The Experimental Study Of Prostate Cancer Imaging Using PSMA Targeting Ultrasound-Magnetic Resonance Imaging Dual Modality Contrast Agents

Objective: The prostate specific membrane antigen?PSMA?is broadly overexpressed on prostate cancer?PCa?cell surfaces.The aim of this study is to develop a dual modality contrast agents of Fe₃O₄ loaded polypeptide-PLGA multifunctional nanobubbles?Fe₃O₄/Polypeptide-PLGA MNBs?encapsulated SF6 gas for ultrasound/magnetic resonance imaging?US/MRI?multi-modality prostate cancer targeted imaging.Method: Fe₃O₄/Polypeptide-PLGA MNBs that show superparamagnetic properties were prepared by a modified double emulsion method.Characterization,in vitro target experiment,in vitro US/MRI imaging experiment and in vitro toxicity experiment were employed to study the stability,specific binding ability,dual modality imaging ability and safety of the contrast agents.In in vivo studies,the optimal concentration of the contrast agents was determined.In in vivo dual-modality imaging studies,MRI was performed before and 2h,6h,12 h,24h post-injection.Relative signal enhancement?RSE?was employed to study the T2 signal change of tumors following the injection of contrast agents.In vivo ultrasound imaging was performed immediately after the injection of contrast agents and AT,TTP and PI was calculated for quantitative analysis.The biodistribution and in vivo toxicity of the contrast agents was further evaluated in nude mice injected with Fe₃O₄/Polypeptide-PLGA MNBs.Result:The average size of the MNBs was 415 nm with Zeta potential of-6.11 m V,spherical shaped,uniformly distributed without aggregation.The Fe₃O₄ particles were successfully loaded and the MNBs possessed superparamagnetic characteristic.Fe₃O₄/Polypeptide-PLGA MNBs offered specific binding with LNCa P cells in vitro,provided positive contrast enhancement on ultrasound images and negative contrast enhancement on T2 MRI images.The contrast agents showed no obvious toxicity in vitro.In in vivo studies,the optimal concentration of the Fe₃O₄/Polypeptide-PLGA MNBs was 12mg/ml.For LNCa P tumor bearing mice injected with Fe₃O₄/Polypeptide-PLGA MNBs,quantitative analysis of MRI T2 images demonstrate that there was obvious increase of RSE post-injection?41.0±1.9%,28.2±6.5%,21.4±5.0% and 16.2±3.6% at 2h,6h,12 h and 24h?,significantly higher than the rest three groups.For in vivo ultrasound imaging,the PI in LNCa P tumors following the injection of Fe₃O₄/Polypeptide-PLGA MNBs was significantly higher than the other three groups?8.9±1.7d B verus 5.6±1.7d B,6.9±1.2d B,7.0±1.4,P<0.05?.Further Prussian blue studies demonstrated the specific binding of LNCa P prostate cancer tissues with Fe₃O₄/Polypeptide-PLGA MNBs.The MNBs was breakdown and metabolized mainly in the reticuloendothelial system and showed no obvious toxicity in vivo.Conclusion: Fe₃O₄/Polypeptide-PLGA MNBs could specifically enhance both ultrasound and MRI images via binding with PSMA expressed on prostate cancer cells.Passage one The in vitro study of Fe₃O₄ loaded polypeptide-PLGA multifunctional nanobubblesPart one The fabrication and characterization of Fe₃O₄ loaded polypeptide-PLGA multifunctional nanobubblesPurpose: To develop a dual modality contrast agents of Fe₃O₄ loaded polypeptide-PLGA multifunctional nanobubbles?Fe₃O₄/Polypeptide-PLGA MNBs?encapsulated SF6 gas for ultrasound/magnetic resonance imaging?US/MRI?dual-modality prostate cancer targeted imaging.Methods: Magnetite loaded Polypeptide-PLGA multifunctional nanobubbles?Fe₃O₄/Polypeptide-PLGA MNBs?that show superparamagnetic properties were prepared by a modified double emulsion method.Dynamic light scattering was employed to study the size distribution and zeta potential of the MNBs.Transmission electron microscope,scanning electron microscope and optical microscope was employed to study the morphology of the MNBs.X-ray diffraction,energy dispersive spectrometer and atomic absorption spectroscopy was employed to confirm the successful loading of iron.Vibrating sample magnetometer was used to study