| With the discovery and applications of nanomaterials,they have developed into a new discipline-Nanotechnology,which mainly focuses on(1)various properties of nanomaterials;(2)control the sizes,shapes and surface properties of nanomaterials by means of various techniques;(3)various nanomaterials and nanodevices showing excellent functions have been designed and successfully used in physics,chemistry,materials,biomedicine and other fields.Especially,application of nanomaterials in biomedicine has received the greatest attention in recent decades,becoming one of the research hotspots.Significant progress has been made in drug delivery,medical imaging,gene delivery,biological molecular detection,and tissue engineering with the aid of nanomaterials,including but not limited to carbon nanotubes,quantum dots,metal nanoparticles(NPs),liposomes,and hyperbranched polymers.Among them,hyperbranched polymer possesses abundant modifiable functional groups,which provide a possibility to be multifunctionalized for NPs.In addtion,hyperbranched polymer can be used as a template to entrap NPs or as a stabilizer to stabilize NPs.Taking hyperbranched polyethylenimine(PEI)as a basic nanoplatform,the thesis mainly focuses on the fabrication of single or dual mode contrast agents,including computed tomography(CT),CT/magnetic resonance(MR),single photon emission computed tomography(SPECT)/CT,with an aim of medical imaging.The thesis also has a discussion on the construction of drug delivery system based on multifunctional PEI,with an aim of disease treatment.Our specific work is listed as follows:(1)PEGylated(polyethylene glycol,PEG)PEI-entrapped gold NPs(Au NPs)for blood pool and tumor CT imaging;(2)Folic acid(FA)-targeted PEI-entrapped Au NPs for targeted CT imaging of tumors;(3)PEGylated PEI-entrapped Au NPs loaded with gadolinium for dual mode CT/MR imaging of blood pool and organs;(4)FA-targeted PEI-entrapped Au NPs loaded with gadolinium for targeted dual mode CT/MR imaging of tumors;(5)Arginine-glycine-aspartic acid(RGD)peptide-targeted PEI-entrapped Au NPs labeled with 99 mTc for targeted dual mode SPECT/CT imaging of liver cancer in situ;(6)Multifunctional PEI-based nanoplatform for targeted anticancer drug delivery to tumors in vivo.Accordingly,it generates the main contents of this paper,exhibited as follows:1)Conventional medical CT contrast agents(etc.,Omnipaque)suffer from many limitations,such as short blood circulation time,not easily controlled imaging time window,heavy toxic side effects,nonspecificity,etc.In contrast,nanoparticulate CT contrast agents display extended blood circulation time,good biocompatibility and targeting after surface modification.Due to the advantage that gold element has better X-ray attenuation property than iodine,Au NPs show a better capacity of CT imaging.In our project,we used PEGylated PEI as a platform to synthesize Au NPs with different Au salt/PEI molar ratios via sodium borohydride reduction chemistry.The formed Au NPs presented relatively small size(1.9-4.6 nm),showed to be quite uniform and had distinct lattices of Au crystals.We also found that the Au NPs to be water-soluble,stable(Au salt/PEI molar ratios less than 300),biocompatible and have prolonged blood circulation half-decay time(t1/2 = 11.2 h).The X-ray attenuation intensity of the Au NPs was confirmed to be higher than that of Omnipaque at the same molar concentration of Au or iodine.The inferior vena cava,left and right renal veins,and iliac artery could be seen clearly after intravenous injection,suggesting that the Au NPs showed good CT contrast enhancement in vivo.Moreover,this CT contrast enhancement had a duration for more than 75 min postinjection.The Au NPs could also been used as a contrast agent for CT imaging of tumor via passive enhanced permeability and retention(EPR)effect.Our experiments of biodistribution and histological evaluation showed that the Au NPs could be metabolized in vivo,demonstrating its good organ compatibility.2)Based on our study above,FA was modified onto the PEI-entrapped Au NPs surface to endow the Au NPs with active targeting specificity.The designed Au NPs with a size of 2.1 nm were well-distributed,stable(Au salt/PEI molar ratios is equal to 200),and noncytotoxic in a studied concentration range.In vitro cellular uptake and CT imaging data showed that the Au NPs were able to target cancer cells overexpressing FA receptors.Importantly,as the CT imaging experiment of a subcutaneous tumor model in vivo showed,CT values in tumor region proved