| Objective1. To research acute toxicity of o-carboxymethyl chitosans ultrasmall superparamagnetic iron oxide nanoparticles (OCMCS-USPIO-NPs) and Folic acid-o-carboxymethyl chitosans ultrasmall superparamagnetic iron oxide nanoparticles (FA-OCMCS-USPIO-NPs) prepared by our group with dextran-superparamagnetic iron oxide nanoparticles (dextran-SPIO-NPs) as a positive control.2. To study pharmacokinetics features of FA-OCMCS-USPIO-NPs and OCMCS-USPIO-NPs by ultraviolet spectrophotometry with dextran-SPIO-NPs as a positive control.3. To study in vivo distribution characteristics of FA-OCMCS-USPIO-NPs and OCMCS-USPIO-NPs with dextran-SPIO-NPs as a positive control by two experiments:one was determineing iron content in organs by ultraviolet spectrophotometry, the other was researching distribution in living rats by magnetic resonance imaging.4. To evaluate imaging results of FA-OCMCS-USPIO-NPs on KB tumor in BACB/C-nu nude mice and the OCMCS-USPIO-NPs on popliteal lymph node metastasis in New Zealand rabbits, respectively. Methods1. Acute toxicity evaluationThis part was brief introduction of synthetic methods of dextran-SPIO-NPs, OCMCS-USPIO-NPs and FA-OCMCS-USPIO-NPs, and studying the acute toxicity of OCMCS-USPIO-NPs and FA-OCMCS-USPIO-NPs mainly. The method of alkaline coprecipitation was used to synthesize dextran-SPIO-NPs, and the synthesis of SPIO-NPs was coated in dextran solution at the same times. Synthesis of OCMCS-USPIO-NPs in two steps:synthesis of SPIO-NPs core first, and then grafting OCMCS on the surface of the core. We can synthesize FA-OCMCS-USPIO-NPs by Grafting folic acid on the OCMCS-USPIO-NPs. Determineing hydrated particle size of three nanoparticles using Malvern-3000HS. KM mice were injected three concentrations (278,347.5 and 434.5 mgFe·kg-1) of three nanoparticles from tail vein respectively and observed death, diet and weight during 14 days.2. Pharmacokinetic evaluationSD rats were injected 1mL of saline and 5.87 and 13.27 mgFe·kg-1 of three nanoparticles from tail vein respectively after fasted 12h (free water), then we gatherd 0.5 mL of blood from the retinal venous plexus at 0.25,0.5,1,2,4,6,8,12,24 h following administration. Puted the blood in EP tube containing heparin, centrifuged 10 min under the speed of 5000 r·min-1 and mensurated iron content in 0.2 mL of plasma. As the active composition of three nanoparticles was iron, we determined iron content in plasma by phenanthroline method. We pipetted 0.2mL of plasma sample accurately into a vial, added 1mL of nitric acid-perchloric acid (3:1, v:v), and evaporated by the heater after digestion 24h at room temperature.1 mL of 3% Hydrochloric acid solution were added into the vial to dissolve Fe3+ completely after the vail was cool, then we transferred the solution to volumetric flask (10mL). We added 1mL of 10% hydroxylamine hydrochloride solution,2mL of 0.15% phenanthroline solution and 5mL of 1 mol·L-1 NaAc solution into the volumetric flask, calibrated using distilled water, and measured the absorbance at the maximum absorption wavelength, then putted the absorbance into the standard curve to calculate the iron content of plasma.3. In vivo distribution evaluation3.1 determination iron content in tissuesWe determined iron content in tissues by phenanthroline method. We weighed 50mg of organization accurately into a vial using analytical balance, added 1mL of nitric acid-perchloric acid (3:1, v:v), and evaporated by the heater after digestion 24h at room temperature.1 mL of 3% Hydrochloric acid solution were added into the vial to dissolve Fe3+ completely after the vail was cool, then we transferred the solution to volumetric flask (10mL). We added 1mL of 10% hydroxylamine hydrochloride solution,2mL of 0.15% phenanthroline solution and 5mL of 1 mol·L-1 NaAc solution into the volumetric flask, calibrated using distilled water, and measured the absorbance at the maximum absorption wavelength, then putted the absorbance into the standard curve to calculate the iron content of plasma.KM mice were injected 1mL of saline in control group and 9.53mg·kg-1 or 19.06mg·kg-1 of three nanoparticles in treatment group from tail vein respectively after fasted 12h (free water), then we executed animals at 2,4,8,16h (each time point 3) following administration. Heart, liver, spleen, lung and kidney of mice were taked, washed with saline, exsiccated with filter paper, weighed 50mg accurately, and measured iron content.Heart, liver, spleen, lung and kidney of mice in control and high concentration group at 16h were fixed in 10% formalin solution, embedded with paraffin, cutted into slices, stained with Prussian blue and photographed under optical microscope.3.2 