Disclosure of Invention
Aiming at the defects existing in the prior art, the invention provides a nano-composite, a preparation method and application thereof, and provides a new way for researching organ transplantation medicines.
The invention solves the technical problems through the following technical scheme: the invention firstly provides a nano-composite which is formed by covalent connection of a high molecular polymer with catalase CAT.
The high molecular polymer is polyethylene glycol (polyethylene glycol, PEG) which is covalently linked around catalase to form a nano-complex.
The nano composite has a spherical structure, the particle size is 10-100 nm, and the surface charge is-0 mV.
The above-mentioned nanocomposites are nanoparticles represented by CAT-PEG by covalent binding of PEG around CAT. PEG surrounding the CAT outer layer enhances enzyme stability and immune evasion.
The invention further provides a preparation method of the nano-composite, which mainly comprises the steps of reacting catalase with polyethylene glycol for 2 hours to obtain a nano-composite crude product; and dialyzing the crude nano-composite product by using PBS solution, removing unreacted polyethylene glycol, and eluting by using PBS to remove non-entrapped catalase so as to obtain the purified nano-composite. The method comprises the following specific steps: dissolving catalase in a phosphate buffer solution with the pH value of 8.0 and 50mM, adding PEG5000-NHS dissolved in dimethyl sulfoxide for reaction for 2 hours to obtain a CAT-PEG crude product, dialyzing the CAT-PEG crude product at the temperature of 4 ℃ for 12 hours by using a dialysis bag with the molecular weight cut-off of 100 kDa in a1 XPBS solution to remove unreacted PEG5000-NHS, and removing unencapsulated CAT through a phenyl sepharose CL-4B column by using 10 XPBS as eluent to obtain purified CAT-PEG.
In the method, the molar ratio of PEG5000-NHS to CAT is 1000:1.
The invention removes CAT which is not connected with PEG through dialysis and column chromatography purification. CAT-PEG retains CAT activity and has better stability and longer in vivo circulation time.
The invention still further provides application of the nano-composite in preparing anti-inflammatory drugs required by organ transplantation.
The invention further provides application of the nano-composite in preparing medicines for treating inflammation caused by ischemia and reperfusion injury in liver transplantation.
The invention still further provides the use of the nanocomposite in the manufacture of a medicament for diseases associated with reactive oxygen species.
The invention further provides a medicament for treating active oxygen related diseases prepared from the nano-composite, which comprises the preparation of medicaments for treating liver fibrosis, liver cirrhosis, bacterial and viral infection and cancer as an active oxygen scavenger.
Studies have shown that CAT-PEG catalyzes toxicity H following intravenous administration2 O2 Decomposition to non-toxic O2 With water, reduce intracellular ROS levels, and combat pathological processes caused by ROS. Thus, PEG covalently bound to the CAT surface can be effective in extending the circulation time of CAT in vivo to deliver CAT to the liver and prevent IRI or for the treatment of other ROS-related diseases during liver transplantation. The nanoparticle can efficiently catalyze and remove ROS represented by hydrogen peroxide in vivo, relieve oxidative stress in cells and reduce injury caused by ROS. Can be used for preventing IRI in organ transplantation. Can also be used as ROS scavenger for the treatment of other ROS related diseases such as liver fibrosis, liver cirrhosis, bacterial and viral infections and cancer.
Detailed Description
Example 1
The nanocomposites were prepared according to the following procedure and further analyzed to investigate the indications.
1. Preparation of CAT-PEG nanocomposite
1.1. Preparation of CAT-PEG
CAT was dissolved in 50mM Phosphate Buffer (PB) at pH 8.0, and PEG5000-NHS (1000:1, n/n, PEG 5000-NHS: CAT) dissolved in DMSO was slowly added to react for 2 hours to give crude CAT-PEG.
1.2. Purification of CAT-PEG
The crude nanocomposite CAT-PEG product was dialyzed against 1 XPBS solution at 4℃for 12 hours using a dialysis bag having a molecular weight cut-off of 100 kDa to remove unreacted PEG5000-NHS. The unreacted CAT was removed by phenyl-sepharose CL-4B column using 10 XPBS as eluent to give purified CAT-PEG.
2. CAT Activity test
And detecting the catalase activity of the prepared CAT-PEG by using a catalase activity detection kit through an ultraviolet spectrophotometry. The method comprises the following specific steps:
1mL of H is taken2 O2 Solution (ph=7.4, 0.1M HEPES buffer, H2 O2 Concentration: 0.03% w/v) in a 1mL quartz cuvette, adding 35 mu L of sample, and uniformly mixing for 5s; the initial absorbance A1 at 240nm and absorbance A2 after 1min were measured immediately at room temperature. Delta a=a1-A2 is calculated.
