Folate Receptor-targeted Laser-activatable Plga Nanoparticles Loaded With Paclitaxel/Indocyanine Green For Multifunctional Theranostics Via Dual-modality Photoacoustic/Ultrasound Imaging And Chemo-/Photothermal Therapy

PART ONE: THE PREPARATION AND CHARACTERIZATION OF FA-PEG-PLGA-PTX@ICG-PFH NANOPARTICLESObject1.A folate receptor-targeted laser-activatable PLGA nanoparticles loaded with paclitaxel/indocyanine green for multifunctional theranostics(Noted: FA-PEG-PLGA-PTX@ICG-PFH NPs)was constructed,and its characterization was detected.2.As a drug delivery vehicle,cytotoxicity and the targeting ability of the FA-PEG-PLGA@ICG-PFH NPs were studied in vitro.Method1.Using the FA-PEG-PLGA polymer,indocyanine green(ICG),perfluorohexane(PFH)and paclitaxel(PTX),the FA-PEG-PLGA@ICG-PFH NPs was constructed via ultrasonic emulsification.The size and zeta potential of the NP were measured by dynamic light scattering(DLS).The morphology of the NP was determined by scanning electron microscopy(SEM).And,the encapsulation efficiency(EE)and loading content(LC)of ICG and PTX were determined.The release behavior of PTX in vitro was assessed by high-performance liquid chromatography(HPLC).Immunofluorescence was applied to assess the immunogenicity of folate on the surface of the FA-PEG-PLGA-PTX@ICG-PFH NPs.The photothermal conversion efficiency of the NPs was detected by thermal imaging camera.The laser activated phase change of the NPs was observed by an inverted microscope.2.Cytotoxicity of the FA-PEG-PLGA@ICG-PFH NPs was assessed by Cell Counting Kit-8(CCK-8).To study the cellular uptake,folate-functionalized NPs(FA-PEG-PLGA@ICG-PFH)and non-folate-functionalized NPs(PEG-PLGA@ICG-PFH)incubated with MDA-MB-231 folate receptor-overexpressing cells,A549 folate receptor-under-expressing cells and MDA-MB-231 cells with antagonized folate receptors.And the cellular uptake was observed under a confocal laser scanning microscopy(CLSM).Results1.Using the ultrasonic emulsification method,FA-PEG-PLGA-PTX@ICG-PFH NP was successfully constructed.The size and the zeta potential of the NPs were measured by Malvern Instruments and determined to be 308±5.82 nm and-22.2±6.4 mV,respectively.Under the SEM,the NPs were generally spherical and dispersed homogeneously.The EE and LC of ICG were(27.15±2.91)% and(1.09±0.12)%.And the EE and LC of PTX were(71.71±7.89)% and(6.69±0.69)%.The HPLC analysis found that the PTX release could be accelerated by the NIR laser irradiation in vitro.It was demonstrated that the immunogenicity of the folate on the surface of the NPs was preserved.It was detected that the NP had the excellent photothermal conversion ability.And the NPs can be rapidly activated into microbubbles by the NIR laser irradiation,which was validated by the microscope.2.The CCK-8 showed that the cell viability of all the treated groups was greater than 80%.The cellular uptake result showed that more folate-functionalized NPs were phagocytosed by MDA-MB-231 folate receptor-overexpressing cells than A549 folate receptor-under-expressing cells.Meanwhile,there were less non-folate-functionalized NPs were phagocytosed by MDA-MB-231 cells.Conversely,as the folate receptors of the MDA-MB-231 cells were preoccupied,the number of folate-functionalized NPs was remarkably decreased in these cells.Conclusion1.A folate receptor-targeted laser-activatable PLGA nanoparticles loaded with paclitaxel/indocyanine green for multifunctional theranostics,FA-PEG-PLGA-PTX@ICG-PFH NPs,was successfully constructed.And its characterization was variously detected.2.The FA-PEG-PLGA@ICG-PFH NPs are eminently biocompatible as a drug delivery vehicle.The cellular uptake result indicates that the folate ligand receptor could play a significant role in mediating the MDA-MB-231 cell efficient phagocytosis of folate-functionalized NPs.PART TWO: THE DUAL-MODALITY PHOTOACOUSTIC/ULTRASOUND IMAGING OF THE FA-PEG-PLGA-PTX@ICG-PFH NANOPARTICLES IN VITRO AND EVALUATION OF THE CHEMO-/PHOTOTHERMAL THERAPY AGAINST MDA-MB-231 CELLSObject 1.Study the imaging ability of FA-PEG-PLGA-PTX@ICG-PFH NPs as the photoacoustic/ultrasound contrast agents in vitro.2.Evaluate the chemo-/photothermal therapeutic effect induced by FA-PEG-PLGA-PTX@ICG-PFH NPs to MDA-MB-231 cells in vitro.Method 1.Different concentration of FA-PEG-PLGA-PTX@ICG-PFH NP solutions,FA-PEG-PLGA@PFH NP solution and PBS were added to phantom agar gel models for photoacoustic(PA)imaging.And the PA signal was analyzed.To evaluate the NP as an ultrasound imaging contrast agent,the NP solution was added to the phantom gel model and irradiated with a NIR laser(808 nm).And ultrasound images were taken by an ultrasonography during irradiation.The quantitative results were measured with US imaging analysis software.2.To evaluate the chemo-/photothermal