Construction Of Thrombus-Targeted Photoacoustic Probe And Specific Enhancement Imaging On Acute Venous Thrombosis

Part Ⅰ:The construction,characterization and identification of molecular probesObjective:The application of near infrared absorption material as exogenous photoacoustic probe has been widely used because it improves PAI sensitivity and deeper tissue penetration.We rationally designed a cyclic Arg-Gly-Asp(cRGD)peptide-modified NIR-absorptive organic semiconducting NPs self-assembled by a sort of amphiphilic perylene-3,4,9,10-tetracarboxylic diimide(PDI)derivatives and successfully realized it as an efficient PA contrast agent for selectively lightening early thrombus in living mice.The purpose of the first part of the experiment is to evaluate the design,characterization and identification of the prepared molecular probe cRGD-PDI NPs.Methods:The synthesis of the perylene imide molecules used to form the nanoparticles is obtained by a four-step chemical reaction.The dibromo perylenetetrahydride is reacted with the long phthalate amine to obtain a symmetrical structure.This step utilizes the long alkyl chain to improve the oil solubility of the peryletetracarboxylic acid,making it more favorable for subsequent modification;followed by the substitution of two bromine atoms in the perylene position of the perylene for the pyrrolidine,so that perylene imide can have near-infrared absorption.In addition,the introduction of polyethylene glycol provides water solubility and biocompatibility to the asymmetric molecule;the nanoparticles are firstly formed by ultrasonication in aqueous solution,and then by chemical bonds of peptide molecules cRGD and cRAD.The outermost layer of cRGD-PDI NPs or cRAD-PDI NPs is a polypeptide molecule with targeted functionality,and a biocompatible PEG molecule whose core is a near-infrared absorption of perylene imide molecule.The molecular probes were subjected to various tests:the UV absorption profiles tested in tetrahydrofuran,the water solubility and stability tested in PBS,the blood stability tested in bovine serum,the half life measured in vivo by PAI,the light stability tested by laser irradiation.MTT assay was used to examine the cytotoxicity of cRGD-PDI NPs.Results:The UV absorption profile of the perylene imide molecule in tetrahydrofuran showed a near infrared absorption peak of 700 nm.The PDI NPs in water showed dark green color and exhibited excellent water-solubility of 10 mg mL-1.cRGD and cRAD were then modified to the PEG surface of PDI NPs using sulfo-SMCC as a linker to investigate its thrombus targeting ability.The obtained cRGD-PDI or cRAD-PDI NPs showed a relatively constant diameter of about 40.0 ± 3.1 nm and 41.2 ± 2.5 nm by TEM.The DLS measurements also revealed that these NPs have a relatively narrow size distribution with a mean size of around 70.3± 2.3 nm and 68.9 ± 3.2 nm(Figure 2c and S13)in phosphate-buffered saline(PBS,0.1 M,pH = 7.4).The size of the PDI NPs observed under TEM is smaller than the DLS result due to their shrinking in the dry state.The ratio of non-cRGD-modified PEG(or non-cRAD-modified PEG)to cRGD-modified PEG(or cRAD-modified PEG)in a NP is calculated from MALDI-TOF MS of about 2:1,indicating a large amount of cRGD or cRAD(10K per NP)on the NP surface that can provide sufficient targeting ability.cRGD-PDI NPs exhibited NIR absorption in aqueous solution with a maximum absorption at 650 nm and a shoulder at 700 nm.The extinction coefficient of PDI NPs at 700 nm was 2.58 × 108M-1cm-1.The photostability of the cRGD-PDI NPs was further tested using a continuous laser irradiation at 700 nm and 8 mJ cm-2 and showed excellent photostability(almost no reduced absorption)during 30 min irradiation.The half-life of the PAI nanoprobe is approximately 22 hours.With the concentration of cRGD-PDI NPs ranging from 0 to 100 μg mL-1,all the cells retained over 90%viability indicating their low cytotoxic effect.Conclusions:Our designed cRGD-modified PDI(cRGD-PDI)NPs present high PA intensity,good stability in light and serum,sufficient blood-circulating half-life,and low cytotoxicity.Therefore,the structure and optical stabilities make cRGD-PDI NPs significant advantages for in vivo PAI.Part Ⅱ:Organic semiconducting nanoparticles as an efficient photoacoustic agent for lightening early thrombus and monitoring thrombolysis in living miceObjective:The purpose of this study was designed to a specific molecular probe targeting to acute venous thrombosis and to evaluate the ability of binding to integrin GPⅡb/Ⅲa in vitro and in vivo and also to monitor the thrombolytic effect of early thrombosis in vivo.Methods:In vitro experiments,platelet plasma was obtained from whole blood by