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Objective 1. Glucan - superparamagnetic iron oxide nanoparticles (dextran-superparamagnetic iron oxide nanoparticles, dextran-SPIO-NPs) as a positive control, the Task Force has prepared a good carboxymethyl chitosan - Ultrasmall paramagnetic iron oxide nanoparticles (o-carboxymethyl chitosans ultrasmall superparamagnetic iron oxide nanoparticles OCMCS-USPIO-NPs) and folic acid - carboxymethyl chitosan - ultra small superparamagnetic iron oxide nanoparticles (Folic acid-o-carboxymethyl chitosans ultrasmall superparamagnetic iron oxide nanoparticles, FA-OCMCS-USPIO-NPs) acute toxicity research. Dextran-SPIO-NPs as a positive control, the use of UV - visible spectrophotometry investigation FA-OCMCS-USPIO-NPs and OCMCS-USPIO-NPs in rats in vivo drug pharmacokinetic characteristics. Dextran-SPIO-NPs as a positive control, two sets of test design study FA-OCMCS-USPIO-NPs and OCMCS-USPIO-NPs in mice tissue distribution characteristics: UV - visible spectrophotometry organization within the iron content and magnetic resonance imaging in vivo investigated drug distribution. 4 using magnetic resonance imaging techniques were evaluated FA-OCMCS-USPIO-NPs BACB / C-nu nude KB cells transplanted tumors and OCMCS-USPIO-NPs the rabbit VX2, New Zealand lymph node metastases contrast effect. Method 1. Evaluation of acute toxicity introduced the positive control dextran-SPIO-NPs and test drugs OCMCS-USPIO-NPs and FA-OCMCS-USPIO-NPs Synthesis, investigated the acute toxicity of two experimental drugs. Synthesis of dextran-SPIO-NPs alkaline co-precipitation method: package was dextran solution while synthetic SPIO-NPs nanoparticles. The two-step synthesis OCMCS-USPIO-NPs: first alkaline coprecipitation method SPIO-NPs core the again SPIO-NPs surface grafting OCMCS of. Synthetic good OCMCS-USPIO-NPs surface grafting FA the-OCMCS-USPIO-NPs to folic acid synthesis. Malvern laser particle size was determined by measuring the the three nanoparticles hydrated particle size. Acute toxicity investigated selection KM mice as experimental subjects, respectively tail vein disposable mice were given three concentrations (278,347.5 and 434.5mgFe · kg-1) Three nanoparticles observed 14d mice death, diet, and weight change situation. 2 the pharmacokinetic evaluations SD rats fasted for 12h (free water), tail vein once given 1mL saline and 5.87 and 13.27 mgFe, · kg-1 three nanoparticles, and after the administration of 0.25, 0.5, , 1,2,4,6,8,12,24 h from the fundus venous plexus blood collection 0.5mL As for the EP tube heparin sodium, 5000r · mmin-1 centrifugal 10min, whichever 0.2mL supernatant Determination of iron content. The the three nanoparticles of the active ingredient is iron, the iron content in the plasma using the phenanthroline method. Plasma samples taken with the precise amount of the pipettor 0.2mL vials Add 1mL nitrate - perchlorate (3:1, v: v), the mixture was digested at room temperature for 24h with plate heater was evaporated to dryness, and after cooling was added 3 the Fe3 ion% hydrochloric acid solution 1mL dissolved vials and transferred to 10mL volumetric flask, respectively, followed by adding 10% hydroxylamine hydrochloride solution 1mL, 0.15% o-phenanthroline solution 2mL, 1 mol · L-1 NaAc solution mL, distilled water calibration, the absorbance was measured at the maximum absorption wavelength, into a standard curve equation, the iron content, and then calculating in accordance with the dilution ratio of the iron content of the plasma. The tissue distribution of school visits 3.1 Organization iron content was determined by the iron content in the tissue phenanthroline method. Using an analytical balance, said take 50mg tissue in vials Add 1mL mixed acid (nitric acid - perchlorate volume ratio 3:1), was digested at room temperature for 24h later plate heater was evaporated to dryness, and after cooling added 3% hydrochloric acid solution the the Fe3 ions the 1mL dissolved vials, and transferred to 10mL volumetric flask, respectively, followed by adding 10% hydroxylamine hydrochloride solution 1mL 0.15% o-phenanthroline solution 2mL of 1 mol L-1 NaAc solution of 5 mL of distilled water calibration UV - visible spectrophotometry at the wavelength of maximum absorption of the absorbance was measured into the standard curve equation, the iron content of iron content in the plasma, and then calculating the dilution ratio. KM mice were fasted for 12h (free water), blank control group tail vein once given 0.2mL saline administered group were from the tail vein once given 9.53mg · kg \tablets, animals were sacrificed at 2,4,8,16 h (3) for each time point, heart, liver, spleen, lung, kidney residual blood in the tissue of normal saline wash, dry filter paper moisture accurately weighed 50mg, tissue iron content was measured at each time point. taken blank group and 16h when high concentrations of the three-administered mice heart, liver, spleen, lung, kidney, fixed in 10% formalin solution for 24 h , embedded in paraffin and sliced, Prussian blue staining in the optical microscope and photographed. 