therapy against MDA-MB-231 cells,experiments were carried out in seven groups(i: FA-PEG-PLGA-PTX@ICG-PFH NPs with laser irradiation;ii: PEG-PLGA-PTX@ICG-PFH NPs with laser irradiation;iii: FA-PEG-PLGA-PTX@ICG-PFH NPs without laser irradiation;iv: FA-PEG-PLGA@ICG-PFH NPs with laser irradiation;v: PTX only;vi: laser only;vii: control).After 24 h incubation,cell viability was measured by CCK-8 assay.Results 1.As a photoacoustic contrast agent,FA-PEG-PLGA-PTX@ICG-PFH NP showed an excellent ability.And PA signal strengthened linearly with an increase in the concentration of the FA-PEG-PLGA-PTX@ICG-PFH NPs.Whereas,Negligible PS signal appeared in the PBS group and FA-PEG-PLGA-PTX@PFH NPs group.With the NIR laser irradiation,the signal of FA-PEG-PLGA-PTX@ICG-PFH NP solution increased in B-mode and Contrast-enhanced ultrasound(CEUS)mode.Especially,the enhancement effect was much more evident in CEUS mode.2.The cell viability was lowest in the FA-PEG-PLGA-PTX@ICG-PFH NPs with laser irradiation group(P<0.05).And there was no significant difference between laser only group and control group(P>0.05).Conclusion1.The FA-PEG-PLGA-PTX@ICG-PFH NP showed the excellent enhancement ability to the dual-modality photoacoustic/ultrasound imaging.It is verified that the NP has the potential be a dual-modality contrast agent for photoacoustic/ultrasound imaging.2.Photothermal therapy(PTT)combined chemotherapy induced by folate-functionalized FA-PEG-PLGA-PTX@ICG-PFH NPs achieved the optimal therapeutic effect to MDA-MB-231 cells in vitro.Meanwhile,the safety of NIR laser irradiation was verified.PART THREE: THE DUAL-MODALITY PHOTOACOUSTIC/ULTRASOUND IMAGING OF THE FA-PEG-PLGA-PTX@ICG-PFH NANOPARTICLES IN VIVO AND EVALUATION OF THE CHEMO-/PHOTOTHERMAL THERAPY TO THE MDA-MB-231 TUMOR-BEARING NUDE MICEObject 1.Evaluate in vivo biocompatibility and biodistribution of the FA-PEG-PLGA-PTX@ICG-PFH NPs.And study the imaging ability of the NPs as the photoacoustic/ultrasound contrast agents in vivo.2.Evaluate the chemo-/photothermal therapeutic effect induced by FA-PEG-PLGA-PTX@ICG-PFH NPs to the MDA-MB-231 tumor-bearing nude mice.Method 1.First,the biocompatibility of FA-PEG-PLGA-PTX@ICG-PFH NPs was detected in vivo.Seven days after injection of FA-PEG-PLAG-PTX@ICG-PFH NP solutions at different doses or saline(control),the liver function,renal function and routine blood tests were detected.Then,the in vivo biodistribution of the folate-functionalized FA-PEG-PLGA-PTX@ICG-PFH NPs and non-folate-functionalized PEG-PLGA-PTX@ICG-PFH NPs were detected by in vivo fluorescence imaging system.Finally,the folate-functionalized NP solution and non-folate-functionalized NP solution were administered by intravenous injection to the tumor-bearing nude mice,respectively.And images of tumor region were captured by the PA imaging system and US imaging system.2.MDA-MB-231 tumor-bearing node mice were randomly divided into seven groups and underwent different treatments(i: FA-PEG-PLGA-PTX@ICG-PFH NPs with laser irradiation;ii: PEG-PLGA-PTX@ICG-PFH NPs with laser irradiation;iii: FA-PEG-PLGA-PTX@ICG-PFH NPs without laser irradiation;iv: FA-PEG-PLGA@ICG-PFH NPs with laser irradiation;v: PTX only;vi: laser only;vii: control).And these treatments were evaluated in multi-aspects to study the effect on the tumor growth.Results 1.There were no significant differences among the FA-PEG-PLAG-PTX@ICG-PFH NP solutions administered groups and control group in liver function,renal function and routine blood tests(P>0.05).The fluorescent signal of the tumor region peaked at 1 h post-injection in folate-functionalized NPs group.In particular,the signal of the tumor region was higher and longer in the FA-PEG-PLGA-PTX@ICG-PFH group than in the PEG-PLGA-PTX@ICG-PFH group(P<0.05).And for the dual-modality photoacoustic/ultrasound imaging of the tumor,the folate-functionalized NPs were superior to the non-folate-functionalized NPs.2.Evaluation from Multi-aspects showed that the FA-PEG-PLGA-PTX@ICG-PFH NPs with laser irradiation group achieved the optimal therapeutic effect(P<0.05).And there was no significant difference between laser only group and control group(P>0.05).Conclusion1.The FA-PEG-PLGA-PTX@ICG-PFH NPs are biocompatible in vivo.The accumulation of the folate-functionalized NPs peaked at 1 h post-injection in the tumor region,which were more and longer than that of non-folate-functionalized NPs.The FA-PEG-PLGA-PTX@ICG-PFH NP as a dual-modality contrast agent for photoacoustic/ultrasound imaging shows an excellent ability to tumor targeted imaging in vivo.2.PTT combined chemotherapy induced by folate-functionalized FA-PEG-PLGA-PTX@ICG-PFH NPs achieved the optimal anti-cancer therapeutic effect in vivo.Meanwhile,the safety of NIR laser irradiation was further verified in vivo.