CN115920083A - Curcumin-loaded apoferritin nanocage and application - Google Patents
Curcumin-loaded apoferritin nanocage and application Download PDFInfo
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- CN115920083A CN115920083A CN202310018804.3A CN202310018804A CN115920083A CN 115920083 A CN115920083 A CN 115920083A CN 202310018804 A CN202310018804 A CN 202310018804A CN 115920083 A CN115920083 A CN 115920083A
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
The invention provides a curcumin-loaded apoferritin nanocage and application thereof. The invention shows that the cell uptake of the apoferritin nanocage carrying curcumin is obviously more than that of free curcumin, and the cell uptake can be further increased under the stimulation of hydrogen peroxide, which shows that the apoferritin nanocage carrying curcumin can be rapidly absorbed by renal tubular epithelial cells of ischemia reperfusion-acute renal injury, and has stronger internalization capability under the molding condition. The curcumin-loaded apoferritin nanocage has the advantages that the curcumin-loaded apoferritin nanocage has kidney targeting capability and high kidney retention capability, can not be accumulated in other tissues such as heart, liver, spleen and lung, and can still retain obvious fluorescent signals after 12 hours, so that the curcumin-loaded apoferritin nanocage can be prevented from being rapidly cleared by the kidney to maintain a long-acting effect, and can be used for reducing an extrarenal effect and simultaneously preventing the apoferritin nanocage from being rapidly cleared by the kidney to maintain a strong and durable treatment effect. Can be applied to the preparation of the medicine for treating ischemia-reperfusion-acute kidney injury.
Description
Technical Field
The invention belongs to a pharmaceutical composition, in particular relates to a apoferritin nano cage loaded with curcumin and application thereof, and discloses the apoferritin nano cage loaded with antioxidant curcumin and having an autonomous renal tendency effect and application thereof in preparation of a medicament for treating acute renal injury caused by ischemia-reperfusion.
Background
Acute kidney injury has a globally associated mortality rate far exceeding that of breast cancer, heart failure or diabetes, which is high for the past 50 years. Acute kidney injury is caused by various causes and is mainly characterized by rapid decline of kidney function in a short time and is accompanied by a series of clinical syndromes with serious complications, the acute kidney injury is rapidly developed, and the death rate reaches 23.0 percent. Ischemia reperfusion is one of the main causes of acute kidney injury, and ischemia reperfusion-acute kidney injury can be caused by major surgery, hemorrhage, severe burn and the like, and the early intervention treatment is particularly important. However, the current clinical treatment means mainly comprise support treatment such as kidney replacement treatment and the like, and an effective drug treatment means is not available.
Studies have shown that excessive reactive oxygen species production during the pathological course of ischemia reperfusion-acute kidney injury is the major cause of renal function injury. When acute kidney injury occurs, mitochondrial respiratory chain is inhibited, and electrons leaking out of the respiratory chain are combined with free oxygen taken in by reperfusion to increase the production of active oxygen; in addition, endogenous antioxidant consumption such as decreased antioxidant enzyme activity, decreased glutathione synthesis, etc. results in a significant decrease in the ability to scavenge oxygen radicals; the two kinds of medicine can cause abnormal rise of active oxygen, and induce renal cell oxidative stress, cell apoptosis and renal function injury. Therefore, antioxidant therapy is an effective approach for drug therapy of ischemia reperfusion-acute kidney injury.
Curcumin is a natural antioxidant, is a natural effective component extracted from the root tuber of curcuma longa of curcuma genus of zingiberaceae family, has various pharmacological activities of tumor resistance, inflammation resistance, bacteria resistance, oxidation resistance and the like, has low toxicity and has better clinical application prospect. The anti-oxidation effect of curcumin is related to the molecular structure of curcumin, and active groups in molecules provide protons in the anti-oxidation process to play an anti-oxidation effect. In recent years, researches show that curcumin can be used for treating acute renal injury through ways of removing oxygen free radicals, reducing the generation of inflammatory cytokines and the like, but has a plurality of defects in clinical application: curcumin has low water solubility, poor dissolution rate, easy metabolism in vivo, short half-life and other factors, so that the bioavailability is very low, and the curcumin lacks the selection specificity to kidney tissues, so that the curative effect is unsatisfactory.
In addition, since renal tubular cells are highly sensitive to iron overload, oxidative damage of renal tubular epithelial cells induced by iron overload is also one of the causes of ischemia reperfusion-acute renal injury. Under physiological conditions, the majority of absorbed iron is transferrin bound Fe 3+ Is present in the form of (a), and then reduced to Fe after cellular uptake 2+ . One part of the iron enters the iron bath, and the other part is stored in the iron storage protein. However, under pathological conditions, iron metabolism is disturbed, resulting in intracellular Fe 2+ Overload, which reacts with lipids through fenton's reaction to produce abundant reactive oxygen species, ultimately leading to cellular iron death. One study showed that the use of a selective iron death inhibitor, fer-1, to inhibit iron death in the pathological course of ischemia reperfusion-acute kidney injury, had a significant protective effect. Another study showed that hepcidin mediates the protective effects of renal ischemia-reperfusion injury by dynamically regulating intracellular iron homeostasis. Therefore, it is also a key strategy to treat ischemia reperfusion-acute kidney injury by interfering with iron overload.