the magnetic properties of the MNBs.Results: The average size of the MNBs was 415 ± 11.4 nm with polydispersity index of 0.142.The particle size distributions do not significantly change in 7 days.The Zeta potential of the MNBs is-6.11 m V.Microscopic images demonstrated that the MNBs exhibited a smooth and spherical morphology,uniformly distributed without aggregation.High resolution Transmission electron microscope showed that the Fe₃O₄ nanoparticles were homogeneously distributed in the contrast agents.The successful loading of Fe₃O₄ particle was confirmed using X-ray diffraction and energy dispersive spectrometer.Atomic absorption spectroscopy demonstrated that the iron content was 12.8ug/mg.Vibrating sample magnetometer indicated that the prepared MMBs possessed the superparamagnetic characteristic.Conclusions: In this study,we successfully synthesized the Fe₃O₄/Polypeptide-PLGA MNBs for dual-mode US/MR imaging of prostate cancer.The characterization analyses indicated that Fe₃O₄/Polypeptide-PLGA MNBs was suitable for US/MR dual modality imaging.Part two The in invitro binding study of Fe₃O₄ loaded polypeptide-PLGA multifunctional nanobubblesPurpose: To study the in vitro specific binding ability of polypeptide-PLGA multifunctional nanobubbles?Fe₃O₄/Polypeptide-PLGA MNBs?.Materials and methods: The in vitro specific binding ability of the polypeptide K?FITC?]CQ[K?Dde?]HHNYLC was first investigated using immunofluorescence.After that,the specific binding experiments of the Fe₃O₄/Polypeptide-PLGA MNBs were divided into four groups: LNCa P cells incubated with Fe₃O₄/Polypeptide-PLGA MNBs,LNCa P cells incubated with Fe₃O₄-PLGA MNBs,antibody blocked LNCa P cells incubated with Fe₃O₄/Polypeptide-PLGA MNBs and PC3 cells incubated with Fe₃O₄/Polypeptide-PLGA MNBs.Prussian blue staining,in vitro magnetic resonance imaging?MRI?,cell attachment studies and immunofluorescence were employed to study the specific binding ability of the Fe₃O₄/Polypeptide-PLGA MNBs.Results: Immunofluorescence confirmed the specific binding ability of the polypeptide K?FITC?]CQ[K?Dde?]HHNYLC.In specific binding experiments of the Fe₃O₄/Polypeptide-PLGA MNBs,Prussian blue staining found the deposition of iron around LNCa P cells when incubated with Fe₃O₄/Polypeptide-PLGA MNBs.No iron deposition was found in the other three groups.In vitro MRI demonstrated the significant lower T2 signal in LNCa P cells incubated with Fe₃O₄/Polypeptide-PLGA MNBs than the other groups.The SNR of the experimental group was 624.6±18.7,significantly lower than the control groups?818.6±23.3,814.4±10.9,790.2±23.7,P<0.05?.Cell attachment studies and immunofluorescence studies confirmed the specific binding of Fe₃O₄/Polypeptide-PLGA MNBs with LNCa P cells.Conclusions: Fe₃O₄/Polypeptide-PLGA MNBs offered specific binding with LNCa P cells in a PSMA dependent manner.Part three The in vitro dual modality imaging study of Fe₃O₄ loaded polypeptide-PLGA multifunctional nanobubblesPurpose: To study the in vitro dual modality imaging ability of polypeptide-PLGA multifunctional nanobubbles?Fe₃O₄/Polypeptide-PLGA MNBs?.Materials and methods: Fe₃O₄/Polypeptide-PLGA MNBs with different concentration were prepared and deionized water was served as controls.Baseline gray-scale imaging modality was used to evaluate the ultrasound imaging ability of Fe₃O₄/Polypeptide-PLGA MNBs.T2 magnetic resonance imaging?MRI?was performed to study the MRI imaging ability of the contrast agents.Carr–Purcell–Meiboom–Gill pulse sequence was employed in order to determine the transverse relaxation time of MNBs.The correlation between Fe concentration and transverse relaxation time was evaluated and transverse relaxation rate?R2?of the MNBs was calculated.Results: In in vitro ultrasound imaging studies,the Fe₃O₄/Polypeptide-PLGA MNBs presented as hyperechoic on grayscale imaging modality,and the echogenicity was increased with the concentration of the MNBs.By contrast,the deionized water was anechoic on grayscale imaging modalities.On T2 weighted MRI,Fe₃O₄/Polypeptide-PLGA