3.2 resonance investigated after the body distribution SD rats fasted for 12h (water ad libitum), anesthetized by intraperitoneal injection with a 10% chloral hydrate. first scan all animals after animal tail vein once given 28μg · kg-1 of dextran-SPIO-NPs, OCMCS-USPIO-NPs or FA-OCMCS-USPIO-NPs, respectively, after administration of 1,2,4 6,8,24 h magnetic resonance scanning head coil line T2 coronal scan, scan sequence parameters are as follows: The spin echo-T2-weighted images (SE-T2WI) scanning sequence, repetition time (TR) and echo time (TE) are 4000ms and 106ms, field of view (field of view, FOV): 12 × 12cm, thickness 2mm. measured using the Image Viewer software liver, lung, kidney T2 signal value (SI), in the background area, select larger the standard deviation of the area to determine the background noise (Standard-deviation of the noise, SD), calculated at each time point of the organizations SNR (signal to noise ratio, SNR), calculated as follows: SNR = SI / SD compare before drug administration and at different time points after the three organ SNR changes. 4. the pharmacodynamic evaluation Holland KB nude mice using a head coil line T2 coronal scan, scan sequence parameters are as follows: spin echo - T2-weighted images (SE-T2WI) scan sequence, repetition time (TR) and echo time (TE) are 4000ms and 85ms, field of view (field of view, FOV): 12 X 12cm, thickness 3mm intraperitoneal injection of 4% chloral hydrate solution anesthetized unenhanced all animal tail intravenous administration of 5.62 mg · ml-1 of the FA-OCMCS-SPIO-NPs solution 0.25ml 3h after an MRI scan. lymph node metastasis in New Zealand rabbits VX2 head coil line T2 coronal scan, scan sequence parameters are as follows: the white fast spin echo-T2-weighted images (FSE-T2WI) scanning sequence, repetition time (TR) and echo time (TE) are 3500ms and 85ms, field of view (field of view, FOV): 10 × 10cm, thickness 3mm with 3% sodium pentobarbital (4 ℃ save) solution ear vein injection of anesthesia after scan all animals, give ear vein 2.00mg · ml1 OCMCS-SPIO- NPS solution 16.80ml administration 12h and then an MRI scan lymph node metastasis of the contrast effect of the tumors in nude mice and New Zealand rabbits were used to SNR (Signal to Noise Ratio, SNR) of contrast to noise ratio (Contrast to Noi SE RATIO, the CNR ) were evaluated. adopted the MRI film-reading software CDViewer relatively uniform before administration of the two groups of experimental animals, after the signal strength of the region of interest (Region of interest, ROI) measurements (signal intense, SI), ROI select local signal, No apparent artifact region three measurements taken SI average nude mice administered before and after tumor SNR calculation, see 3.2 Method under rabbit lymph node metastasis before administration, after CNR is calculated as follows: CNR = the | SInormal SIcancer | / SDbackground, SInormal, SIcancer and SDbackground lymph normal tissue signal, the standard deviation of metastatic lymph nodes signal and background noise. HE staining KB tumors in nude mice and rabbit VX2 tumor and lymph node metastases were made to determine tumor formation and metastasis situation; administration do after the KB tumor and rabbit VX2 tumor and lymph node metastases Prussian blue stained sections to determine FA-OCMCS-SPIO-NPs the KB tumor targeting and OCMCS-SPIO-NPs rabbit VX2 lymph node metastasis tumor contrast effect results dextran-SPIO-NPs OCMCS-USPIO-NPs and FA-OCMCS-USPIO-NPs hydrated particle size were 125nm and 38.2nm and 41.4nm. acute toxicity results showed that the FA-OCMCS-USPIO -NPs and OCMCS-USPIO-NPs the LDso are greater than 434.5 mgFe · kg-1, less than the positive control drug dextran-SPIO-NPs LDso (greater than 347.5 mgFe · kg-1). two test drugs group, all animals were no obvious toxicity; dextran-SPIO-NPs group in the medium dose group survival animals apparent toxicity. 2. pharmacokinetic experiments, the basal plasma iron concentration changes with time and fluctuations (1h when burst release peak, followed by a slow decline in hours after 5mg · L-1 about fluctuations initial level restore 24h), so three nanoparticle concentration in plasma, respectively, after the administration of plasma iron concentration minus the blank plasma iron concentration results showed that the smaller particle size of the two test drugs longer in vivo half-life (t1 / 2) (greater than 7 hours), and greater area under the concentration-time curve, in vivo retention time (MRT) was significantly extended. 