Ferritin is one of the major intracellular iron storage proteins, consisting of heavy and light chains, and plays a central role in iron homeostasis. When iron is overloaded, ferritin can have a strong destructive redox activity of the excess Fe 2+ Transported into the cavity and oxidized to form harmless Fe 3+ Then Fe 3+ Migration, nucleation, mineralization and formation of iron nuclei. This ferritin-mediated iron metabolism can protect renal tubular epithelial cells from iron overload-induced oxidative stress injury. Studies have shown that mice with a kidney proximal tubule-specific ferritin heavy chain subunit knockout have a more significant mortality rate and more severe functional kidney damage, further suggesting that ferritin eggs with ferroxidase activityWhite is necessary for iron transport and iron toxicity limitations. In addition, the inner cavity of ferritin may also be desferrized to prepare a desferricin nanocage that may encapsulate various therapeutic agents.
In addition, the size of the protein nanoparticles can affect their biodistribution in vivo. Studies have shown that larger protein nanoparticles typically accumulate more in the liver and spleen, while nanoparticles with a particle size of 10nm and below accumulate more in the kidney. The kidney, which is the main organ for removing foreign substances from the body, filters approximately one-fourth of the cardiac blood output, and small proteins are transported to the kidney for removal after entering the blood circulation. Therefore, the apoferritin nanocages with a particle size of around 8nm are considered to be a natural kidney-targeting nanocarrier.
Disclosure of Invention
The invention aims to provide a curcumin-loaded apoferritin nanocage, which consists of curcumin and the apoferritin nanocage, wherein the mass percentage ranges of the substances are as follows: the apoferritin nanocage is 82.44% -91.40%, and curcumin is 8.60% -17.56%, wherein the types of ferritin comprise Ma Pitie protein, human liver ferritin, and Ma Pi cationic ferritin.
The second purpose of the invention is to provide the application of the apoferritin nanocage carrying curcumin in the preparation of the medicine for treating ischemia-reperfusion-acute kidney injury. The cell uptake experiment result shows that the cell uptake of the apoferritin nanocage group carrying curcumin is obviously more than that of the free curcumin group, and the cell uptake can be further increased under the stimulation of hydrogen peroxide, which indicates that the apoferritin nanocage carrying curcumin can be rapidly absorbed by renal tubular epithelial cells of ischemia reperfusion-acute kidney injury. The in vivo distribution results show that the curcumin-loaded apoferritin nanocages can effectively target the injured kidney tissues without accumulating in other tissues such as heart, liver, spleen, lung and the like, and significant fluorescence signals are still remained in the mouse kidney after 12 hours, which indicates that the curcumin-loaded apoferritin nanocages can be prevented from being rapidly cleared by the kidney and maintain the long-acting effect. The cell efficacy results show that compared with the free curcumin group, the apoferritin nanocage group carrying curcumin shows remarkable treatment effects on various indexes of cell survival rate, oxidative stress level and inflammation level. Histopathology and mitochondrial ultramicro results show that compared with free curcumin, the apoferritin nanocage loaded with curcumin is obviously improved in the renal tubular necrosis, hyaline cast and cell shedding conditions, the mitochondrial swelling conditions and the cristae structure recovery after being used for the treatment of mice subjected to ischemia reperfusion-acute renal injury modeling. Immunohistochemical staining results of nitrotyrosine as a sensitive marker of oxidative stress show that the number of nitrotyrosine positive tubules and the degree of positive reaction are obviously reduced after the curcumin-loaded apoferritin nanocage is used for treating mice subjected to ischemia-reperfusion-acute kidney injury modeling. Immunohistochemical staining results of CD68, the most reliable marker for macrophages, represent the level of inflammation and show that curcumin-loaded apoferritin nanocages have the best anti-inflammatory effect, returning to almost normal levels.
According to the invention, the unique cage-shaped space structure and reversible self-assembly capability of the apoferritin nanocages are utilized to load the antioxidant curcumin, so that the properties of the curcumin such as solubility, stability and hemolysis are improved. Meanwhile, as the kidney is used as a main organ for clearing foreign matters in vitro, the small molecular protein can be transported to the kidney to be cleared after entering blood circulation. The apoferritin nanocage has a kidney passive targeting effect and high kidney retention capacity, and can play a powerful and lasting role in the damaged part. The curcumin and the apoferritin nanocages can respectively reduce active oxygen and absorb overloaded iron to cooperatively play an antioxidation role, so that the condition of iron overload is relieved, and the pathological process of ischemia-reperfusion-acute kidney injury is reversed. The curcumin-loaded apoferritin nanocage represents a potential kidney-targeting nano platform and can be used for the synergistic treatment of ischemia-reperfusion-acute kidney injury.