In vivo distribution study by MRI SD rats were anesthetized with 10% chloral hydrate solution by intraperitoneal injection after fasted 12h (free water). We Scaned all the animals before administration, then injected 28μg·kg-1 of dextran-SPIO-NPs, OCMCS-USPIO-NPs or FA-OCMCS-USPIO-NPs from tail vein respectively. All the animals were scaned at 1,2,4,6,8,24 h after injection by MRI. We scaned at coronal section with head coil to get the T2 signal value, and scan sequence parameters were as follows:We used spin echo-T2 weighted (SE-T2WI) sequences, repetition time (TR) and echo time (TE) were 4000ms and 106ms, field of vision (Field of view, FOV) was 12×12cm, slice thickness was 2mm. The T2 signal value (SI) of liver, lung and kidney and standard deviation of background noise (SD) were measured using the Image Viewer software, then calculated and compared signal to noise ratio (SNR) of three tissues at different time points. The formula is:SNR=SI/SD.4. pharmacodynamic evaluationKB tumor-bearing nude mice were scaned at coronal section with head coil to get the T2 signal value, and scan sequence parameters were as follows:We used spin echo-T2 weighted (SE-T2WI) sequences, repetition time (TR) and echo time (TE) were 4000ms and 85ms, field of vision (Field of view, FOV) was 12×12cm, slice thickness was 3mm. We anesthetized all the animals with 4% chloral hydrate solution by intraperitoneal injection and Scaned them before administration. KB tumor-bearing nude mice were injected 5.62 mg·ml-1 of the FA-OCMCS-SPIO-NPs solution (0.25mL) from tail vein respectively, and scaned following 3h.New Zealand white rabbits with lymph node metastasis of VX2 tumor were scaned at coronal section with head coil to get the T2 signal value, and scan sequence parameters were as follows:We used fast spin echo-T2 weighted (FSE-T2WI) sequences, repetition time (TR) and echo time (TE) were 4000ms and 85ms, field of vision (Field of view, FOV) was 12×12cm, slice thickness was 3mm. We anesthetized all the animals with 3% pentobarbital sodium (4℃save) solution by ear vein injection and Scaned them before administration. New Zealand white rabbits with lymph node metastasis of VX2 tumor were injected 2.00 mg·ml-1 of the OCMCS-SPIO-NPs solution (0.25mL) from tail vein respectively, and scaned following 12h.Using SNR and contrast to noise ratio (CNR) evaluated contrast effect of KB tumor-bearing nude mice and New Zealand white rabbits with lymph node metastasis of VX2 tumor separately. The T2 SI of Region of interest (ROI) before and after administration and SD of background noise were measured using the Image Viewer software. We selected ROI with a homogeneous signal and measured SI three times. The average of SI was used finally to calculate and the SNR of KB tumor and CNR of lymph node metastasis of VX2 tumor. Calculation method of SNR see paragraph 3.2. The formula of CNR is CNR=| SInormal-SIcancer |/SDbackground-SInormal, SIcancer and SDbackground were SI of normal lymph node, SI of lymph node metastasis and background noise standard deviation.We maked the HE staining of KB tumor in nude mice and VX2 tumor and lymph node metastases in New Zealand white rabbits to determine the formation of the tumor and the situation of metastasis. We maked the Prussian blue staining of KB tumor in nude mice and VX2 tumor and lymph node metastases in New Zealand white rabbits to determine the imaging results of the FA-OCMCS-SPIO-NPs on the KB tumor and OCMCS-SPIO-NPs on lymph node metastasis of VX2 tumor.Results1. The hydrated particle size of dextran-SPIO-NPs, FA-OCMCS-USPIO-NPs and OCMCS-USPIO-NPs was 125,38.2 and 41.4nm. Acute toxicity results showed that the LD50 of FA-OCMCS-USPIO-NPs and OCMCS-USPIO-NPs was greater than 434.5 mgFe·kg-1 which was less than it of dextran-SPIO-NPs (greater than 347.5 mgFe·kg-1). All the animals in two test groups were not found obvious toxicity, but significant toxicity was appeared in middle and high dose groups of dextran-SPIO-NPs.2. Because the basical plasma iron concentration fluctuated at the different times in pharmacokinetic study (one peak of plasma iron concentration was appeared at 1h, then the concentration was decreased slowly and fluctuated in the vicinity of 5mg·L-1 from 4h. It resumed the initial level at 24h), plasma drug concentration of three nanoparticles was expressed by the differentials between plasma iron concentration in treatment groups and blank plasma iron concentration. The results showed that two test drugs with smaller size had longer half-life (t1/2 was more than 7h), larger area under the curve and the mean residence time (MRT) extended significantly.3. Determination of iron content in five organs