to be much higher when injected with the FA-targeted Au NPs than when injected with the nontargeted Au NPs at the same time points,suggesting that the FA-mediated active targeting was able to significantly enhance the tumor particle accumulation,resulting in enhanced tumor CT imaging.Further,the Au NPs we synthesized are able to be cleaned out of the mouse body,demonstrating their excellent biocompatibility in vivo.3)Owing to the fact that single mode CT imaging is difficult to meet the higher sensitivity and accuracy of cancer diagnosis,in this project,chelation of gadolinium(Gd)ions was used for T1-weighted MR imaging and internal entrapped Au NPs for CT imaging,in order to realize dual mode CT/MR imaging applications.The specific idea is: PEI sequentially modified with m PEG and Gd chelator(DOTA)was used as a template to synthesize Au NPs,followed by Gd(III)chelation and acetylation of the remaining PEI surface amines.The formed Au NPs with a size of 4.0 nm were well-distributed,colloidally stable and biocompatible.Owing to the positive coexistence effect of Au and Gd elements within one NP system,the Au NPs had a better X-ray attenuation property.However,the strong interaction between Gd(III)ions and the entrapped Au NPs would interfere proton exchange rate,and correspondingly,the r1 relaxivity would slightly decrease,but still up to 0.89 m M-1s-1,compared to r1 relaxivity of PEI.Ac-m PEG-DOTA(Gd)(1.0 m M-1s-1).For CT imaging of mice after intravenous injection with the particles,only veins could be observed distinctly at a small administration dose,while both veins and arteries could be clearly seen at a large administration dose with high resolution.For MR imaging of mice after intravenous injection of the particles,veins and arteries were detected simultaneously even at a small administration dose,and images with better resolution were only obtained with a larger administration dose.4)On the basis of researches above,FA were conducted to target PEI-entrapped Au NPs loaded with Gd(III)for targeted dual-mode CT/MR imaging applications.Cellular uptake data revealed that the particles could target cancer cells overexpressing FA receptors.In addition,the CT values or MR signal of tumor region injected with the FA-targeted Au NPs were much higher than those treated with the nontargeted Au NPs at the same time points,suggesting that the FA-mediated active targeting could significantly enhance the tumor particle accumulation,resulting in enhanced tumor CT and MR imaging.5)CT and MR imaging both belong to structure imaging,which could just obtain anatomic information of focal region.While PET and SPECT,as functional imaging,can get information of physiology and biochemistry of tumor.If structural imaging and functional imaging are merged together,we are able to get more comprehensive information of focal region,which would do favor to improve accuracy of disease diagnosis.Therefore,with these work,PEI was then not only modified with DTPA to label 99 mTc for SPECT imaging,but also entrapped by Au NPs for CT imaging.Meantime,PEI was modified with RGD peptide to endow the particles with targeting property and further to observe its active targeting function.The Au NPs with a diameter of 2.6 nm showed to be well-distributed,colloidally stable and biocompatible.HCC-LM3 cells(a line of human hematoma cells)could overexpress avb3 integrin as illustrated by cell immunohistochemistry,were able to specifically uptake the Au NPs.In vivo CT and SPECT imaging results showed that the particles displayed good contrast enhancement of liver cancer region,and could target to liver cancer region in situ.6)Besides the fact that PEI-based NPs can be applied as a contrast agent for disease diagnosis,in the last part,PEGylated PEI was used as an effective vehicle to encapsulate an anticancer drug doxorubicin(DOX)for targeted cancer therapy in vivo.We showed that the formed complexes,with each PEGylated PEI encapsulating 6.9 DOX molecules(DOX loading efficiency is 69%),were water dispersible,and could release DOX in a sustained manner with a higher release rate at an acidic p H condition(p H 5.0,51%)than at PBS solution(p H 7.4,31.5%).Furthermore,the complexes displayed specific therapeutic efficacy to cancer cells in vitro and antitumor activity in vivo.Simultaneously in vivo toxicity evaluation showed that the complexes had good organ compatibility.The multifunctional PEI we designed may hold great promise to be used as an effective vehicle for targeted cancer chemotherapy applications. |