According to the formula CAT (U/mL) = [ ΔA×V inverse total ≡ε×d ] ×106 ]Peroxycat activity was calculated as V-sample ≡t=678×Δa.
3. Characterization of CAT-PEG
3.1. Potential of nanocomposite
The nanocomposite 1mL dialyzed in PB solution was subjected to dynamic light scattering test with Malvern Zetasizer Nano ZSE instrument, and the zeta potential of the nanocomposite was obtained.
3.2. Transmission electron microscopy of nanocomposites
The preparation process of the projection electron microscope (Transmission electron microscope, TEM) samples is as follows: firstly, 10 mu L1 mg/mL of nano-composite solution is dripped onto a TEM copper grid with a carbon film, and the mixture is kept stand for 5 minutes, then redundant samples are sucked by filter paper, after dyeing for 2 minutes by using a 1% phosphotungstic acid solution with the pH of 7.0, the dyeing agent is washed by deionized water, and the mixture is dried for TEM observation.
4. In vivo experiments in mice
4.1. Mouse raising conditions
BALB/C (SPF) male mice, 8 weeks old, weighing 25-30g, were purchased from Beijing Fukang Biotech Co., ltd. Feeding conditions: SPF-class animal laboratory, constant temperature (22-25 deg.C) and constant humidity (55+ -5%) are used for raising.
4.2. Nanocomposite time profile
The mice were injected intravenously with CAT and CAT-PEG samples (n=3) via the tail vein, respectively. The dose of each sample was set at 1mg/kg. Blood samples were collected at 0.1, 1, 2, 4, 6, 12, 24, 36 and 48 hours post injection. Serum was separated from blood by centrifugation at 4,000 x g for 10 min twice. The activity of catalase is monitored by monitoring H during degradation2 O2 Is determined by the rate of decrease of (a). 1mL of H is taken2 O2 Solution (ph=7.4, 0.1M HEPES buffer, H2 O2 Concentration: 0.03% w/v) in a 1mL quartz cuvette, adding 35 mu L of serum sample, and uniformly mixing for 5s; the initial absorbance A1 at 240nm and absorbance A2 after 1min were measured immediately at room temperature. Delta a=a1-A2 is calculated.
The peroxycat activity was calculated according to the formula CAT (U/mL) = [ Δa×v inverse total ∈×d×106] +.v sample +.t=678×Δa.
4.3. Establishment of Lipopolysaccharide (LPS) induced acute liver injury model of mice and test of therapeutic effect
Mice were randomly divided into 3 groups, namely PBS group, CAT-PEG group. 3 mice per group.
Each group of mice is injected with 100 MuL PBS by tail vein respectively; CAT group, CAT-PEG. CAT activity of each group was 5000U/mL. After intravenous injection, each group was intraperitoneally injected with LPS (10 mg/kg). Mice were sacrificed 6 hours after injection and blood and organ samples were collected for further analysis. As a blank, blood and organ samples from another 3 mice not injected with LPS were taken. All mice were perfused with PBS prior to organ collection. The blood sample was centrifuged at 4000 rpm for 15 minutes and the supernatant (serum) was collected and used for further measurement. Serum AST, ALT levels were assessed using a glutamic-pyruvic transaminase (AST) colorimetric activity assay kit, according to the manufacturer's instructions.
4.4. Establishment of mouse liver ischemia reperfusion injury model and treatment effect test
Mice were randomly divided into 3 groups, namely PBS group, CAT-PEG group. 3 mice per group. 3 mice per group. Each group of mice is injected with 100 MuL PBS by tail vein respectively; CAT group, CAT-PEG. CAT activity of each group was 5000U/mL. After 6h tail vein injection, a mouse model of 70% hepatic ischemia-reperfusion (25 min/8 h) injury was established. Briefly, fasted mice (24 hours) were anesthetized and shaved, and then carotid arteries were exposed with a midline cervical incision. The atraumatic clip was then passed over the portal vein, hepatic artery and bile duct above the branch next to the right branch to block approximately 70% of hepatic blood flow for 25 minutes. The clip is then withdrawn and the abdomen closed to allow reperfusion. Mice were sacrificed 8 hours after ischemia and blood and organ samples were collected for further analysis. As a blank, blood and organ samples from 3 additional mice without ischemia-reperfusion procedure were taken. All mice were perfused with PBS prior to organ collection. The blood sample was centrifuged at 4000 rpm for 15 minutes and the supernatant (serum) was collected and used for further measurement. The organ was used for H & E staining for further observation. Plasma AST, ALT was evaluated following the same procedure in the acute hepatitis model section.
