Novel WS₂-Ga³⁺-PEG-peptide Nanosheets Binding Glypican-3 For In Vivo Bimodal MR/Photoacoustic Imaging Guided Photothermal Therapy

Research backgroundHepatocellular carcinoma?HCC?is one of the most common malignancies,and the third most frequent cause of cancer-related deaths worldwide.For the character of high invasive ability and latent clinical manifestation of HCC,most of patients are diagnosed in the middle or late stage without surgery opportunities,leading to a poor prognosis.Current diagnosis method contains serological examination,traditional imageological examination and biopsy examination,but all of them have their own deficiency.Prevalent tumor marker alpha-fetoprotein?AFP?test of blood sample which is employed as a clinical screening test lacks sufficient sensitivity and specificity.Traditional enhanced spiral Computed Tomography?CT?scanning and Magnetic Resonance Imaging?MRI?also lacks specificity,because comparison of liver tumor enhancement patterns at various time points after intravenous contrast administration is limited.And in some cases,they are enable to distinguish between liver cirrhosis nodule and small hepatocellular carcinoma.As to DSA and biopsy test,which are invasive examination,limited by time consuming and increase tumor-seeding risks.A sensitive and specific approach therefore is required urgently to satisfy early diagnosis HCC,improve the prognosis and reduce morbidity.Recently,Glypican-3?GPC3?is found as a biomarker and therapeutic target to HCC,according to the study,72%of HCCs express GPC3,whereas this protein was not detected in normal hepatocytes,cirrhotic liver or benign lesions.Contrast to another typical HCC serum marker AFP,which has higher false positive rate?14.3%to 35.0%?in benign liver diseases,some studies recommended combination of circulating GPC3,GPC3 mRNA and AFP to improve the accuracy of HCC diagnosis,and performed an ideal outcome.In clinical biopsy test,immunohistochemistry study with GPC3 has been used to confirm HCC pathological diagnosis when malignant nature of liver lesion is hard to identify with benign lesion.GPC3 is a member of the glypican family of proteoglycans that are attached to the cell surface by a glycosyl-phosphatidylinositol anchor.It has been shown that mutations in GPC3 or knockdown of its function can inhibit HCC growth,demonstrating the significant role of GPC3 in HCC development.Furthermore,GPC3 protein anchors at the cell membrane so that it is more effective to specifically combine by nanoparticles.As a result,administrating a probe which specifically binds with the GPC3 protein,can target the HCC cell exclusively in the procedure of diagnosis and therapy.In recent study,anti-GPC3 antibody and aGPC3-F?ab'?2 fragments were utilized to target GPC3.Though the monoclonal antibody binds to the GPC3 high affinity,the larger size of the anti-GPC3 antibody and aGPC3-F?ab'?2 fragments would lead a bad effect such as suboptimal imaging pharmacokinetics,poor tumor penetration and increase immunogenicity.To address this problem,we utilized a peptide identified by screening a phage display peptide libary which has been derrmonstrated its specific and affinity ability to GPC3 to perform our study regarding target GPC3 protein.Peptide-based molecular imaging probes have the advantages of low molecular weight,ease of modification and low cost for scale up,and the phage display peptide library has been proven to be an effective tool to identify protein-binding ligands by screening with purified target.Transition metal dichalcogenides?TMDCS?,which constructs with metal atoms and chalcogen atoms,have been explored in many different fields.And it is promising for applications in biomedicine field.Certain types TMDCS such as WS₂ and Bi2Se3 can be utilized as a computed tomography?CT?agents,meanwhile by attaching a magnetic element