Preparation And Properties Of Gadolinium-based Macromolecular MRI Contrast Agents With Targeting Capacity Based On Poly (Glycerol)

Early diagnosis of tumors is extremely vital for controlling and treating the pathological tissues.Magnetic reson ance imaging(MRI),as imaging technology in vivo,has been widely used in clinic due to its high spatio-temporal resolution,and non-invasive and non-radiative procedure.Generally,the difference of magnetic resonance(MR)signal between normal and pathological tissues is small,leading the low sensitivity when diagnosing the tumor with MRI.Therefore,it is necessary to introduce the MRI contrast agents to increase the magnetic resonance signal in lesion location.Nowadays,the most widely used contrast agents in clinic are small molecules,which suffer from low relaxivity(r1),short circulation in blood,insufficient imaging window and no specificity.As a result,these contrast agents could not realize the accurate diagnosis of tumor at early stage.The strategy of conjugating small-molecule gadolinium chelates to macromolecular carriers to fabricate macromolecular contrast agents could significantly increase the rotational correlation time(?R),resulting in efficient improvement of r1,Moreover,the circulation time in blood could be improved due to the increase of molecular size.Based on the enhanced permeability and retention effect(EPR),the macromolecular contrast agents would accumulate in the tumor region,which could efficiently improve the MR signal.Additionally,the signal would be further improved through appropriate modification of targeting ligands on macromolecular contrast agents.Considering all the contents mentioned above,together with the requirements on biocompatibility for macromolecular carriers,poly(glycerol)with excellent biocompatibility was employed to construct the macromolecular carriers for designing several kinds of gadolinium-based macromolecular contrast agents for tumor-targeted imaging,including linear polymer,hyperbranched polymer,polymeric micelles and globular macromolecule.Furthermore,the properties in regard to MRI were systematically investigated,and the main contents were as follows:1.Conjugating gadolinium chelates and folic acid(FA)ligand through the hydroxy groups in linear poly(glycerol),the linear macromolecular MRI contrast agent with targeting specificity was constructed.In details,poly(ethylene glycol)methyl ether(mPEG)was used as an initiator to synthesize the poly(glycerol),and the polymer mPEG-PG was obtained.Furthermore,the hydroxy groups of poly(glycerol)were partially modified to azido groups,which were further reacted with alkynyl modified lysine dendron(alkynyl-G2)through "click chemistry" reaction.Finally,the amine groups were conjugated with Gadolinium Diethylenetriamine Pentaacetic Acid(Gd-DTPA)and FA ligand.In comparison with the commercial MRI contrast agent Gd-DTPA,the macromolecular contrast agent exhibited a higher relaxivity of 7.04 mM-1s-1.Simultaneously,there were no obvious cytotoxicity and toxicity in vivo.Moreover,the macromolecular contrast agent showed significant targeting specificity to tumor cells and tumor tissue.In vivo imaging demonstrated the enhanced contrast at tumor region and the relative long imaging window.2.Employing poly(?-caprolactone)(PCL)as a core,and both poly(glycerol)modified with gadolinium chelates and poly(ethylene glycol)conjugated with FA ligand as a shell,the mixed micelle used for tumor-targeted MRI was designed.Short chain mPEG was first used to initiate the polymerization of ethoxyethyl glycidol ether(EEGE)to achieve the mPEG-PEEGE.Then,the terminal hydroxy was employed to initiate the polymerization of?-caprolactone.The resulting diblock copolymer mPEG-PEEGE-b-PCL was treated to remove the protected groups and the diblock copolymer mPEG-PG-b-PCL was obtained.After modifying partial hydroxy groups of poly(glycerol)block with azido groups,the alkynyl-functionalized gadolinium chelates(alkynyl-DOTA(Gd))were conjugated,leading to the gadolinium chelate-modified diblock copolymer mPEG-PG(DOTA(Gd))-b-PCL.In addition,utilizing alkynyl-functionalized poly(ethylene glycol)to polymerize ?