Preparation And Evaluation Of The Anti-Breast Cancer Efficacy Of Novel RGD-modified Curcumin-loaded Chitosan/Perfluorohexane Nanocapsules

PART Ⅰ Preparation and characterization of novel RGDmodified curcumin loaded chitosan/perfluorohexane nanocapsulesObjective To prepare a novel curcumin(Cur)loaded chitosan/perfluorohexane(CS/PFH)nanocapsules modified by RGD(RGD-CS/PFH-Cur).To evaluate the physicochemical properties and stability of the nanocapsules in vitro.To investigate the Cur release of the novel nanocapsules under ultrasonic irradiation.Methods Cur loaded chitosan/perfluorohexane(CS/PFH-Cur)nanocapsules were prepared by a nano-emulsion approach,and then RGD modified curcumin loaded chitosan/perfluorohexane(RGD-CS/PFH-Cur)nanocapsules were prepared by surface peptide crosslinking method.Preparation of novel RGD-CS/PFH-Cur nanocapsules labeled with Rhodamine B.The physical and chemical properties of the new RGD-CS/PFH-Cur nanocapsules and CS/PFH-Cur nanocapsules,such as particle size,morphology,polydispersity index(PDI),zeta potential,were detected by light microscope,electron microscope and Malvin laser particle sizer.The structural characteristics of CS/PFH-Cur nanocapsules and RGD-CS/PFH-Cur nanocapsules were analyzed by Fourier transform infrared spectroscopy.The entrapment efficiency and drug loading of CS/PFH-Cur nanocapsules and RGDCS/PFH-Cur nanocapsules were detected respectively.Free curcumin,CS/PFH-Cur nanocapsule and RGD-CS/PFH-Cur nanocapsules were added to PBS solution respectively,incubated at 37℃ and gently shaken,and the stability of curcumin in each group was compared after 24 hours.To determine the effect of 1-MHz ultrasonic irradiation on the release of curcumin from the new RGD-CS/PFH-Cur nanocapsules and CS/PFH-Cur nanocapsules.Results The novel nano RGD-CS/PFH-Cur prepared was a spherical body with shell core structure,which is relatively uniform in size and had no obvious aggregation.Its average particle size was(531.20 ± 68.97)nm,the PDI was 0.27±0.03,and the zeta potential was+19.20 ±1.08 mV;The average particle size of CS/PFH-Cur nanocapsules was(458.67 ±98.96)nm,the PDI was 0.26 ±0.04,and the zeta potential was+15.20±0.44 mV.The Fourier transform infrared spectra of CS/PFHCur nanocapsules and RGD-CS/PFH-Cur nanocapsules showed that the characteristic peaks of curcumin mostly overlapped or merged with the chitosan peaks.Curcumin transmission peaks at about 1242 cm-1 were observed in the spectra of these nanocapsules,confirming the existence of curcumin in these samples.The v(C-N),δ(C-C-N),8(O-H),v(C=O-Amide Ⅱ band),and vas(CH2)groups of RGD exhibited respective absorption peaks at 1065 cm-1,1238 cm-1,1466 cm-1,1631 cm-1,and 2925 cm-1,that were present in the spectrum of RGD-CS/PFH-Cur-NCs confirming the presence of RGD in these nanocapsules.The entrapment efficiency and drug loading of CS/PFH-Cur nanocapsules were 72.19± 2,85%and 9.62±0.36%respectively,while the corresponding values of RGD-CS/PFH-Cur nanocapsules were 70.25 ±2.56%and 9.31 ± 0.33%.After being added to PBS solution,inccrbated at 37℃ and gently shaken for 24 hours,the stability of curcumin contained in CS/PFH-Cur nanocapsules and RGD-CS/PFH-Cur nanocapsules was significantly better than that of free curcumin.CS/PFH-Cur nanocapsules and RGD-CS/PFH-Cur nanocapsules were incubated at 37℃ and almost no curcumin was released from these nanocapsules within 7 minutes without ultrasonic irradiation;However,under ultrasonic irradiation,71.4%curcumin in CS/PFH-Cur nanocapsules and 77.7%curcumin in RGD-CS/PFH-Cur nanocapsules were released within 4 minutes.Conclusion The prepared CS/PFH-Cur nanocapsules and RGD-CS/PFH-Cur nanocapsules are spherical in shape,uniform in size.FTIR analysis confirmed that curcumin was present in both prepared nanocapsules and RGD was present in RGDCS/PFH-Cur nanocapsules.Both nanocapsules have good entrapment efficiency and drug loading capacity.The encapsulated curcumin has relatively good stability,and can rapidly release curcumin under ultrasonic irradiation,which lays a foundation for the next anti-tumor research of carrying drugs.PART Ⅱ Imaging study and evaluation of anti-breast cancer efficacy of novel