to acquire magnetic resonance imaging?MRI?.Photoacoustic imaging?PAI?is a novel technology that largely overcomes the spatial resolution and depth limits of traditional optical imaging.It is a hotpot that various PA agents has been developed for medical research,TMDCs also have potential to be an ideal PA agents.Herein,utilizing the paramagnetic property of Ga³⁺ and strong near-infrared region?NIR?absorbance of WS₂,we synthesized a MRI and PAI bimodal nanosheets.For acquiring satisfied MR and PA imaging effects at the same time,we fabricated the new nanosheets by bottom-up method,we used this method to dope with Ga element in our interest ratio..Meanwhile,peptide that specific targets to GPC3 protein was adhered to the nanosheets,that constituted a positive targeting HCC nanosheets WS₂-Ga³⁺-PEG-peptide.Photothermal Therapy?PTT?usually employs NIR light-absorbing agents to generate heat from optical energy,causing thermal ablation of cancer cells.TMDCs was first demonstrated as a PTT agents for in vitro-photothermal-killing of cancer cells in vitro at the cell culture level in 2013.Following that,successive research reports concerning TMDCs for PTT ablation of cancer in vitro and in vivo have been published.However,the in vivo applications of TMDCs,particularly with conjugating certain tumor ligand to increase specific targeting ability remain to be explored,the PTT effects after systemic administration in animal remain to be evaluated.Materials and Methods.Synthesis and modification of the nanosheetslmmol WC16 and GaCl3 mixture composed at 7:3 ratio was mixed with 20ml oleylamine and 10ml 1-octadecene in a 50ml three neck flack.The flack was heated to 150'C to remove the water and oxygen with magnet stirring in nitrogen protection in 30 minutes.After then the solution temperature was risen up to 300?,and kept that for 30 minutes with nitrogen protection.Sulfur solution had been prepared before that 2mmol S powder dissolved in 5ml olyalamine.Herein added it slowly into 300?solution participating in reaction then kept it for another 30 minutes.After the solution was cooled down to room temperature,approximately 30ml anhydrous ethanol was added to precipitate the nanosheets.Hexane was employed to disperse the nanosheets and wash out the oleylamine.For the PEGylation of nanosheets,the nanosheets in hexane solvent were add to water-containing excess of NH2-PEG-COOH in a beaker and stirred overnight using magnetic stirring.The nanosheets were collected by centrifuging the solution to remove the supernatant,the nanosheets(WS₂-Ga³⁺-PEG)were soluble in water.To link the peptide to WS₂-Ga³⁺-PEG,the nanosheets and peptide were mixture in a molecular ratio of 2:1 and oscillated overnight.WS₂-Ga³⁺-PEG-peptide was collected after centrifuging the mixture to remove the supernatant.Characterization of the probeThe microstructure was obtained by TEM?JEM 1011,JEOL Co.Ltd,Japan?.The absorbance spectra of the probe was obtained by using UV-Vis-NIR spectrophotometer.Utilizing the specific spectra,the stability in FBS?fetal bovine serum,Gibco Corporation,NY,USA?,phosphate-buffered saline?PBS?and culture media?DMEM,Gibco Corporation?was performed by testing the spectra variation after incubating in serum at different time points?0,6,12,24,48 hours?.The MR signal intensity of the different concentrations?0,1,2,3,5 mg/ml?of probe was measured in phantom.Likewise the PA signal intensity of the nanosheets was measured though injecting different concentrations?0.1,1.1 2.2 5.5 mg/ml?WS₂-Ga³⁺-PEG-peptide su'bcutaneously in one mouse.To explore the PTT efficiency of WS₂-Ga³⁺-PEG-peptide,different concentration of nanosheets?100d?g/ml,300?g/ml,500?g/ml?was exposed to 2W/m2 intensity 808nm laser for 5 minutes,the temperature variation was recorded by IR thermal camera?Ti27,FLUCK,WA,USA?.Cell culture experimentHepG2 murine