The invention provides an antioxidant curcumin-loaded apoferritin nanocage. On one hand, the passive targeting property of the apoferritin nanocages to the kidney can realize targeted delivery of curcumin and reduce extrarenal effects. And the hydrophobic core of the apoferritin nanocage can be loaded with curcumin, so that the solubilization of curcumin is realized, and the bioavailability is improved. On the other hand, curcumin and apoferritin nanocages can synergistically play an anti-oxidation role by reducing active oxygen and absorbing overloaded iron respectively, so that the condition of iron overload is relieved, and the pathological process of ischemia-reperfusion-acute kidney injury is reversed. Therefore, the curcumin-loaded apoferritin nanocages may be a kidney-targeting nanoplatform for the synergistic treatment of ischemia reperfusion-acute kidney injury.
Drawings
FIG. 1 is a TEM image of curcumin-loaded desferriferous Ma Pitie protein nanocages (example 1).
FIG. 2 is the results of cellular uptake of curcumin-loaded desferriferous Ma Pitie protein nanocages (example 8).
FIG. 3 is the results of in vivo distribution of curcumin-loaded desferriferous horse spleen ferritin nanocages (example 8).
FIG. 4 is a graph of the effect of curcumin-loaded desferrioxamers Ma Pitie protein nanocages on the expression of inflammatory factors, oxidative stress factors (example 8).
Figure 5 is the histopathological, mitochondrial ultrastructural and immunohistochemical results for curcumin-loaded desferri Ma Pitie protein nanocages (example 8).
Detailed Description
The invention is further explained by the accompanying drawings and examples.
Example 1 preparation of apoferritin nanocages and curcumin-loaded apoferritin Ma Pitie nanocages
1. Preparation of apoferritin nanocage from horse's spleen
The horse spleen ferritin nano cage with iron removed is prepared by demineralizing horse spleen ferritin through reduction reaction and iron chelation. Sodium acetate buffer was prepared by dissolving 41g of sodium acetate in 5L of purified water and adjusting the pH to 5.0 with 1M hydrochloric acid. 5ml of Ma Pitie protein solution (CAS No.: 9007-73-2, product No.: F4503) was diluted to 50mg/ml with 30ml of sodium acetate buffer and transferred to a dialysis bag with a cut-off molecular weight of 8000-14000 Da. The dialysis bag was placed in 1L of sodium acetate buffer solution and subjected to a deoxidation treatment for 1 hour. 2ml of thioglycolic acid were slowly added subsurface in the dark. The whole reaction is carried out in a dark deoxygenated state, the buffer is replaced every 30min, and thioglycolic acid is added again. After about 4-6 buffer replacements, the color of the fluid in the dialysis bag changed from deep red to colorless, at which time the iron core in the Ma Pitie protein was removed. The liquid in the dialysis bag was then sterile filtered through a 0.22 μm filter and dialyzed against deionized water for two days to obtain a desferriequine splenin nanocage. The effect of the apo-ferritin on particle size, as measured by dynamic light scattering, was minimal, with both ferritin and apoferritin nanocages having a particle size of about 9nm. The residual iron content in the apoferritin nanocages was 4.17 ± 0.15% as determined by inductively coupled plasma mass spectrometry (ICP-MS). The result of transmission electron microscopy is shown in fig. 1, in which the iron core in ferritin is removed and the ferritin nanocages still maintain a hollow spherical structure. The successful preparation of the apoferritin nanocages is proved by the experiments.
2. Preparation of curcumin-loaded deferrization Ma Pitie protein nanocage
Curcumin was encapsulated into the cavity of a desferriferous Ma Pitie protein nanocage by a subunit disassembly/recombination strategy based on pH change. First, the pH of the solution of apoferritin nanocages was adjusted to 12.0 by the addition of an appropriate volume of 1M sodium hydroxide, at which time the subunits of apoferritin nanocages were considered to have depolymerized. After 5min, 60. Mu.l curcumin in aqueous sodium hydroxide (50 mg/ml) was added. The pH was quickly adjusted to 7.0 using 1M hydrochloric acid, at which time the solution changed color from light yellow to dark red to orange yellow. The resulting solution was stirred at room temperature for 2 hours to facilitate protein assembly. Then centrifugating (4000rpm, 5min) to remove free curcumin, and obtaining the curcumin-loaded deferrization Ma Pitie protein nanocage. The obtained protein suspension is ultrafiltered, centrifugally concentrated to about 1ml, placed in a refrigerator at the temperature of 80 ℃ below zero for 4 to 6 hours, and then freeze-dried for 12 hours to obtain yellow powder. The particle size of the drug loaded by the dynamic light scattering instrument is increased to about 14nm, the result of the transmission electron microscope is shown in figure 1, the particle size of the apoferritin nano cage is increased, and the result is consistent with the data of the dynamic light scattering instrument. The curcumin drug loading rate can be measured by a fluorescence spectrophotometer to be 17.56 percent.
The data prove that the curcumin-loaded deferral Ma Pitie protein nano cage is successfully prepared, wherein the mass percentages of the substances are as follows: the ferritin nanocage is 82.44% and curcumin is 17.56%.