RGD-CS/PFH-Cur nanocapsulesObjective To evaluate the enhancement ability of RGD-CS/PFH-Cur nanocapsules by ultrasound in vitro.To study the targeting ability of fluorescently labeled RGDCS/PFH-Cur nanocapsules to breast cancer MDA-MB-231 cells in vitro.To evaluate the cytotoxicity of RGD-CS/PFH-Cur nanocapsule to MDA-MB-231 breast cancer cells in vitro.To investigate the safety,ultrasonic enhancement and antitumor ability of these novel targeted fluorescent drug-loaded nanocapsules in nude mouse breast cancer models in vivo.Methods The in vitro ultrasound enhanced development ability of CS/PFH-Cur nanocapsules and RGD-CS/PFH-Cur nanocapsules were evaluated by medical color ultrasound scanner,and their ultrasound enhanced development effect in subcutaneous implanted tumor model of nude mice bearing tumor was evaluated.The ability of red fluorescent rhodamine B labeled CS/PFH-Cur nanocapsules and RGDCS/PFH-Cur nanocapsules to target MDA-MB-231 breast cancer cells was evaluated by confocal laser scanning microscopy.MTT assay was used to evaluate the ability of cytotoxic MDA-MB-231 breast cancer cell death caused by free curcumin,CS/PFH-Cur nanocapsules and RGD-CS/PFH-Cur nanocapsules.The safety and organ toxicity of CS/PFH-Cur nanocapsules and RGD-CS/PFH-Cur nanocapsules in nude mouse MDA-MB-231 breast cancer model were evaluated by blood biochemical examination and pathological tissue sections.The ultrasound-enhanced imaging effect of the two kinds of nanocapsules in the tumor-bearing nude mice model was evaluated by using a medical color Doppler ultrasound scanner.The tumor-bearing nude mice were divided into blank control group,free curcumin group,ultrasonic irradiation(US)group,CS/PFH-Cur nanocapsule group,RGD-CS/PFHCur nanocapsule group,and RGD-CS/PFH-Cur nanocapsules+US group to evaluate the antitumor efficacy of RGD-CS/PFH nanocapsules under ultrasound irradiation.Results CS/PFH-Cur nanocapsules and RGD-CS/PFH-Cur nanocapsules are uniformly fine dot echo in two-dimensional gray mode and uniformly enhanced hyperecho in enhancement mode.Both of them show good enhancement ability for ultrasonic scanning,and there is no significant difference between the enhancement effects of the two(P=0.578).Using confocal laser scanning microscope,red fluorescent nanocapsules accumulated significantly more in MDA-MB-231 breast cancer cells treated with targeted RGD-CS/PFH-Cur nanocapsules than MDA-MB231 breast cancer cells treated with non targeted CS/PFH-Cur nanocapsules.The IC50 values of free curcumin group,CS/PFH-Cur nanocapsule group and RGD-CS/PFHCur nanocapsule group measured by MTT were 26.23 ±2.76 respectivelyμg/mL、15.95 ±1.57 μg/mL and 10.65 ±0.97 μg/mL,there was significant difference among the three groups(P<0.05).The MDA-MB-231 model of nude mice bearing tumor was successfully established.The results were the same as those of in vitro ultrasound enhanced development test.CS/PFH-Cur nanocapsules and RGDCS/PFH-Cur nanocapsules had good ultrasound enhanced development ability in nude mice,but the enhancement effect of RGD-CS/PFH-Cur nanocapsules was better than that of CS/PFH-Cur nanocapsules.After CS/PFH-Cur nanocapsules and RGDCS/PFH-Cur nanocapsules were injected into nude mice bearing tumors,the results of blood tests and the pathological pictures of the heart,liver,kidney,lung and spleen showed that the two nanocapsules were safe and reliable in vivo without obvious toxic and side effects.Grouped treatment of tumor-bearing nude mice,the tumorbearing nude mice in the RGD-CS/PFH nanocapsule+US group had the slowest growth compared with the other groups,and there was a statistical significance,indicating that the combination of RGD-CS/PFH-Cur nanocapsules with US could enhance the anti-tumor effect in vivo.Conclusion RGD-CS/PFH-Cur nanocapsules have good capabilities of ultrasoundenhanced imaging in vitro and in vivo.These nanocapsules are safe and reliable,have no obvious toxic and side effects,and can effectively target and kill breast cancer,which provides a theoretical basis for the drug delivery strategy of targeted nanocapsules,and lays a foundation for further research in the field of clinical targeted treatment of cancer.