hepatocarcinoma cell line was cultured in Dulbecco's Modified Eagle Medium?Gibco Corporation?at 37? in a 5%C02 atmosphere.For testing the toxicity,cells were seeded in the 96 wells plate for culturing 24 hours with density of 1×104 cells per well,after that different concentration of WS₂-Ga³⁺-PEG-peptide were added into the 96 wells for incubating another 24 hours.Then the CellTiter 96???AQueousOne solution reagent?Promega Biotech Corporation.Ltd,Beijing,PR China?were incubated for 4 hours based to the standard protocol to determine the numbers of viable cells,measured with multi-mode microplate reader?Biotec SynergyTM HT,USA?.For testing the uptake of the probe,WS₂-Ga³⁺-PEG-peptide and PBS were incubated with HepG2 cells respectively for certain time.Then washed with PBS,we acquired the quantified uptake data of the nanosheets with multi-mode microplate reader at different time point after co-incubation utilizing the specific NIR spectrum of the WS₂-Ga³⁺-PEG-peptide.For in-vitro PTT assay,HepG2 cells cultured in 96 wells were exposed to 808nm NIR laser?Hi-Tech Optoelectronics Co,CHINA?in different power density?0.4,0.8,1.2,1.6,2.0 W/m2?24 hours after incubating with PBS and 100?g/ml WS₂-Ga³⁺-peptide respectively.MTT assay also conducted after PTT and stained with Calcein AM for 15 minutes,imaged by fluorescence microscope?Leica M205F,Leica Microsystems Inc,Buffalo Grove,Germany?to reveal the cell viability.Tumor modelBalb/c nude 5-6 weeks male mice were obtained from Vital River Corporation?Beijing,China?.The subcutaneous HepG2 tumor bearing mice were formed by subcutaneous injection of 1×106 HepG2 cells suspension onto the back of each mouse.And the orthotopic HepG2 liver tumor bearing mice were performed by injecting amount of 1×106 cells in 30?-50?l solution mixed with matrigel?Basement Membrane Matrix,Growth Factor Reduced,Phenol Red-free,BD Biosciences,NJ,USA?into hepatic subcapsular place space of liver.HepG2 cells bearing mice were randomly divided to 2 groups.Control Group:i.v.injection with PBS?n=5?.Test Group:i.v.injection with WS₂-Ga³⁺-PEG-peptide?n=5?.To explore the biodistribution of WS₂-Ga³⁺-PEG-peptide we conjugated the fluorescent dye Cy5.5 with the probe and monitored the changes in the whole-body image using the IVIS system?IVIS Spectrum,Perkin Elmer Co.Ltd,AZ,USA?after the intravenous administration of nanosheets into the tumor-bearing BALB/c mice.To evaluate the diversity in tumor homing property of WS₂-Ga³⁺-PEG-peptide and WS₂-Ga³⁺-PEG in vivo,both the nanosheets were administrated to the mice and their biodistribution was determined.MRI and PAI in-vivoT1-weighted MRI imaging was conducted by BioSpec 94/30 USR?BRUKER Corporation,Germany?with a small animal coil.T1-weighted MRI was using the following parameters:slice thickness 0.8mm,slice spacing 0.1mm,matrix 256*256,FOV 2.2cm*2.2cm.PA imaging was performed by using MOST inversion 128?iThera medical company,Munich,German?.HepG2 cells bearing mice were i.v injected with WS₂-Ga³⁺-PEG-peptide?200?l,2mg/ml?24 hours before both MR and PA scan.The quantified MR T1-weighted relaxivity and PA signal intensity value in tumor region is compared between test group and control group.In vivo PTT therapy24 hours after i.v.injection WS₂-Ga³⁺-PEG-peptide?200?l,2mg/ml?,all mice were performed PTT with intensity of 2W/m2 for 5 minutes by 808nm NIR laser,while same dose of PBS were injected in control groups mice.The therapy temperature during PTT procedure was measured by IR thermal camera?TI26,FLUCK Corporation?.Tumor lengths and widths were measured by calipers each 2 days calculating the tumor volume as:Volume=length*?width?2/2.In addition,10 days after treatment,tumor tissues in PTT group and post-ablation tissues in control group were dissection to make paraffin sections for H and E staining pathological examination applied standard