| BackgroundIn recent years,molecular imaging has become increasingly important for the diagnosis and treatment of diseases,and a large number of key molecular targets have been selected to determine the development process of the disease,and qualitative and quantitative studies are carried out for the targets.As the earliest imaging technology used in molecular imaging,nuclear medicine imaging is one of the few for the clinical application of molecular imaging technology.The continuous development of nanotechnology has promoted the development of nuclear medicine diagnosis and treatment,especially in the selective delivery of biologically active substances to disease sites.Since the surface of the dendritic nanoparticles PAMAM has a large number of active groups,to which a plurality of radionuclides and targeting molecules can be linked by a bifunctional chelating agent,a highly contrastable diagnostic image can be formed at low concentration.Recently,the application of PAMAM in tumor diagnosis and treatment has attracted the attention of more researchers.At present,this direction has been developed as a new choice in the study of nano-medicine.It is a research in drug transportation system and pharmaceutical science hotspot.The binding of dendrimers to specific peptides can significantly increase the targeting efficiency of drug and enzyme delivery.RGD as an anti-tumor drug targeting vector,the current related research has been quite mature.Through the specific binding with ?v?3 protein,RGD can increase the lesion local drug concentration,reduce the adverse effects of anti-tumor drugs.At the same time,MAL-PEG-NHS,which has a bifunctional group,is used as a "bridge" for nanocarriers and targeting polypeptides,and can link RGD to PAMAM.In previous studies,many researchers have modified PAMAM with PEG to improve their biocompatibility,and to carry out drug delivery and release,as well as tumor imaging.The addition of PEG not only reduces the toxicity of PAMAM carriers,but also enhances the hydrophilicity of the drug delivery system.131I is the classical radionuclide for radiotherapy,with a half-life of 8.04 days,a ? ray emitted at 131 I decay,and an energy of 0.608 MeV for internal irradiation,while? rays can be developed in vitro simultaneously.Based on its excellent biochemical properties,application for decades,and continuous development of new drugs,such as some monoclonal antibodies radiolabeling,131 I has been playing an important role in bio-targeted therapy.131 I labeling technology is quite mature.The chemical synthesis methods,directed labeling methods inculde Iodogen method and chloramine-T method based.Based on the successful application of 131 I in clinical and basic research,The direct labeling of chloramine T was selected for 131 I radiolabeling of nano-carrier to make it a therapeutic drug targeting tumor.In this study,131I-RGDyC-PEG-PAMAM complex was synthesized by using the dendritic nanoparticles(PAMAM)as the nanocarrier and RGD as the tumor-directed molecular modified drug carrier.The target molecule was labeled with radionuclide 131 I.Compared with the traditional single tree-like carrier,this new type of polymer carrier can simultaneously achieve treatment,imaging and targeting.The research results will also have a certain clinical value.This study is divided into four parts.The first part is the synthesis of radioactive iodine labeled precursor RGDyC-PEG-PAMAM and its quality detecting;the second part is radioactive iodine labeling of the targeting of nano-carrier,and its quality detecting;the third part is the study of biological activity of nano-probes,including study on cell level and animal level.And finally explore the possibility of applying it to targeted tumor imaging and diagnosis and treatment of drugs in vivo.Objective1.Chemical synthesis of the iodine precursor RGDyC-PEG-PAMAM.And its quality identification such as chemical properties,physical properties were detected to evaluate whether it can be used for the next