centrifugation using fresh citrate-containing blood and then activated by ADP(20 μmol/mL)and thrombin receptor activating peptide(30 μmol/L)Platelet,the constructed molecular probe was incubated with activated platelets for repeated washing to receive PA spectroscopy.In vivo experiments,5%FeC13 was used to induce the model of non-complete obstructive venous thrombosis in the neck vein of mice.Then,the images of American,MRI and PAI were evaluated,and the morphological changes were observed.PAI was measured after challenge,and its in vivo specific targeting ability was evaluated.Finally,the PAI was used to dynamically monitor the thrombolytic effect by molecular probe.Animals were sacrificed at each stage after the end of the experiment for pathological examination.Results:In vitro specificity binding assessment of cRGD-PDI NPs to activated platelets:After treatment with the green cRGD-PDI NPs,the previous colorless platelets appeared green color while the PBS and cRAD-PDI group still retained colorless.The PA spectra of the related platelets showed that the maximum PA intensity peak of the cRGD-PDI group was at 700 nm which was the same as cRGD-PDI NPs in aqueous solution.We have successfully established FeC13-induced murine non-occlusive model of jugular vein thrombus.In comparison with the normal jugular venous lumen,an ambiguous protrusion on the wall which belongs to the wall-adherent thrombus was found in the lumen with early thrombus by US.In such,luminal blood appeared as black or dark grey and early thrombus as white grey.We could easily visualize the jugular vein by T2-weighted MRI because MRI afforded more detailed anatomic reference information and higher spatial resolution than US.However,no clear evidence for thrombus formation by MRI was observed in the blood vessel having thrombus.The thrombus formation was clearly observed as a loss of PA signal by PAI.In living mice,the PA intensity of early thrombus significantly increased after tail-vein injection of cRGD-PDI NPs,but as controls,no enhanced PA signal were observed in the thrombus via tail vein injection with cRAD-PDI NPs and PBS.When cRGD-PDI NPs(3.33 mg mL-1,0.3 mL)as a contrast agent was tail-vein injected for PAI,we found that in contrast to the enhanced PA signal(4536 ± 121)in the early thrombus,no enhanced PA signal(1056 ± 96)was observed in the old thrombus region during 48 h after NPs injection.Consequently,the monitoring ability of cRGD-PDI NPs to the therapeutic effect of thrombolysis on early thrombus was investigated.In this experiment,cRGD-PDI NPs was first injected in vein until an enhanced PA signal in the thrombus region was observed after 6 h injection.Then 50 000 international units(IU)human urokinase(thrombolytic agents)was injected via the tail vein for thrombolytic therapy.After 6 h NPs injection,the vessel around the lightened thrombus showed irregular margin in PAI.While the related profile gradually turned smooth only after 30 min injection of thrombolytic agents and became similar to the normal vessel after 1 h injection.Different with the remained strong PA signal of untreated early thrombus after 48 h NPs injection,the weakened PA signal after thrombolytic therapy was nearly the same as the normal vessel.Pathological examination showed non-obstructive venous thrombosis model was successfully established.Next,the corresponding result of immunohistochemistry showed that GPIIb/IIIa was expressed abundantly in the luminal thrombus of the test group,less in the blocked thrombus,and seldom in the control jugular vein.H&E staining of liver,spleen and kidney,in which PDI NPs accumulated,showed no apparent damage in cellular structures and no obvious inflammation of major organs.Conclusions:The above results indicated that the constructed organic molecular probe was highly efficient and precisely targeted to GPIIb/Ⅲa receptor of activate the platelet and in vivo PAI successfully "ignited" early venous thrombosis.And the constructed molecular probe cRGD-PDI NPs has unique properties:high PA signal intensity,high biocompatibility and light stability,and high affinity to GPⅡb/Ⅲa receptor of activated platelet,which can be distinguish early venous thrombosis from old thrombus.Combined with the excellent 3D characteristics of PAI,such a lightening PAI effect by cRGD-PDI NPs successfully provided the accurate information including the profile,the size and conformation,and the spatial distribution of early thrombus,which may timely monitor the obstructive degree of thrombus in the blood vessel and the thrombolysis effect.Undoubtedly,the unique PAI property of cRGD-PDI NPs in lightening thrombus paved an efficient way for accurate diagnosis of early thrombus.