protocol and then observed with microscope?Leica?.Results and DiscussionThe physical properties of WS₂-Ga³⁺-PEG-peptide was showed in?figure l?.The absorbance spectra was high ranged from 700nm-1000nm wave lengths in NIR spectra,the absorbance summit was approximate at 954nm?Figure 1A?,and following incubating with FBS,PBS and culture medium for 48 hours,the absorbance spectra of the probe still persisted in the original curve and in the stable absorbance unit,indicating that the probe physical property was stable in FBS,PBS and culture medium,thus presenting a favorable biological compatibility what can be employed in subsequent in-vivo experiment.The TEM showed the typical WS₂-Ga³⁺-PEG-peptide with diameters of 100nm?Figure 1D?.For the high absorbance in a wide spectral range in NIR,the probe was such potential to be a photothermal agent that it was irradiated by 808nm laser.An obvious concentration depend temperature increases was observed,the real-time temperature data were recorded by IR thermal camera?Figure 1C?.The enhanced temperature of the WS₂-Ga³⁺-PEG-peptide in highest concentration solution?50?g/ml?climbed up nearly 40 C from normal room temperature within a short 5 minutes.In comparison to PBS exposed to same photothermal condition,the measured temperature maintained at room temperature.It proved that WS₂-Ga³⁺-PEG-peptide was an excellent PTT agent.PAI is a new molecular imaging technique obtained significant interest and is expected to broad applications in biomedical treatment,especially prior in tumor diagnosis,due to the enhancement of imaging penetration depth as well as spatial resolution.It provides greater ability to detect haemoglobin,lipids and other light-absorbing agents.So the higher vessel density in the tumor could be revealed as a clearer region,especially when high light-absorbing agents accumulated in tumor.Herein,we investigated the use of WS₂-Ga³⁺-PEG-peptide as a PA agent with its high NIR absorbance.After subcutaneous injecting different concentrations of the nanosheets on the back of one mouse,then PA intensity value were measured and calculated respectively by MSOT.It thought to be more persuadable than measured in certain container because the test was administered in living animal.As?FigurelE?showed,PA signal intensity enhanced sharply with the concentration increase.It demonstrated WS₂-Ga³⁺-PEG-peptide may be a promising contrast agent for PA imaging.Magnetic Resonance Imaging?MRI?is one of the most commonly used imaging tools for clinical tumor diagnosis due to its high resolution.Agents containing paramagnetic Gd3+ are popular as a T1 contrast in MR imaging.To explore the possibility of WS₂-Ga³⁺-PEG-peptide applying as a MRI agent,the T1-weighted relaxivity was measured similarly under different concentrations.An obvious concentration-dependent T1 relaxivity increase showed in?Figure 1F?,WS₂-Ga³⁺-PEG-peptide exhibited excellent imaging effect both in MRI and PAI,it required an optimal ratio of WS₂:Ga³⁺ in WS₂-Ga³⁺-PEG-peptide.If the proportion of Ga³⁺ was too large,it would affect the PAI and PTT effect.On the contrary,if proportion of Ga³⁺ was too small,the MRI effect would not be satisfied.Therefore the result demonstrated we successfully balanced the ratio of W and Ga element in former bottom-up synthesis method,7:3 of W:Ga was thought to be an optimal ratio proven by previous study[4].WS₂-Ga³⁺-PEG-peptide was qualified for a proper bimodal MRI and PAI agent.Combined the both imaging advantages,MRI can used to determine the macroscopic outline and location of the malignancy,and PAI can provide higher spatial resolution in optical way.How to define the border of HCC during operation remains a medical challenge.Since the obvious shortage of intraoperative MRI than optical methods,such as relative strict instruments requirement,administration