step 131 I radiolabeling..2.The 131I-RGDyC-PEG-PAMAM radioactive nanoprobe was prepared by labeling RGDyC-PEG-PAMAM nanocomposite with 131 I,and its labeling rate,stability and lipo-hydro partition coefficient were identified to evaluate whether it could be used in the next step Biology experiment.3.To evaluate the targeting effect of 131I-RGDyC-PEG-PAMAM nanocomposites on tumor cells in vitro,and the effect of radiactive inhibition.To lay the foundation for further study of tumor imaging and anti-tumor in tumor-bearing mice.4.The drug distribution and imaging effect of 131I-RGDyC-PEG-PAMAM nanoprobe at the animal level were evaluated.MethodsThe first part: the preparation of the radiolabeled precursor RGDyC-PEG-PAMAM(1)Chemical synthesis: The maleimide groups on the bifunctional PEG(NHS-PEG-MAL)react with the mercapto groups on RGDyC under weak acid conditions.40mg(17.22?mol)of bifunctional PEG(NHS-PEG-MAL)was weighed and added to sodium acetate buffer(NaAc-HAc 1ml 0.1M pH6.0)containing cyclic RGDyC(4mg,6.72?mol).The reaction was carried out for 30 s with magnetic stirring.The mixed product in the previous reaction was rapidly transferred to a boric acid(borax-NaOH)buffer(1ml 0.05 M pH9.0)in which PAMAM nanomolecules(4.2mg,0.15?mol)were dissolved and reacted under magnetic stirring for 12 h.After 12 h,the pH of the reaction mixture was adjusted to 7.0 and then ?-mercaptoethanol(2?l,28?mol)was added to quench the unreacted maleimide group.After 1h,the mixture was purified by ultrafiltration(Amicon Ultra-4,MWCO10000;4500 rpm,15 min,9times)to remove free PEG and RGDyC,and the solution of the purified product was lyophilized to give the product RGDyC-PEG-PAMAMA as a pale yellow solid.(2)The basic property identification: weigh 5mg RGDyC-PEG-PAMAMA dissolved in 0.6ml heavy water(D2O),nuclear magnetic resonance spectroscopy was used to detect the material characterization.After 1mg of RGDyC-PEG-PAMAMA was diluted with double distilled water,the material characterization was characterized by UV,spectroscopythe particle size distribution and potential were measured by nano-particle size and Zeta potential analyzer,and the morphological size and distribution were observed by transmission electron microscopy(TEM).The second part: the preparation of radioactive nano-probe 131I-RGDyC-PEG-PAMAM(1)Chemical synthesis: chloramine-T method was used for radionuclide 131 I labeling.1.5mg of RGDyC-PEG-PAMAMA was weighed and dissolved in 100?l of PBS buffer(pH7.4).Then,2mCi(74MBq,200?l)Na131I solution was added,followed by addition of chloramine-T 300?l(5mg/ml).300ul(5mg/ml)sodium metabisulfite solution was added to terminate the reaction,complete the labeling.(2)The basic property identification:(1)The labeling rate was determined by Instant Thin Layer Chromatography(ITLC).The stationary phase is: ITLC-SG chromatography paper,and the developing solvent is methanol.Take 2?l of reaction solution on ITLC-SG paper(10mm × 150mm),spread in methanol,naturally dry in air.In the expansion system,the free 131 I was moved to the front of the chromatography paper with the solvent Rf of 0.9 to 1.0;and the marker 131I-RGDyC-PEG-PAMAMA remained at the origin and Rf was 0.0-0.1.The chromatogram was scanned with a Bioscan thin-layer radioactivity scanner and the radioactivity labeling rate(%)was calculated.(2)Stability test: 100?l labeled product 131I-RGDyC-PEG-PAMAMA was added to 1ml of fresh bovine serum and 1ml of PBS,incubated in a 37 ? water bath for 2,12,24,and 72 hours.A small amount of samples were taken at different time points and the radiochemical purity was determined by Instant Thin Layer Chromatography.