X-ray imaging from gold.It can play the necrosis imaging and vector detection role in tumor.The study results will provide new platform for drug delivery to tumor necrosis.Methods:1.2 mg gold,100 ug hypericin and 5 mg mPEG-PCL were dissolved in tetrahydrofuran(THF),after effective agitation,2 m L 5%dextrose was added into the reacting system.Then the system was moved to chemical hood for THF evaporation overnight.After adjusting the volume to 2 m L with nanopure water,the nanoparticles were obtained following 0.22 um filtration.The characterization was performed using digital light scatting system(DLS),cryo-TEM,dark field microscopy and UV-vis spectrophotometry to investigate the size,surface charge,PDI,morphology and encapsulating efficiency.The stability was conducted by recharacterization of nanoparticles after storage in 4 centigrade in a month.2.Nanoparticles with difference gold concentrations(500、250、125、62.5 ug/m L),PBS and 25%DMSO in PBS were incubated with 2.5%red blood cells for 3 h in 37℃.Hemocytometry was used to investigate the integrate cells to evaluate hemolysis safety.Nanoparticles with difference gold concentrations(1000、500、250、125、62.5、31.25 ug/m L)incubated with A2780N cells seeding in 96 wells,RPMI1640 medium and 25%DMSO as the negative and positive control respectively,Calcein-AM method was used to evaluate the cytotoxicity.Nanoparticles with gradient gold concentration(0-3000 ug/mL)and iohexol as the control were used to evaluate X-ray imaging in Micro-CT.The fluorescence imaging and cellular internalization were investigated in A2780N cells incubated with PNP@AuNP-Hyp for 36 h(hypericin concentration of 10 ug/m L)via fluorescence microscopy.Cellular necrosis model was created by dry ice freeze method,PNP@AuNP-Hyp with gold concentration of 100 ug/m L was added to evaluate necrosis affinity.3.Two nude mice(six week)were employed and divided to treated and control group.2×10~6 A2780N cells were implanted in the flank of nude mice,and the tumor necrosis model was developed when the tumor diameter of 1.5cm.One mice underwent tail vein injection of PNP@AuNP-Hyp of gold dose of 8mg/kg.The control mice was injected 200 uL saline.Fluorescence and X-ray imaging were observed at different time points.30 min after another injection,the tumor was harvest,and 2 mm slice was made to evaluate the necrosis affinity.Then cryo-section and HE staining were conducted to observe the hypericin and necrosis distribution respectively.Various organs including heart,liver,spleen,lung,kidney,pancreas and brain between treated and control mice were analyzed in histological level to evaluate safety in vivo.Results:1.PNP@AuNP-Hyp was successfully developed via solvent evaporation method.The hydrodiameter is 103.9±1.74 nm,PDI=0.121±0.017,Zeta potential=-17.3±4.14.2.Cryo-TEM and dark field microscopy revealed a monodisperse spherical morphology.3.UV-vis spectrophotometry showed PNP@AuNP-Hyp of specific hypericin absorbance peak and slope of gold.4.After making standard calibration curves of hypericin and gold,the hypericin encapsulation efficiency was93.4%in PNP@AuNP-Hyp and gold of 71.3%.5.The hydrodiameter decreased very small after one month storage in 4 centigrade with no change of PDI and zeta potential,and 10.4%gold and 8.8%hypericin loss during storage.6.PNP@AuNP-Hyp demonstrated no hemolysis effect,it is safer than 25%DMSO.7.In cytotoxicity evaluation,died cells increased with the increase of PNP@AuNP-Hyp concentration,cell survival rate was 100.2±13.5%in nanoparticles with gold of 250ug/mL,59.8±18.2%in nanoparticles with gold of 500 ug/m L,and 7.0±0.7%in 25%DMSO as the control.8.PNP@AuNP-Hyp demonstrated satisfactory X-ray imaging by micro-CT and fluorescence imaging in microscopy after incubation with A2780N cells,the red fluorescence of hypericin colocalized in cytoplasm in cells.In cell death assay,PNP@AuNP-Hyp mainly distributed in dead cells area.9.After tail vein injection of PNP@AuNP-Hyp in mice bearing A2780N tumor,the tumor fluorescence generally increased to top at 30 min,and X-ray density arrived the highest at 5 min post injection.Fluorescence and X-ray imaging returned to background at 24 h post injection.10.The tumor was harvested after injection again at 30 min post injection,2mm tumor section was performed.The center showed necrosis in optic view,which colocalized with strong hypericin fluorescence and relative high X-ray density.11.Histological analysis including cryosection and paraffin section demonstrated PNP@AuNP-Hyp mainly distributed in necrotic zones.12.HE staining analysis between the treated and control mice showed there was no significant difference in harvested organs.Conclusion:1.PNP@AuNP-Hyp can be formulated by solvent evaporating method,which is easily manipulated.The nanoparticles have an uniform disperse with spherical shape.Its stable profile and rational size can be used for tumor.2.PNP@AuNP-Hyp is a safe delivery platform for hypericin,which is superior to DMSO as the delivery medium.It preserves X-ray imaging of gold and fluorescence imaging and necrosis affinity of hypericin.3.PNP@AuNP-Hyp exhibits fluorescence and X-ray imaging in nude mice bearing A2780N tumor,which can play multimodality imaging monitor role for tumor necrosis.Moreover,the satisfactory necrosis affinity of nanoparticle provides new ideas for further necrosis targeting researches. |
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