of high dose of Gd may result in false-positive enhancement,PAI will be a promising surgery navigation tool to apply in HCC.As a result,the WS₂-Ga³⁺-PEG-peptide agent is promising to apply in surgery navigation,further in vivo examination is expected.Meanwhile WS₂-Ga³⁺-PEG-peptide is an excellent PTT agent,then it can focus diagnosis and therapy in the same time,pro.vide much diversified therapy method for clinical choice.To explore the cytotoxity of WS₂-Ga³⁺-PEG-peptide,standard methyl thiazolyl tetrazolium?MTT?assay was carried out to determine the relative viability of hepatocarcinoma cell line HepG2.After incubated with the probe at different concentrations,,no significant cytotoxity was observed at the highest concentration 0.1mg/ml?Figure 2A?.For PTT experiment,various power densities of 808nm laser were carried out under same concentration?0.1 mg/ml?of WS₂-Ga³⁺-PEG-peptide which incubated with HepG2 cells 24 hours.The relative cell viability decreased starting at 1.6 W/m2 power density,especially when employed to 2W/m2?Figure 2B?.A similar result was also observed in Calcein AM fluorescent staining method.The green light spots showed in Figure 2C represented the live cells,the density of live cells apparently come to scarcity with increase of laser power density.It demonstrated that WS₂-Ga³⁺-PEG-peptide as a PTT agency is effective,and the effective laser power density should more than be 1.6 W/m2,especially high up to 2.0 W/m2.In WS₂-Ga³⁺-PEG-peptide uptake test,WS₂-Ga³⁺-PEG-peptide was incubated with HepG2 cells.As shown in Figure 2D,WS₂-Ga³⁺-PEG-peptide absorbance intensity,tested by multi-mode microplate reader,was significantly higher than H2O from the beginning after co-incubation for one hour,and the gap got larger with incubating time growing.This demonstrated that the HepG2 cells had uptaken WS₂-Ga³⁺-PEG-peptide successfully,and the uptaken amount of probe was time-dependent.To explore the biodistuibution of WS₂-Ga³⁺-PEG-peptide and to compare it with WS₂-Ga³⁺-PEG,the changes in the whole-body image were monitored using the IVIS system after the intravenous administration of the nanosheet.Strong fluorescent signals were emitted from the body 10 min after administration in the nanosheet administrated mice.After 1h,the observed fluorescent signals began to shift from the liver to tumor region and therefore the tumor tissue was distinguished from peripheral normal tissues.However,the fluorescent signals in the WS₂-Ga³⁺-PEG-peptide-administrated mice sustained significantly longer and stronger during the 48h observation period.This indicates that WS₂-Ga³⁺-PEG-peptide has a tumor accumulation property stronger than that of WS₂-Ga³⁺-PEG,due to its ability to target GPC3.Utilizing the optical and magnetic properties of WS₂-Ga³⁺-PEG-peptide,a bimodal MR/PA imaging for HepG2 tumor bearing mice was conducted.Balb/c nude mice bearing HepG2 subcutaneous tumor were i.v injected with WS₂-Ga³⁺-PEG-peptide?2mg/ml 200?l?,then imaged 24 hours after injection by BRUKER BioSpec 94/30 USR MR instrument and iThera MOST inversion 128 PA instrument.A high tumor contrast was observed after WS₂-Ga³⁺-PEG-peptide injection both in MR and PA imaging?Figure 3A and 3B?.The MR signal intensity value increased from 5065.8±517.3 to 9375.6±528.2,and the PA signal intensity value increased from 69.6±1.75 to 142.4±3.3.The quantified data was shown in Figure 3C.It demonstrated in vivo imaging level that WS₂-Ga³⁺-PEG-peptide possessed favorable targeting capacity to HepG2 tumor after systemic administration and it promising come to be an ideal contrast agent for MRI and PA imaging.MR inspection was recognized as first imaging diagnosis choice to HCC in clinical practice presently.Thurs we also conducted HepG2 orthotopic tumor-bearing mice MR imaging experiment?n=5?.Likewise 2mg/ml WS₂-Ga³⁺-PEG-peptide 200t,g was