(3)Determination of lipo-hydro partition coefficient: 500?l n-octanol and 480?l PBS were added to the centrifuge tube at the same time,and 20?l of 131I-RGDyC-PEG-PAMAMA in PBS was added to the tube,sealed with gelatin film,shocked evenly then dealed with high-speed centrifugation(10000 rpm)till n-octanol and PBS balanced.100?l of each sample from the organic phase and the aqueous phase were placed in two centrifuge tubes,respectively,and their radioactivity counts were measured.Repeat the sample three times.The lipo-hydro partition coefficient was calculated as follow: log P = log(cts N-octanol / cts PBS).The third part: the bioactivity experiment(1)Cell culture: Human lung adenocarcinoma A549 cells were cultured in an incubator at 37°C in a 5%CO2,in RPMI 1640 culture medium containing 10% fetal bovine serum,penicillin(100IU/mL)and streptomycin(100IU/m L).Replace the culture medium every two days.When the cells are covered with a culture bottle,separated it with 0.25% trypsin / 0.02% EDTA and 0.01 mol/L PBS(pH7.4)buffer,the cells were incubated for further cell culture,passage,and cryopreservation.(2)Cell uptake and elution experiments:(1)Untake experiment: add 5×104 A549 cells in each hole of the 24-well plate,culturing overnight till to adherence.The cells were divided into two groups.10?Ci / well of 131I-RGDyC-PEG-PAMAMA and 131I-serum-free medium were added and incubated at 37°C for 1,2,4 and 6 hours.Followed by rinsing with the frozen PBS buffer for 3 times.Finally,the cells were digested with trypsin and the cell suspensions were collected and counted with a ? counter.After attenuation correction,Cell uptake data were dealed with a percentage of added dose as cell binding rate.Three parallel samples were designed.(2)Elution experiment: add 5×104 A549 cells in each hole of the 24-well plate,culturing overnight till to adherence.The cells were divided into two groups,and 10?Ci / well of 131I-RGDyC-PEG-PAMAMA and 131I-serum-free medium were added and incubated at 37°C for 2h to bind to the cells fully.Then remove the culture medium,the cells were rinsed with frozen PBS 3 times,then add serum-free medium and incubated for 1,2,4,6h.Followed by rinsing with frozen PBS buffer for 3 times.Finally,the cells were digested with trypsin and the cell suspensions were collected and counted with a ? counter.Cell uptake data were dealed with a percentage of added dose as cell retention rate.Three parallel samples were designed.(3)Inhibitory effect of drugs: A549 cells(1 × 103)were placed in 96-well plates and divided into 4 groups,which were 131I-RGDyC-PEG-PAMAM group,RGDyC-PEG-PAMAM group,131I-group,and PBS control group.Each group was set up nine wells,a blank control hole.MTT assay was used to detect the viability of A549 cells after 24,48 and 72 hours.(4)Apoptosis: A549 cells(2 × 105)were placed in 6-well plates and divided into 131I-RGDyC-PEG-PAMAM group,131I-group and blank control group.Set up 3 wells.Flow cytometry(FCM)was used to detect the apoptosis of cells induced by the nanoparticles.(5)The animal model of nude mice bearing tumor was established: the animal model of A549 cell bearing nude mice was established,and the transplanted tumor was constructed by axillary subcutaneous cell suspension injection.(6)The radioactive distribution of 131I-RGDyC-PEG-PAMAM in different organs of normal mice.(1)131I-RGDyC-PEG-PAMAM in vivo injection: first,2% KI solution of drinking water was given to the tumor-bearing mouse model to inhibite the thyroid,continued until the end of the experiment to reduce iodine uptake of thyroid tissue.(2)The distribution of radiopharmaceuticals in different organs of mice at different times after injection of drugs was detected.(7)Vivo experiments of 131I-labeled nano-drug carrier in tumor-bearing mice:(1)131I-RGDyC-PEG-PAMAM in vivo injection: first,2% KI solution of drinking water was given to the tumor-bearing mouse model to inhibite the thyroid,continued until the end of the experiment to reduce iodine uptake of thyroid tissue.