Example 2 preparation of Deferralid spleen ferritin nanocages and curcumin-loaded Deferralid Ma Pitie protein nanocages
1. Preparation of apoferritin nanocages
The horse spleen ferritin demineralization nano cage with iron removed is prepared by carrying out reduction reaction and iron chelation on the horse spleen ferritin demineralization nano cage. Sodium acetate buffer was prepared by dissolving 41g of sodium acetate in 5L of purified water and adjusting the pH to 5.0 with 1M hydrochloric acid. 5ml of Ma Pitie protein solution (CAS No.: 9007-73-2, product No.: F4503) was diluted to 50mg/ml with 30ml of sodium acetate buffer and transferred to a dialysis bag with a cut-off molecular weight of 8000-14000 Da. The dialysis bag was placed in 1L of sodium acetate buffer solution and subjected to a deoxidation treatment for 1 hour. 2ml of thioglycolic acid were slowly added subsurface in the dark. The whole reaction is carried out in a dark deoxygenated state, the buffer is replaced every 30min, and thioglycolic acid is added again. After about 4-6 buffer replacements, the color of the fluid in the dialysis bag changed from deep red to colorless, at which time the iron core in the Ma Pitie protein was removed. The liquid in the dialysis bag was then sterile filtered through a 0.22 μm filter and dialyzed against deionized water for two days to obtain a desferriequine splenin nanocage. The effect of the apo-ferritin on particle size, as measured by dynamic light scattering, was minimal, with both ferritin and apoferritin nanocages having a particle size of about 9nm. The residual iron content in the apoferritin nanocages was 8.15 ± 2.13% as determined by inductively coupled plasma mass spectrometry (ICP-MS). The result of the transmission electron microscope shows that the iron core in the ferritin is removed, and the ferritin nano cage still maintains a hollow spherical structure. The successful preparation of the apoferritin nanocages is proved by the experiments.
2. Preparation of curcumin-loaded deferrization Ma Pitie protein nanocage
Curcumin was encapsulated into the cavity of a desferriferous Ma Pitie protein nanocage by a subunit disassembly/recombination strategy based on pH change. First, the pH of the solution of apoferritin nanocages was adjusted to 2.0 by adding an appropriate volume of 1M hydrochloric acid solution, at which time the subunits of apoferritin nanocages were considered to have depolymerized. After 5min, 60. Mu.l curcumin in aqueous sodium hydroxide (50 mg/ml) was added. The pH was quickly adjusted to 7.0 using 1M sodium hydroxide solution, at which time the solution changed color from light yellow to dark red to orange yellow. The resulting solution was stirred at room temperature for 2 hours to facilitate protein assembly. Then centrifugating (4000rpm, 5min) to remove free curcumin, and obtaining the curcumin-loaded deferrization Ma Pitie protein nanocage. The obtained protein suspension is ultrafiltered, centrifugally concentrated to about 1ml, placed in a refrigerator at the temperature of 80 ℃ below zero for 4 to 6 hours, and then freeze-dried for 12 hours to obtain yellow powder. The particle size of the drug loaded is increased to about 12nm according to the measurement of the dynamic light scattering instrument, and the result of the transmission electron microscope shows that the particle size of the apoferritin nano cage is increased, and the result is consistent with the data of the dynamic light scattering instrument. The curcumin drug loading rate can be measured by a fluorescence spectrophotometer to be 12.05%.
The data prove that the curcumin-loaded deferral Ma Pitie protein nano cage is successfully prepared, wherein the mass percentages of the substances are as follows: the apoferritin nanocage is 87.95% and curcumin is 12.05%.
Example 3 preparation of Dermatophagoides pteronyssinus ferritin nanocages and curcumin-loaded Dermatophagoides Ma Pitie protein nanocages
1. Preparation of apoferritin nanocages
The horse spleen ferritin demineralization nano cage with iron removed is prepared by carrying out reduction reaction and iron chelation on the horse spleen ferritin demineralization nano cage. Sodium acetate buffer was prepared by dissolving 41g of sodium acetate in 5L of purified water and adjusting the pH to 5.0 with 1M hydrochloric acid. 5ml of Ma Pitie protein solution (CAS number: 9007-73-2, product number: F4503) was diluted to 50mg/ml with 30ml of sodium acetate buffer and transferred to a dialysis bag with a molecular weight cut-off of 8000-14000 Da. The dialysis bag was placed in 1L of sodium acetate buffer solution and subjected to a deoxidation treatment for 1 hour. 2ml of thioglycolic acid were slowly added subsurface in the dark. The whole reaction is carried out in a dark deoxygenated state, the buffer is replaced every 30min, and thioglycolic acid is added again. After about 4-6 buffer replacements, the color of the dialysis bag changed from deep red to colorless, at which time the iron core in the Ma Pitie protein was removed. The fluid in the dialysis bag was then sterile filtered through a 0.22 μm filter and dialyzed against deionized water for two days to obtain a desferriequine splenin nanocage. The effect of the deferrization of ferritin on particle size, as measured by dynamic light scattering instruments, was small, with ferritin and apoferritin nanocages having a particle size of about 9nm. The residual iron content in the apoferritin nanocages was 9.79 ± 1.56% as determined by inductively coupled plasma mass spectrometry (ICP-MS). The result of the transmission electron microscope shows that the iron core in the ferritin is removed, and the ferritin nano cage still maintains a hollow spherical structure. The successful preparation of the apoferritin nanocages is proved by the experiments.