i.v injected 24 hours before MR scan,most of orthotopic tumor lesion T1-weighted MRI showed no distinctions between pre-injecting WS₂-Ga³⁺-PEG-peptide and after injection.Except for one case as shown in Figure 4,as small size as approximate 1cm diameter orthotopic liver tumor could be observed much clearer in MR imaging after WS₂-Ga³⁺-PEG-peptide injection.But it could not be inspected before WS₂-Ga³⁺-PEG-peptide injection,owning to the early stage tumor was so small in size.As WS₂-Ga³⁺-PEG-peptide was more potent to adhere to GPC3 protein which anchors on He-pG2 cells,the tumor might uptake more nanosheets that contribute a stronger T1 weighted MRI signal.So it demonstrated again in orthotopic liver tumor model that WS₂-Ga³⁺-PEG-peptide successfully targeted HepG2 cells liver tumor.Furthermore,since at the early stage of HCC,the tumor size was so small and tumor differentiation level was relative not beyond to the normal or cirrhotic liver tissue,thus positive rate of ordinary MR inspection result might decrease in those cases.The inspection sensitivity may decreased from 82%-93%to 31%-69%,in smaller tumors less than 20mm.In our experiment,we could inspected small tumor approximate to 1cm after applying the novel nanosheets,so it probably promising to inspect early small HCC according to the result contrast to the ordinary MR imaging.However,the sample amount in our experiment was not large enough to convince,future more experiments are required to focus on WS₂-Ga³⁺-PEG-peptide applying on this early stage HCC.After the administration of WS₂-Ga³⁺-PEG-peptide into the HepG2 subcutaneous tumor-bearing mice,the location and spatial structure of the tumor appear to be clear and precise in the MR and PA images.Supported by the ideal imaging effect,high tumor accumulation,high NIR absorbance and strong PTT efficacy of WS₂-Ga³⁺-PEG-peptide nanosheets in vitro experiments,we then carried out the further in vivo PTT experiment.Subcutaneous HepG2 tumor bearing mice were divided into 2 groups.Control Group?n=5?and Test Group?n=5?were injected with PBS and WS₂-Ga³⁺-PEG-peptide respectively,then exposed to 808nm NIR laser at the power density of 2W/m2 for 5 minutes.For the test group,the tumor completely eliminated 24 hours after PTT leaving black scars at the surface of tumor site?Figure 5a?,Under estimated volume variation of subcutaneous tumor with calipers in the following 10 days,no tumor recurred in our observation period?Figure 5b?.Antitumor efficacy was assessed by H&E staining pathological slices.Tumor cells from test group showed severe destruction as evidence by changed cell shapes,increased vacuoles,condensed nuclei observed from micrograph?Figure 5c?.While for PBS injected mice,tumor volumes increased gradually day by day,and H&E staining slices showed no remarkable tumor damage signs.The results suggested that an excellent PTT efficacy of WS₂-Ga³⁺-PEG-peptide,illuminated that it was a powerful agent for in vivo photothermal ablation of HCC.In summary,we have successfully developed a new photothermal theranostic agent based on TMDCs which possess specific targeting capacity to HCC,by conjugating HCC membrane protein GPC3 homing ligand on the nanosheets.It is found that WS₂-Ga³⁺-PEG-peptide exhibit favorable compatibility in physiological environment and show no observable toxicity in vitro and in vivo experiment.In addition,it can serve as an excellent MRI and PA imaging agent,MRI imaging is used to determine the macroscopic outline of the tumor,PA imaging can further provide higher spatial resolution in optical way,and distribution information of nanosheets.In vivo PTT experiment,WS₂-Ga³⁺-PEG-peptide showed as a high effective photothermal agent,which enabled excellent NIR-induced tumor ablation effect.It is promising come to be an ideal nano-material treating on HCC diagnosis and therapy.