(2)SPECT imaging of tumor-bearing mice at different time points after different injection of different drugs.ResultsPart 1: the preparation of the radiolabeled precursor RGDyC-PEG-PAMAMThe synthesized radiolabeled precursor RGDyC-PEG-PAMAM was yellowish and identified as the target product by nuclear magnetic resonance spectroscopy(400MHz,D2O)and UV absorption spectroscopy.The average particle size of RGDyC-PEG-PAMAMA was 32.67 nm The TEM images show that the synthesized RGDyC-PEG-PAMAMA is a spherical nanocomposite with an overall diameter of about 22 nm.Part 2: the preparation of radioactive nano-probe 131I-RGDyC-PEG-PAMAM131I labeled RGDyC-PEG-PAMAM labeling rate> 95%,and the specific activity was 0.08mCi/mg.The results showed that the nano-probe in different solution(fetal bovine serum,PBS)has good stability at different time points in 72 h.The log P =-1.59 ± 0.09 of the nanoprobe was determined by the lipo-hydro partition coefficient test,indicating that the molecular probe had obvious hydrophilicity.Part 3: bioactivity experiment(1)Cell uptake and elution experiments: compared to the free 131I-,131I-RGDyC-PEG-PAMAM nanoprobe was able to bind specifically to A549 cells,and the binding rate of A549 cells was further improved with the prolongation of incubation time.In the cell elution experiments,the drug 131I-RGDyC-PEG-PAMAM group showed a small amount of elution of the drug in the cells over time,but the change was less pronounced and the free 131I-has been at a low level.(2)In vitro cell inhibition and apoptosis test results showed that 131I-RGDyC-PEG-PAMAM had some inhibitory effect on A549 cells and had a certain relationship with the time of action.The longer the time,the more inhibitation the cells.In the promotion of apoptosis,compared to the free 131I-,the effect of 131I-RGDyC-PEG-PAMAM is stronger,which is related to the drug dose.At high dose,the nano-probe on A549 have a certain apoptosis and radiation killing effect.(3)Animal experiments(1)The distribution of 131I-RGDyC-PEG-PAMAM in normal mice after injection showed that the kidney and liver had the higher uptake,followed by spleen,intestine and thyroid,and the uptake of the organs such as blood,heart,muscle and bone were very low,indicating that the metabolic excretion of drug was mainly through the liver and kidney.(2)131I-RGDyC-PEG-PAMAM was injected into different tumor-bearing mice by different modes of administration.The SPECT imaging was performed at different time.The pharmacokinetic characteristics and tumor targeting characteristics of drug probes 131I-RGDyC-PEG-PAMAM in nude mice bearing A549 tumor were analysed.The results showed that the tumor imaging was good.Compared with the free 131I-group in the same condition,the nano-probe had a longer residence time in the tumor,and 131I-was soon excreted out of body,indicating that the combination of nano-probe and tumor has a good affinity,and a specific combination.Especially the intratumoral injection,the drug in the tumor aggregation and drug retention time are superior to the tail vein injection.In addition,the untake of drugs in the liver and kidney is the higher,the untake of muscle is low,similar to the distribution of the body.ConclusionsThe precursor compound RGDyC-PEG-PAMAM can be successfully prepared by "one-pot two-step" method and click chemical reaction.The chemical synthesis method is simple,rapid and efficient.The 131I-RGDyC-PEG-PAMAM radioactive probes were synthesized by direct labeling of chloramine-T,and high labeling rate,good stability and hydrophilicity were obtained.The results of cell experiments in vitro showed that 131I-RGDyC-PEG-PAMAM had some effect on growth inhibitory,apoptosis-promoting and radiation-killing of A549 cells.The distribution of 131I-RGDyC-PEG-PAMAM nanoprobe showed that the mainly excretion pathways were the renal and liver metabolism,and the uptake of other organs was low,which was consistent with the distribution of SPECT imaging.The results of tumor SPECT imaging showed that 131I-RGDyC-PEG-PAMAM could well accumulate in the tumor site of the tumor-bearing mice,and had a good tumor targeting,which laid a good foundation for the anti-tumor effect in tumor-bearing mice.In general,the radioactive probe 131I-RGDyC-PEG-PAMAM has the potential as a tumor-targeted SPECT imaging and treatment. |
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