2. Preparation of curcumin-loaded deferrization Ma Pitie protein nanocage
Curcumin was encapsulated into the cavity of a desferriferous Ma Pitie protein nanocage by a subunit disassembly/recombination strategy based on pH value changes. First, the pH of the solution of apoferritin nanocages was adjusted to 10.0 by the addition of an appropriate volume of 1M sodium hydroxide, at which time the subunits of apoferritin nanocages were considered to have depolymerized. After 5min, 90. Mu.l curcumin in aqueous sodium hydroxide (50 mg/ml) was added. The pH was quickly adjusted to 7.0 using 1M hydrochloric acid, at which time the solution changed color from light yellow to dark red to orange yellow. The resulting solution was stirred at room temperature for 2 hours to facilitate protein assembly. Then centrifugating (4000rpm, 5min) to remove free curcumin, and obtaining the curcumin-loaded deferrization Ma Pitie protein nanocage. The obtained protein suspension is ultrafiltered, centrifugally concentrated to about 1ml, placed in a refrigerator at the temperature of 80 ℃ below zero for 4 to 6 hours, and then freeze-dried for 12 hours to obtain yellow powder. The particle size of the drug loaded by the dynamic light scattering instrument is increased to about 15nm, and the result of a transmission electron microscope shows that the particle size of the apoferritin nano cage is increased, and the result is consistent with the data of the dynamic light scattering instrument. The curcumin drug loading rate can be measured by a fluorescence spectrophotometer to be 16.79%.
The data prove that the curcumin-loaded deferral Ma Pitie protein nano cage is successfully prepared, wherein the mass percentages of the substances are as follows: the ferritin nanocage is 83.21% and curcumin is 16.79%.
Example 4 preparation of Desferricin Nanocamp and curcumin-loaded Desferricin Ma Pitie protein Nanocamp
1. Preparation of apoferritin nanocages
The horse spleen ferritin demineralization nano cage with iron removed is prepared by carrying out reduction reaction and iron chelation on the horse spleen ferritin demineralization nano cage. Sodium acetate buffer was prepared by dissolving 41g of sodium acetate in 5L of purified water and adjusting the pH to 5.0 with 1M hydrochloric acid. 5ml of Ma Pitie protein solution (CAS No.: 9007-73-2, product No.: F4503) was diluted to 50mg/ml with 30ml of sodium acetate buffer and transferred to a dialysis bag with a cut-off molecular weight of 8000-14000 Da. The dialysis bag was placed in 1L of sodium acetate buffer solution and subjected to a deoxidation treatment for 1 hour. 2ml of thioglycolic acid were slowly added subsurface in the dark. The whole reaction is carried out in a dark deoxygenated state, the buffer is replaced every 30min, and thioglycolic acid is added again. After about 4-6 buffer replacements, the color of the dialysis bag changed from deep red to colorless, at which time the iron core in the Ma Pitie protein was removed. The fluid in the dialysis bag was then sterile filtered through a 0.22 μm filter and dialyzed against deionized water for two days to obtain a desferriequine splenin nanocage. The effect of the deferrization of ferritin on particle size, as measured by dynamic light scattering instruments, was small, with ferritin and apoferritin nanocages having a particle size of about 9nm. The residual iron content in the apoferritin nanocages was 14.36 ± 2.68% as determined by inductively coupled plasma mass spectrometry (ICP-MS). The result of the transmission electron microscope shows that the iron core in the ferritin is removed, and the ferritin nano cage still maintains a hollow spherical structure. The above experiments demonstrate the successful preparation of apoferritin nanocages.
2. Preparation of curcumin-loaded deferrization Ma Pitie protein nanocage
Curcumin was encapsulated into the cavity of a desferriferous Ma Pitie protein nanocage by a subunit disassembly/recombination strategy based on pH change. First, the pH of the solution of apoferritin nanocages was adjusted to 12.0 by the addition of an appropriate volume of 1M sodium hydroxide, at which time the subunits of apoferritin nanocages were considered to have depolymerized. After 10min, 90. Mu.l curcumin in aqueous sodium hydroxide (50 mg/ml) was added. The pH was quickly adjusted to 8.0 using 1M hydrochloric acid, at which time the solution changed color from light yellow to dark red to orange yellow. The resulting solution was stirred at room temperature for 2 hours to facilitate protein assembly. Then centrifugating (4000rpm, 5min) to remove free curcumin, and obtaining the curcumin-loaded de-ferred Ma Pitie protein nano cage. The result of a transmission electron microscope shows that the ferritin nanocage is loaded with the drug, and the diameter of the nanocage is increased. The obtained protein suspension is ultrafiltered, centrifugally concentrated to about 1ml, placed in a refrigerator at the temperature of 80 ℃ below zero for 4 to 6 hours, and freeze-dried for 12 hours to obtain yellow powder. The particle size of the drug loaded is increased to about 10nm according to the measurement of the dynamic light scattering instrument, and the result of the transmission electron microscope shows that the particle size of the apoferritin nano cage is increased, and the result is consistent with the data of the dynamic light scattering instrument. The curcumin drug loading rate can be measured by a fluorescence spectrophotometer to be 14.54 percent.