3 The five organs of the iron content measurement results show that the three contrast agents are mainly distributed in the liver, spleen and parts, but compared to dextran-SPIO-NPs, regardless given the low concentrations or high concentrations of the drug, liver, spleen FA -OCMCS-USPIO-NPs and OCMCS-USPIO-NPs phagocytosis was significantly reduced, and the phagocytosis of the FA-OCMCS-USPIO-NPs was also significantly less than OCMCS-USPIO-NPs, this may be because after grafting folate nanoparticles having a target isotropic, the concentration of the target tissue or participate in the metabolism of folic acid pathway, reduces liver, spleen metabolized amount of these conclusions can be clear from the organs of the three drugs Prussian blue stained sections to see the results of magnetic resonance the investigated liver, lung and kidney, the three organs T2 signal value, in descending order liver kidney gt; liver gt; lung after administration of dextran-SPIO-NPs group signal decreased significantly, indicating liver engulfed the nanoparticles reduces the SNR value; kidney SNR values ??decreased significantly larger particle size dextran-SPIO-NPs easier metabolism in vitro. lungs as well as the two test groups liver time point SNR mean almost no change in kidney SNR value 24h dextran-SPIO-NPs group and] FA-OCMCS-USPIO-NPs group restored to the level before the administration, but OCMCS-USPIO-NPs group only restore to the administration 2-4h, a longer period of time in the body, OCMCS-USPIO-NPs retained 24h when there are drugs excreted from the body, FA-OCMCS-USPIO-NPs' recovery to the administration of former may be due to the grafting folic acid particle size increased to speed up the process of metabolism in the body. 4. pharmacodynamic results show that the tail intravenous the FA-OCMCS-USPIO-NPs in nude KB tumor T2 signal significantly reduced (FA-OCMCS-USPIO-NPs on the KB tumor folate receptor targeting), give ear vein after the rabbit OCMCS-USPIO-NPs popliteal lymph node normal part of T2 signal value decline (macrophages within the lymph nodes can to engulf OCMCS-USPIO-NPs), while cancerous area T2 signal and before the administration, almost no change (macrophages lose functionality can no longer be swallowed nanoparticles) Statistical results show that before the administration, after KB tumor SNR will have a significant difference (t = 11.596, P = 0.007 ), VX2 lymph node metastasis tumor of the CNR have significantly with the difference (t = 10.586, P = 0.009), illustrate the effect of two contrast agents significantly. Prussian blue staining slices Figure Description KB tumor have FA-OCMCS-USPIO-NPs distribution (there are blue color particles), VX2 popliteal lymph node metastases in normal tissue distribution of OCMCS-USPIO-NPs (blue particles), the cancerous tissue without nanoparticles (blue particles). Conclusions 1. acute toxicity study results show that the FA- OCMCS-USPIO-NPs and OCMCS-USPIO-NPs acute toxicity than dextran-SPIO-NPs, this may be a particle size (less than 50nm) caused far less than the positive control drug (greater than 100nm) of the test drug, and also might be better than coated material OCMCS biocompatibility dextran, leading to decline in vivo toxicity. the pharmacokinetic results illustrate the positive control of the particle size dextran-SPIO-NPs into the body quickly liver, spleen phagocytic metabolism, in vivo half-life and AUC is smaller, while the small particle size OCMCS-USPIO-NPs and FA-OCMCS-USPIO-NPs partially escape the phagocytosis of the liver, spleen, and maintain a longer time and a higher concentration in the blood for tumor targeted contrast basis after administration of liver, spleen and phagocytosis of dextran-SPIO-NPs significantly higher than the FA-OCMCS USPIO-NPs and OCMCS-USPIO-NPs description of the small particle size of the two tests the medicine part to escape the liver, spleen macrophages phagocytosis, both can be distributed to other parts of the body angiography and lay the foundation; magnetic resonance distribution in the body study three kinds of nanoparticles confirmed liver metabolism of dextran-SPIO- NPs phagocytosis, two equal doses of test drug is not swallowed, no distribution of the three drugs in the lungs, are excreted from the kidneys. 4 the pharmacodynamic results show that, FA-OCMCS-USPIO-NPs with folate receptor KB cells in nude mice transplanted tumor targeting imaging of New Zealand rabbit VX2 OCMCS-USPIO-NPs popliteal lymph node metastases in role judgment tumor size, to provide the basis for effective judgment of metastatic foci.
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