4.5. H & E staining
Sequentially placing the slices into xylene I20 min-xylene II 20 min-absolute ethanol I5 min-absolute ethanol II 5min-75% ethanol 5min, and washing with tap water. And then placing the slices into hematoxylin dye liquor for dyeing for 3-5min, washing with tap water, differentiating the differentiation liquor, washing with tap water, returning blue liquor, returning blue, and washing with running water. Then, the slices are dehydrated in gradient alcohol of 85% and 95% for 5min respectively, and then are dyed in eosin dye solution for 5min. Then, sequentially placing the slices into absolute ethyl alcohol I5 min-absolute ethyl alcohol II 5 min-absolute ethyl alcohol III 5 min-dimethyl I5 min-dimethyl II 5min for transparency, and sealing the slices with neutral resin. And finally, microscopic examination and image acquisition and analysis are carried out.
5. Experimental results
The results of this example are as follows:
FIG. 1 compares the zeta potential, where native CAT is negatively charged, and CAT-PEG is adjusted to-0 mV by covalently attaching PEG (FIG. 1). Transmission Electron Microscopy (TEM) imaging showed that CAT-PEG (fig. 2) had a particle size of about 25 nm, which is significantly larger than natural CAT (< 10 nm). FIG. 3 shows that the unencapsulated CAT is completely removed by a purification step such as dialysis and chromatography.
For the therapeutic use of the nanocomposite CAT-PEG, the pharmacokinetics of CAT-PEG in mice were first studied. FIG. 4 shows that CAT-PEG circulates in vivo for a much longer period than natural CAT. This prolonged circulation allows CAT-PEG to accumulate hepatocytes. Liver transplantation is the most common organ transplant and accounts for more than 30% of the total cases, but nevertheless the five-year survival rate of recipients is still less than 75%. The efficacy of CAT-PEG on IRI during liver transplantation was then examined in a liver ischemia model. BALB/c mice were injected intravenously with PBS, CAT and CAT-PEG, respectively. 6 hours after sample injection, microsurgery was performed continuously to establish liver damage by 70% partial ischemia-reperfusion. During reperfusion, the ROS produced oxidize fat deposited in hepatocytes and produce excessive amounts of superoxide, leading to membrane rupture and release of intracellular and membrane-bound enzymes, including aspartate Aminotransferase (AST) and alanine Aminotransferase (ALT), into the blood, which are indicators of liver damage. To monitor liver damage in the liver ischemia model, AST and ALT levels in blood were measured 8 hours post-surgery. As shown in fig. 5 and 6, AST and ALT levels were 866.9 and 183.8 mU/mL, respectively, for CAT-PEG treated mice. These are significantly lower than PBS and CAT injected mice, indicating effective protection against ROS-induced liver injury. In addition, hematoxylin and eosin (H & E) staining was also tested for ROS-induced damage in liver biopsies. As observed in histological examination of the liver, severe vacuolation was observed in liver tissue of mice treated with PBS and CAT, whereas mice treated with CAT-PEG showed a morphology similar to that of blank mice and no lesions in the liver, indicating no reduction in lipid peroxidation. Cell damage (fig. 9). These results demonstrate that CAT-PEG is effective in protecting ROS-induced liver IRI during ischemia reperfusion.
In addition to IRI during organ transplantation, abnormally high levels of ROS are also involved in pathogen-induced inflammatory diseases, tissue degeneration and tissue fibrosis. Thus, scavenging intracellular ROS is of great importance for the prevention and treatment of these diseases. To explore the use of CAT-PEG in other ROS-induced liver diseases, we assessed its role in bacterial endotoxin LPS-induced acute hepatitis models. We treated mice with acute hepatitis by intravenous injection of PBS, CAT and CAT-PEG, and then collected their blood and liver tissue to compare the efficacy of the three treatment regimens. Similar to the results in the IRI model, CAT-PEG reduced to some extent LPS-induced AST (fig. 7) and ALT levels (fig. 8) compared to PBS and CAT treatment, indicating its ability to protect the liver from ROS. A significant reduction in immune cell infiltration was observed in the livers of mice treated with CAT-PEG compared to mice treated with PBS or CAT (fig. 10). These results indicate that CAT-PEG has superior protection against ROS-induced liver injury in general ROS-related liver disease.
In addition to the implementations described above, other implementations of the invention are possible. All technical schemes formed by equivalent substitution or equivalent transformation fall within the protection scope of the invention.