-CL,the alkynyl-PEG-b-PCL was synthesized.After further conjugation with azido-modified folic acid(N3-FA),another diblock copolymer terminally modified with targeting ligand FA-PEG-b-PCL was prepared.Finally,these two kinds of diblock copolymers self-assembled to the mixed micelles with a diameter of about 85 nm in water.The macromolecular contrast agent exhibited an r1 of 14.01 mM-1s-1 at 0.5 T,low cytotoxicity,and targeting specificity to tumor cells and tumor tissue.Additionally,the contrast agent provided strongly positive contrast effect in MRI for tumor.3.Gadolinim chelates and FA ligand were simultaneously conjugated to poly(glycerol)-based triblock copolymers,which self-assembled in water to form micellar MRI contrast agent with targeting specificity.Short chain PEG was used to polymerize EEGE to obtain polymer OH-PEEGE-OH,which further initiated the polymerization of ?-CL to achieve the triblock copolymer PCL-PG-PCL.After removing protected groups,triblock copolymer with poly(glycerol)block in middle was prepared.Afterward,partial hydroxy groups in poly(glycerol)were modified with azido groups,and alkynyl-functionalized gadolinium chelate(alkynyl-DOTA(Gd))and FA(alkynyl-FA)ligand were both conjugated to the poly(glycerol)block by "click chemistry" reaction.Finally,the micellar contrast agent with a diameter of approximate 250 nm formed in water through the self-assembly of this triblock copolymer,and showed an r1 of 14.71 mM-1s-1 at 0.5 T.The micellar contrast agent could specially target to tumor cells and tumor tissue,exhibited no cytotoxicity.Moreover,in vivo MRI demonstrated the favorable contrast effect of tumor tissue,clearly differentiating the margin of solid tumor.4.One-pot synthesis was used to prepare hyperbranched poly(glycerol),whose peripheral hydroxy groups were conjugated with gadolinium chelates and FA ligand through"click chemistry" reaction.In an easy and efficient procedure,the tumor-targeted macromolecular MRI contrast agent was constructed.Short chain PEG was employed and slowly added to polymerize the glycerol,and hyperbranched poly(glycerol)with a certain molecular weight was achieved.After modifying some hydroxy groups with azido groups,and further conjugating with alkynyl-functionalized gadolinium chelate and FA ligand in the click reaction,tumor-targeted macromolecular contrast agent hPG-PEG-hPG-g-DOTA(Gd)+FA was finally achieved.The macromolecular contrast agent exhibited a higher ri(9.59 mM-1s-1 compared with small-molecule one,no toxicity to cells and organs in vivo,and obvious targeting specificity to tumor cells and tumor tissue.MRI for tumor in vivo demonstrated that tumor region treated with the macromolecular contrast agent showed prolonged and highly enhanced contrast effect.5.Tumor-targeted macromolecular contrast agent with biodegradability was constructed through conjugating gadolinium chelates to globular poly(glycerol)with disulfide bond and modification of FA ligand in periphery.Adamantanemethanol was first used to synthesize the linear poly(glycerol)Ad-PG,whose partial hydroxys were conjugated with disulfide bond-contained linker,simultaneously possessing a terminal alkynyl group.After conjugating with azido-functionalized gadolinium chelates(N3-DOTA(Gd))via"click chemistry"reaction,Ad-PG-g-s-s-DOTA(Gd)was achieved.At the same time,the linear poly(glycerol)with terminal FA ligand Ad-PG-FA was prepared.Furthermore,polyamidoamine(PAMAM)dendrimer modified with ?-cyclodextrin was employed as a core,Ad-PG-g-s-s-DOTA(Gd)and Ad-PG-FA in a certain ratio were conjugated through the host-guest interaction,leading to the biodegradable macromolecular MRI contrast agent based on poly(glycerol)for tumor-targeted imaging.The macromolecular contrast agent possessed an r1 of 8.39 mM-1s-1,not induced any toxicity to cells and organs in vivo,and presented excellent targeting specificity to tumor cells and tumor tissue.In addition,based on the cleavage of disulfide bond,the gadolinium chelates could break away from the macromolecular carrier,demonstrating the potential biodegradability.Moreover,in vivo MRI of tumor with the macromolecular contrast agent showed the rapid and clear detection of tumor tissue,and the Gd3+ion in vivo could be fast excreted after the examination.