MNBs presented as hypo-signal.There was a trend toward increasing Fe concentration with decreasing T2 signal intensity.In comparison,the deionized water exhibited high signal intensity on T2 images.A significant linear fit was obtained between the concentration and the 1/T2 with R2 = 46.58 m M-1s-1.Conclusions: Fe₃O₄/Polypeptide-PLGA MNBs were able to enhance both ultrasound and MRI images in vitro.Part four The in invitro toxicity study of Fe₃O₄ loaded polypeptide-PLGA multifunctional nanobubblesPurpose: To study the in vitro toxicity of polypeptide-PLGA multifunctional nanobubbles?Fe₃O₄/Polypeptide-PLGA MNBs?.Materials and methods: Fe₃O₄/Polypeptide-PLGA MNBs with different concentrations were incubated with LNCa P and PC3 cells.After 24 h,48 h and 72 h of incubation,MTT assay was performed to determine the cell viability.Results: The cell viability was over 90% in both LNCa P and PC3 cells incubated with Fe₃O₄/Polypeptide-PLGA MNBs for 24 h.After 48 h and 72 h of incubation,the cell viability remains over 90%.One-way ANOVA revealed that there were no significant differences among the cell viability between different subgroups.Conclusions: Fe₃O₄/Polypeptide-PLGA MNBs showed no significant cytotoxicity effects.Passage Two The in vivo study of Fe₃O₄ loaded polypeptide-PLGA multifunctional nanobubblesPart one The in vivo imaging for the optimal concentration of Fe₃O₄ loaded polypeptide-PLGA multifunctional nanobubblesPurpose: To study the optimal concentration of polypeptide-PLGA multifunctional nanobubbles?Fe₃O₄/Polypeptide-PLGA MNBs?for in vivo imaging.Materials and methods: LNCa P tumor bearing mice were injected with Fe₃O₄/Polypeptide-PLGA MNBs of different concentration.MRI was performed before and 2h,6h,12 h post-injection.Relative signal enhancement?RSE?was employed to study the T2 signal change of tumors following the injection of contrast agents.In vivo ultrasound imaging was also performed immediately after the injection of contrast agents with the optimal concentration obtain in MRI studies.Results: For LNCaP tumor bearing mice injected with Fe₃O₄/Polypeptide-PLGA MNBs,quantitative analysis of MRI T2 images demonstrate that there was obviousincrease of RSE post-injection.The RSE was correlated with the concentration of contrast agents.Contrast agents with concentration of 12mg/ml resulted in highest RSE,the RSE was 19.9 ± 3.4%,34.3 ± 3.6% and 30.9 ± 1.4% respectively at 2h,6h and 12 h post injection.And this concentration was also suitable for in vivo ultrasound imaging since LNCa P tumor presented as homogenous enhancement in contrast enhanced modality.Conclusions: The contrast enhancement of LNCa P tumor on MRI was correlated with the concentration of contrast agent.Contrast agents with concentration of 12mg/ml were suitable for both MRI and ultrasound imaging.Part two The in vivo dual modality imaging of Fe₃O₄ loaded polypeptide-PLGA multifunctional nanobubblesPurpose: To study the in vivo dual modality targeted imaging ability of polypeptide-PLGA multifunctional nanobubbles?Fe₃O₄/Polypeptide-PLGA MNBs?.Materials and methods: The in vivo imaging studies were divided into four groups: LNCa P tumor bearing mice injected with Fe₃O₄/Polypeptide-PLGA MNBs,LNCa P tumor bearing mice injected with Fe₃O₄-PLGA MNBs,antibody blocked LNCa P tumor bearing mice injected with Fe₃O₄/Polypeptide-PLGA MNBs and PC3 tumor bearing mice injected with Fe₃O₄/Polypeptide-PLGA MNBs.MRI was performed before and 2h,6h,12 h post-injection.Relative signal enhancement?RSE?was employed to study the T2 signal change of tumors following the injection of contrast agents.In vivo ultrasound imaging was performed immediately after the injection of contrast agents and AT,TTP and PI was calculated for quantitative analysis.After in vivo imaging,mice were euthanized;tumor tissues were harvested for histological analyses including HE,immunohistochemistry and Prussian blue staining.Results: For LNCa P tumor bearing mice injected with Fe₃O₄/Polypeptide-PLGA MNBs,quantitative analysis of MRI T2 images demonstrate