The data prove that the curcumin-loaded deferrization Ma Pitie protein nano cage is successfully prepared, wherein the mass percentages of the substances are as follows: the apoferritin nanocage is 85.46% and curcumin is 14.54%.
Example 5 preparation of De-ferrated human liver ferritin nanocages and curcumin-loaded De-ferrated human liver ferritin nanocages
1. Preparation of iron-removed human liver ferritin nanocage
The iron-removed human liver ferritin nanocage is prepared by demineralizing human liver ferritin through reduction reaction and iron chelation. Sodium acetate buffer was prepared by dissolving 41g of sodium acetate in 5L of purified water and adjusting the pH to 5.0 with 1M hydrochloric acid. 5ml of human liver ferritin solution (CAS number 9007-73-2, product number F6754) was diluted to 50mg/ml with 30ml of sodium acetate buffer solution, and transferred to a dialysis bag with a cut-off molecular weight of 8000-14000 Da. The dialysis bag was placed in 1L of sodium acetate buffer solution and subjected to a deoxidation treatment for 1 hour. 2ml of thioglycolic acid were slowly added subsurface in the dark. The whole reaction process is carried out in a dark deoxygenated state, the buffer solution is replaced every 30min, and thioglycolic acid is added again. After about 4-6 buffer replacements, the color of the dialysis bag changed from deep red to colorless, at which time the iron core of the human liver ferritin was removed. The liquid in the dialysis bag was then sterile filtered through a 0.22 μm filter and dialyzed against deionized water for two days to obtain a desferriferous human liver ferritin nanocage. The iron-removing effect of ferritin measured by dynamic light scattering instrument has little effect on the particle size, and both ferritin and apoferritin nanocages have particle size of about 10nm. The residual iron content in the apoferritin nanocages was 7.27 ± 1.37% as determined by inductively coupled plasma mass spectrometry (ICP-MS). The result of a transmission electron microscope shows that the iron core in the ferritin is removed, and the ferritin nano cage of the iron-removed human liver still maintains a hollow spherical structure. The experiments prove the successful preparation of the iron-removed human liver ferritin nanocage.
2. Preparation of curcumin-loaded desferriferous human liver ferritin nanocage
Curcumin was encapsulated into the cavity of the desferriferous human hepatic ferritin nanocages by a subunit disassembly/recombination strategy based on pH change. First, the pH of the solution of desferriferous human hepatic ferritin nanocages was adjusted to 12.0 by the addition of an appropriate volume of 1M sodium hydroxide, at which time the subunits of the desferriferous human hepatic ferritin nanocages were considered to have depolymerized. After 5min, 70. Mu.l curcumin in aqueous sodium hydroxide (50 mg/ml) was added. The pH was quickly adjusted to 7.0 using 1M hydrochloric acid, at which time the solution changed color from light yellow to dark red to orange yellow. The resulting solution was stirred at room temperature for 2 hours to facilitate protein assembly. Then centrifuging (4000rpm, 5min) to remove free curcumin, and obtaining the curcumin-loaded deferrization human liver ferritin nanocage. The obtained protein suspension is ultrafiltered, centrifugally concentrated to about 1ml, placed in a refrigerator at the temperature of 80 ℃ below zero for 4 to 6 hours, and then freeze-dried for 12 hours to obtain yellow powder. The particle size of the drug loaded is increased to about 18nm according to the measurement of the dynamic light scattering instrument, and the result of the transmission electron microscope shows that the particle size of the apoferritin nano cage is increased, and the result is consistent with the data of the dynamic light scattering instrument. The curcumin drug loading rate can be measured by a fluorescence spectrophotometer to be 8.95 percent.
The data prove that the curcumin-loaded deferral Ma Pitie protein nano cage is successfully prepared, wherein the mass percentages of the substances are as follows: the apoferritin nanocage is 91.05% and curcumin is 8.95%.
