Design Of Dendrimer-based Hybrid Nanoplatforms For Tumor Theranostic Applications

Today with the continuous development of molecular imaging technology and nanomedicine study,there is still a great challenge about how to organically integrate two functions above to achieve “theranosis”.And at present medical imaging agents and molecular medicine have several disadvantages,such as the short blood half-life,bad stability and biocompatibility of contrast agent,hydrophobic property,side effect towards normal cells and tissue,nonspecificity towards lesion site of small molecule chemical drugs.Therefore we need to find a suitable nanoplatform to combine the diagnosis function of contrast agents and therapy effect of chemical drug,and to improve the drawbacks of medical imaging agents and chemical drugs to form multifunctional theranostic probe for early diagnosis and treatment of cancer.Based on the unique physical and chemical properties of dendrimers,the surface abundant functional groups which can be conjugated by drugs or modified by functional agents,the interior cavity can be utilized to encapsulate molecular drug and metal nanoparticles.Here we used dendrimers as platform to build functional theranostic hybrid nanoparticles and characterized them.The study indicated the formed theranostic nanosystem can not only improve the solubility and biocompatibility of molecular anti-cancer drugs,but also prolong the blood circulation time of dugs in vivo and prolong their imaging times.At the same time,we can also modify the targeting agents on the surface of nanosystem to improve specific binding towards cancer cells and achieve targeted diagnosis and therapy.Therefore based on dendrimer,the formed theranostic hybrid nanoparticle will provide a new idea to explore new types of multifunctional nanoparticles which own tumor diagnosis and therapy functions.The research contents are as follows:1)According to the unique physical and chemical properties of dendrimers,generation 5 PAMAM dendrimer?G5.NH₂?was utilized as theranostic nanoplatform.Through surface modification with fluorescein isothiocyanate?FI?,polyethylene glycol?PEG?-modified anti-cancer drug a-tocopheryl succinate??-TOS?,and PEGylated targeting molecular folic acid?FA?,then entrapping gold nanoparticles in their interior to form multifunctional dendrimer-entrapped gold nanoparticles?Au DENPs?for targeted CT imaging and chemotherapy of tumor.The proton nuclear magnetic resonance spectroscopy?¹H NMR?result indicated that the formed Au DENPs conjugated with about 9.8 ?-TOS molecules per dendrimer.Transmission electron microscope?TEM?test demonstrated that the average diameter of the formed Au DENPs was 3.3 nm and the formed Au DENPs had the relatively narrow size distribution and excellent monodispersity.Furthermore the formed Au DENPs own a good stability under different p H?p H = 5,6,7,8?,temperature?4 ?,37 ?,50 ??and solvent conditions.Flow cytometric analysis and confocal microscopic test proved that the FA modification enabled the multifunctional Au DENPs to specifically bind the cancer cells overexpressing FA receptors,achieving targeted CT imaging of cancer cells in vitro and tumors in vivo.The conjugation of ?-TOS enabled the formed Au DENPs to own the targeted therapeutic functions in vitro and in vivo,furthermore the therapeutic efficiency is much higher than that of free ?-TOS both in vitro and in vivo.Based on conclusion that the FA-targeted,?-TOS-conjugated multifunctional Au DENPs owned higher cell therapeutic efficiency than that of ?-TOS in vitro we built above,and in order to further investigate the therapeutic mechanism and study whether the nanoplatform can be used for other kinds of tumor system,we utilized the Arg-Gly-Asp?RGD?peptide as targeting agent to modify on the surface of ?-TOS conjugated multifunctional Au DENPs.The study indicated the formed Au DENPs also owned relatively uniform size and excellent stability in vitro.By testing the intracellular reactive oxygen species?ROS?to demonstrate the RGD targeted,?-TOS conjugated multifunctional Au DENPs can induce cancer cells to produce more ROS than that of free ?-TOS.Annexin V-FITC/PI double staining test demonstrated the RGD targeted,?-TOS conjugated multifunctional Au DENPs could induce much more early apoptotic cells and dead cells than that of free ?-TOS.Due to the RGD modification,the formed ?-TOS conjugated Au DENPs are also able to target cancer cells overexpressing avb3 integrin and specifically inhibit the growth of the cancer cells.The formed targeted nanoplatform had higher X-ray attenuation than that of clinical iodine-based contrast agent,omnipaque and afforded targeted CT imaging of cancer cells in vitro.2)G5.NH₂ was utilized as nanoplatform to modify with gadolinium chelator?DOTA-NHS?,PEGylated targeting agent?PEG-FA?on the surface,followed by chelating gadolinium and acetylation of the remaining dendrimer terminal amines.Then thus formed nanocomplexes were used to encapsulate doxorubicin?DOX?within the dendrimer interior to form a theranostic nanosystem.The study demonstrated that the formed theranostic nanosystem can encapsulate 8.5 DOX molecules per dendrimer and own good stability under different pH conditions.Moreover the formed theranostic nanosystem can sustainedly release DOX.The FA modification enables efficient targeting of the particles towards cancer cells overexpressing FA receptors,and thus effective targeted MR imaging and chemotherapy of cancer cells in vitro.3)We utilized partially acetylated multifunctional dendrimers with FA modification as a nanoplatform?G5.NHAc-FA?to covalently conjugate DOX by acid-sensitive cis-aconityl linkage.We then used the formed G5.NHAc-FA-DOX as templates to entrap gold nanoparticle to form a new theranostic nanosystem.The formed Au DENPs conjugated with 9 DOX molecules per dendrimer,with an Au core size of 2.76 nm are stable under different p H and temperature conditions.The formed FA-targeted DOX-conjugated Au DENPs follow an acid-triggered release manner with a higher DOX release rate under an acidic p H condition.Apart from demonstrating certain therapeutic efficacy of cancer cells in vitro,the FA modification enables the particles to efficiently target cancer cells overexpressing FA receptors?FAR?for CT imaging of the cancer cells in vitro.4)According to the dendrimer-based diagnostic nanosystem and chemotherapeutic nanosystem we built before,we further built radiochemical theranostic nanosystem loaded with radioactive iodine-131(¹³¹I).Based on the properties of ¹³¹I which own both ?-ray emission?364 ke V,81.7%?for single-photon emission computed tomography?SPECT?imaging and ?-ray emission?0.606 Me V,89.9%?for radiotherapy,G5.NH₂ were sequentially conjugated with 3-?4'-hydroxyphenyl?propionic acid-OSu?HPAO?and FA linked with polyethylene glycol?PEG?,followed by acetylation modification of the dendrimer remaining surface amines and labeling of radioactive iodine-131(¹³¹I)to build up a radiochemical theranostic system.¹H NMR result demonstrated that G5.NH₂ dendrimers conjugated with approximately 9.4 HPAO moieties per dendrimer are noncytotoxic at a concentration up to 20 ?M before ¹³¹I labeling.Thanks to the modified FA ligands,the formed theranostic nanocarriers are able to target cancer cells overexpressing FA receptors.Radiochemical purity test showed that the formed ¹³¹I labeled multifunctional dendrimers owned good stability.The radiochemical purity is 92.84% until 27 h.The tumor growth curve and survival rate demonstrated that the ¹³¹I-labeled multifunctional dendrimers have excellent radiotherapy efficiency.The SPECT imaging data in vivo indicate that the formed nanomaterials are able to specifically target to tumors overexpressing FA receptors for targeted SPECT imaging.