that there was obvious increase of RSE post-injection?41.0±1.9%,28.2±6.5%,21.4±5.0% and 16.2±3.6% at 2h,6h,12 h and 24h?,significantly higher than the rest three groups.The highest RSE was observed 2h post-injection.For in vivo ultrasound imaging,the PI in LNCa P tumors following the injection of Fe₃O₄/Polypeptide-PLGA MNBs was significantly higher than the other three groups?8.9±1.7d B verus 5.6±1.7d B,6.9±1.2d B,7.0±1.4,P<0.05?,no significant difference among the AT and TTP between the four groups.In histopathological analysis,none of the tumors in this study were hemorrhagic;suggesting the T2 signal reduction of the tumor might be attributed to the uptake of Fe₃O₄/Polypeptide-PLGA MNBs.Immunohistochemistry confirmed elevated PSMA expression within LNCa P tumor tissue and negative PSMA expression within PC3 tumor tissue.In LNCa P tumor bearing mice injected with Fe₃O₄/Polypeptide-PLGA MNBs,obvious deposition of iron was observed using Prussian blue staining.In other three control groups,we found no obvious deposition of iron within the tumor tissue.Conclusions: Fe₃O₄/Polypeptide-PLGA MNBs could specifically enhance both ultrasound and MRI images via binding with PSMA expressed on prostate cancer cells.Part three The biodistribution study of Fe₃O₄ loaded polypeptide-PLGA multifunctional nanobubblesPurpose: To study the biodistribution of polypeptide-PLGA multifunctional nanobubbles?Fe₃O₄/Polypeptide-PLGA MNBs?.Materials and methods: ICP-AES?inductively coupled plasma atomic emission spectroscopy?was employed to assess the iron concentration of the main organs of the mice before and 2h,6h,12 h,24h after the injection of Fe₃O₄/Polypeptide-PLGA MNBs.MR T2 weighted imaging was performed to study the signal change of the liver,spleen and kidney before and 2h,6h,12 h,24h after the injection of Fe₃O₄/Polypeptide-PLGA MNBs,and the mean signal intensity?Smean?was compared.The iron deposition in the liver,spleen and kidney was evaluated using Prussian blue staining.Results: The ICP-AES anlysis showed the deposition of Fe₃O₄/Polypeptide-PLGA MNBs mainly in spleen,liver and lung during 2h to 24 h post-injection.The signal intensity was reduced in the liver and spleen after the injection of Fe₃O₄/Polypeptide-PLGA MNBs.No significant signal change was observed in the kidney.The Smean of the liver and spleen was decreased after the injection of Fe₃O₄/Polypeptide-PLGA MNBs,and further Prussian blue staining confirmed iron deposition in these two organs.Conclusions: Fe₃O₄/Polypeptide-PLGA MNBs was breakdown and metabolized mainly in the reticuloendothelial system.Part four The in vivo toxicity study of Fe₃O₄ loaded polypeptide-PLGA multifunctional nanobubblesPurpose: To study the in vivo toxicity of polypeptide-PLGA multifunctional nanobubbles?Fe₃O₄/Polypeptide-PLGA MNBs?.Materials and methods: The nude mice were randomly divided into experimental group and control group.The mice in the experimental group were injected with Fe₃O₄/Polypeptide-PLGA MNBs via retro-orbital vein.The control group did not receive any treatment.The well-being?reflex,alertness,breathing,feeding,feces,and urine?of all mice was observed for 15 days postinjection and body weights of the mice were also recorded.Blood samples were collected 15 days post-injection for liver function and renal function analysis.Upon completion of the blood collection,the mice were sacrificed and main organs were collected for pathologic analysis.Results: The well-being?reflex,alertness,feeding,feces,and urine?of all mice was not changed after the injection of Fe₃O₄/Polypeptide-PLGA MNBs.No significant differences were found of the body weight between from the experimental group and the control group.The liver function and renal function in mice injected with Fe₃O₄/Polypeptide-PLGA MNBs were similar with the control group.Histological analysis demonstrated that all investigated organs of the mice were found to preserve the same structures as those of the control group.Conclusions: Fe₃O₄/Polypeptide-PLGA MNBs showed no significant toxicity in vivo.