Example 6 preparation of equine splenic cationic ferritin nanocages and curcumin-loaded equine splenic cationic ferritin nanocages
1. Preparation of Ma Pi cation ferritin nano cage
Ma Pi cationic ferritin nanocages were prepared by demineralization of equine splenic ferritin by reduction and iron chelation. Sodium acetate buffer was prepared by dissolving 41g of sodium acetate in 5L of purified water and adjusting the pH to 5.0 with 1M hydrochloric acid. 5ml Ma Pi cationic ferritin solution (CAS number: 9007-73-2, product number: F7879) was diluted to 50mg/ml with 30ml sodium acetate buffer and transferred to dialysis bag with a molecular weight cut-off of 8000-14000 Da. The dialysis bag was placed in 1L of sodium acetate buffer solution and subjected to a deoxidation treatment for 1 hour. 2ml of thioglycolic acid were slowly added subsurface in the dark. The whole reaction process is carried out in a dark deoxygenated state, the buffer solution is replaced every 30min, and thioglycolic acid is added again. After about 4-6 buffer replacements, the color of the dialysis bag fluid changed from deep red to colorless, at which time the iron core of Ma Pi cationic ferritin was removed. The fluid in the dialysis bag was then sterile filtered through a 0.22 μm filter and dialyzed against deionized water for two days to yield Ma Pi cationic ferritin nanocages. The effect of the deferrization of ferritin on particle size, as measured by dynamic light scattering instruments, was small, with ferritin and apoferritin nanocages having particle sizes of about 8nm. The residual iron content in the apoferritin nanocages was 11.24 ± 0.37% as determined by inductively coupled plasma mass spectrometry (ICP-MS). The result of a transmission electron microscope shows that the iron core in the ferritin is removed, and the deferrization Ma Pi cationic ferritin nano cage still maintains a hollow spherical structure. The above experiments demonstrate the successful preparation of apoferritin nanocages from spleen cations.
2. Preparation of Ma Pi cationic ferritin nanocage carrying curcumin
Curcumin was encapsulated into the cavity of Ma Pitie cationic ferritin nanocages by a subunit disassembly/recombination strategy based on pH change. First, the pH of Ma Pi cationic ferritin nanocage solution was adjusted to 12.0 by the addition of an appropriate volume of 1M sodium hydroxide, at which time the subunits of Ma Pi cationic ferritin nanocage were considered to have depolymerized. After 5min, 70. Mu.l curcumin in aqueous sodium hydroxide (50 mg/ml) was added. The pH was quickly adjusted to 7.0 using 1M hydrochloric acid, at which time the solution changed color from light yellow to dark red to orange yellow. The resulting solution was stirred at room temperature for 2 hours to facilitate protein assembly. Then centrifuging (4000rpm, 5min) to remove free curcumin, and obtaining Ma Pi cationic ferritin nanocages loaded with curcumin. The obtained protein suspension is ultrafiltered, centrifugally concentrated to about 1ml, placed in a refrigerator at the temperature of 80 ℃ below zero for 4 to 6 hours, and then freeze-dried for 12 hours to obtain yellow powder. The particle size of the drug loaded is increased to about 10nm according to the measurement of the dynamic light scattering instrument, and the result of the transmission electron microscope shows that the particle size of the apoferritin nano cage is increased, and the result is consistent with the data of the dynamic light scattering instrument. The curcumin drug loading rate can be measured by a fluorescence spectrophotometer to be 10.65%.
The data prove that the curcumin-loaded deferral Ma Pitie protein nano cage is successfully prepared, wherein the mass percentages of the substances are as follows: the apoferritin nanocage is 89.35% and curcumin is 10.65%.
Example 7 preparation of Cy 5-labeled deferral Ma Pitie protein nanocages
The horse spleen ferritin nano cage with iron removed is prepared by demineralizing horse spleen ferritin through reduction reaction and iron chelation. Sodium acetate buffer was prepared by dissolving 41g of sodium acetate in 5L of purified water and adjusting the pH to 5.0 with 1M hydrochloric acid. 5ml of Ma Pitie protein solution is diluted to 50mg/ml with 30ml of sodium acetate buffer solution and transferred to a dialysis bag with a molecular weight cut-off of 8000-14000 Da. The dialysis bag was placed in 1L of sodium acetate buffer solution and subjected to a deoxidation treatment for 1 hour. 2ml of thioglycolic acid were slowly added subsurface in the dark. The whole reaction is carried out in a dark deoxygenated state, the buffer is replaced every 30min, and thioglycolic acid is added again. After about 4-6 buffer replacements, the color of the dialysis bag changed from deep red to colorless, at which time the iron core in the Ma Pitie protein was removed. The fluid in the dialysis bag was then sterile filtered through a 0.22 μm filter and dialyzed against deionized water for two days to obtain a desferriequine splenin nanocage. The effect of the apo-ferritin on particle size, as measured by dynamic light scattering, was minimal, with both ferritin and apoferritin nanocages having a particle size of about 9nm. The residual iron content in the apoferritin nanocages was 4.17 ± 0.15% as determined by inductively coupled plasma mass spectrometry (ICP-MS). The result of a transmission electron microscope shows that the iron core in the ferritin is removed, and the deferrization Ma Pi cationic ferritin nano cage still maintains a hollow spherical structure. The Cy5 fluorescence labeling protein kit is used for successfully labeling the ferremarginine nano cage with fluorescence through steps of coupling reaction, room-temperature light-shielding reaction, ultrafiltration centrifugation and the like, and the Cy5 labeled ferremarginine nano cage is successfully prepared.