showed that three contrast agents mainly existed in liver and spleen, but the quantity of FA -OCMCS-USPIO-NPs and OCMCS-USPIO-NPs was swallowed by liver and spleen was less than dextran-SPIO-NPs whether in high or low concentration groups. Moreover, the phagocytosis of FA-OCMCS-USPIO-NPs was smaller than OCMCS-USPIO-NPs, which may be due to the graft of folic acid making FA-OCMCS-USPIO-NPs possessing targeting; it made FA-OCMCS-USPIO-NPs focuse on tissues with folic acid receptor. It is also probably because FA-OCMCS-USPIO-NPs involves in the metabolism of folate pathway, which reduces metabolism from liver and spleen. These conclusions could be seeing in the Prussian blue staining of organs clearly.The resaults of In vivo distribution of three nanoparticles in liver, lung and kidney by MRI showed that the basical T2 signal value of three organs was different, from high to low was kidney, liver and lung. Liver signal in dextran-SPIO-NPs group decreased significantly after administration, as liver swallowed dextran-SPIO-NPs making SNR values decrease. SNR values of kidneys in dextran-SPIO-NPs group decreased significantly, as larger particle size of dextran-SPIO-NPs was easily metabolized out of the body. Mean SNR values of lung in three groups and liver in two test groups had little change. SNR values of kidneys in dextran-SPIO-NPs and FA-OCMCS-USPIO-NPs groups were returned to the normal level at 24h, but it only returned to the level of 2-4h after administration in OCMCS-USPIO-NPs group, as OCMCS-USPIO-NPs retained in the body longer time and excreted from body at 24h after administration. SNR values of kidneys in FA-OCMCS-USPIO-NPs groups were returned to the normal level may be due to graft of folic acid enlargeing the size and speeding up the body metabolic process.4. Pharmacodynamic results showed that the T2 signal of KB tumor in nude mice reduced after intravenous injection of FA-OCMCS-USPIO-NPs (FA-OCMCS-USPIO-NPs could target KB tumor with folate receptors). The T2 signal of normal part in the VX2 popliteal lymph node metastasis in New Zealand white rabbits reduced after intravenous injection of OCMCS-USPIO-NPs (macrophages in lymph node could swallow OCMCS-USPIO-NPs), while the cancerous part had no change compared with preinjection (macrophages losed function and could not swallow nanoparticles). Statistical results show that SNR value of KB tumor had significant difference (t=11.596, P=0.007) and CNR value of VX2 popliteal lymph node metastasis had significant difference (t=10.586, P=0.009) illustrating the imaging effect of two contrast agents was obvious. Prussian blue staining showed that there were FA-OCMCS-USPIO-NPs in the KB tumor and OCMCS-USPIO-NPs in normal part in the VX2 popliteal lymph node metastasis (having blue stain), but there was no OCMCS-USPIO-NPs in cancerous part in the VX2 popliteal lymph node metastasis (having on blue stain).Conclusions1. The results of acute toxicity study showed that the acute toxicity of FA-OCMCS-USPIO-NPs and OCMCS-USPIO-NPs was lower than dextran-SPIO-NPs, which is probably because the particle size of two test drugs (less than 50nm) was much smaller than the positive drug (greater than 100nm) or the biocompatibility of coated materials (OCMCS) was better than dextran decreasing the toxicity.2. pharmacokinetic results indicated that positive drug (dextran-SPIO-NPs) with large particle size was quickly swallowed and metabolized by liver and spleen in vivo owning shorter half-life and smaller AUC, while OCMCS-USPIO-NPs and FA-OCMCS-USPIO-NPs with small particle size could partly escape from phagocytosis of liver and spleen maintaining longer time and higher concentration in vivo. It provided a basis for target-imaging cancer.3. The phagocytosis of liver and spleen of dextran-SPIO-NPs was more significant than FA-OCMCS-USPIO-NPs and OCMCS-USPIO-NPs after administration, which illustrated OCMCS-USPIO-NPs and FA-OCMCS-USPIO-NPs with small particle size could partly escape from phagocytosis of liver and spleen providing a basis for imaging all of the body. The resaults of In vivo distribution of three nanoparticles by MRI confirmed the phagocytosis of dextran-SPIO-NPs in liver and spleen, but no phagocytosis of two test drugs. Three drugs were not found in lung and excreted from the kidney.4. pharmacodynamic results show that FA-OCMCS-USPIO-NPs could target-imaging KB tumor transplanted in nude mice whose surface had folate receptors and OCMCS-USPIO-NPs could used to judgment of cancer size in the VX2 popliteal lymph node metastasis in New Zealand white rabbits providing a basis for judgment of metastatic focus. |
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