Example 8 application of curcumin-loaded desferriferous equine splenic ferritin nanocages to treatment of ischemia reperfusion-acute renal injury
1. Cell uptake study of curcumin-loaded desferri Ma Pitie protein nanocage
Example 1 was prepared. Tubular epithelial cells of kidney were cultured at 2X 10 4 The density of individual cells/well was placed in a 24-well plate with coverslips placed in advance and incubated overnight. Then, the curcumin-loaded apoferritin nanocages, free curcumin (final concentrations of curcumin were 6.4 μ g/ml each) were added to the corresponding wells. After incubation for 4h, cells were fixed and mounted. Cellular internalization of curcumin-loaded apoferritin nanocages and free curcumin by renal tubular epithelial cells was studied using confocal laser microscopy. The result is shown in fig. 2, the cell internalization of the apoferritin nanocage group loaded with curcumin is obviously better than that of the free curcumin group, and the cell uptake capacity is obviously increased under the stimulation of hydrogen peroxide. The hydrogen peroxide stimulated cell selectivity may be due to strong membrane lipid peroxidation and oxidative stress, resulting in an increase in the selective permeability of the plasma membrane. The experimental result shows that the apoferritin nano cage carrying curcumin can be quickly internalized by renal tubular epithelial cells of ischemia reperfusion-acute kidney injury and enter the cells to play a role.
2. In vivo distribution study of curcumin-loaded deferrization Ma Pitie protein nanocages
Example 1, example 6 was prepared. ICR mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital (50 mg/kg) and were clamped to the bilateral renal pedicles to cause renal ischemia. Mice were placed on a 37 degree celsius heating table to maintain body temperature during ischemia. After 30min, the two clamps were released and an AKI mouse model was established. Cy 5-labeled ferritin nanoparticles were prepared to assess their distribution in vivo. Immediately after the AKI model was established, mice were injected with cy 5-labeled apoferritin nanocages and healthy mice were used as controls. Mice were sacrificed 4 or 12 hours post injection to obtain vital organs including heart, liver, spleen, lung and kidney, and fluorescence visualized with an in vivo imaging system. In vivo distribution results are shown in fig. 3, the curcumin-loaded apoferritin nanocages can effectively target damaged kidney tissue without accumulating in other tissues such as heart, liver, spleen, lung, etc. And after 12 hours, a significant fluorescence signal still remained in the mouse kidney, which indicates that the curcumin-loaded apoferritin nanocage can avoid being rapidly cleared by the kidney to maintain a long-acting effect.
3. Cell efficacy study of curcumin-loaded deferral Ma Pitie protein nanocage
Example 1 was prepared. The ischemia reperfusion-acute kidney injury model was established 30 minutes after the mice were clamped by bilateral renal arteries, and the mice were sacrificed 2 days after administration and the kidneys were collected and homogenized. The oxidative stress level is detected by using an active oxygen, lipid peroxide malondialdehyde and superoxide dismutase kit, and the inflammation level is detected by using an elisa kit interleukin-6 and a tumor necrosis factor. The results are shown in fig. 4, compared with the free curcumin group, the apoferritin nano-cage group carrying curcumin shows remarkable treatment effect on each index of oxidative stress level and inflammation level.
4. Histopathology, mitochondrial ultrastructure and immunohistochemical research of curcumin-loaded deferral Ma Pitie protein nanocage
Example 1 was prepared. Immunohistochemistry and HE staining are carried out, and part of kidney is taken and placed in glutaraldehyde for sample preparation and transmission electron microscope observation. The results are shown in fig. 5, compared with free curcumin, the apoferritin nanocages loaded with curcumin are used for treating mice modeled after ischemia-reperfusion-acute renal injury, the renal tubular necrosis, the transparent casting mold and the cell shedding of the renal structure are obviously improved, the morphology of the mitochondrial cristae is reduced or disappeared, the mitochondrial swelling is also obviously improved, and the structure of the cristae is recovered. Immunohistochemical staining results of nitrotyrosine as a sensitive marker of oxidative stress show that the number of nitrotyrosine positive tubules and the degree of positive reaction are obviously reduced after the curcumin-loaded apoferritin nanocage is used for treating mice subjected to ischemia-reperfusion-acute kidney injury modeling. Immunohistochemical staining results of CD68, the most reliable marker for macrophages, represent the level of inflammation and show that curcumin-loaded apoferritin nanocages have the best anti-inflammatory effect, returning to almost normal levels.
Claims (5)
1. A curcumin-loaded apoferritin nanocage is characterized by consisting of curcumin and the apoferritin nanocage, wherein the mass percentage ranges of the substances are as follows: the apoferritin nano cage is 82.44% -91.40% and curcumin is 8.60% -17.56%.
2. The apoferritin nanocage loaded with curcumin as claimed in claim 1, wherein apoferritin is selected from Ma Pitie protein, human hepatic ferritin or equine splenic cationic ferritin.
3. A curcumin-loaded apoferritin nanocage according to claim 1 or 2 for use in the manufacture of a medicament for the treatment of ischemia reperfusion-acute kidney injury.
4. The use according to claim 3, wherein the curcumin-loaded apoferritin nanocages are rapidly taken up by tubular epithelial cells and have a greater capacity to internalize under molding conditions.
5. The use of claim 3, wherein the curcumin-loaded apoferritin nanocage has potent kidney targeting and high kidney retention, reducing extrarenal effects while avoiding rapid clearance by the kidney and maintaining potent and long lasting therapeutic effect.
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