CN113350520A - Hydrogen sulfide controlled-release brain targeting nano system for protecting nervous system after cardiac arrest and preparation method thereof - Google Patents
Hydrogen sulfide controlled-release brain targeting nano system for protecting nervous system after cardiac arrest and preparation method thereof Download PDFInfo
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Abstract
The invention belongs to the field of medicinal preparations, relates to a brain targeting nano system, and particularly relates to a hydrogen sulfide controlled release brain targeting nano system for protecting a nervous system after cardiac arrest and a preparation method thereof. The mesoporous iron oxide nanoparticles of the nano system can avoid a mononuclear phagocytic system and keep stable for a long time in systemic circulation; the assembled lactoferrin LF can be combined with lactoferrin receptors which are distributed on the surface of the brain capillary endothelial cells in a large number, so that receptor-mediated brain targeted transfer is realized; the nano particles have good safety and small cytotoxicity, can efficiently play a role in protecting the nervous system with low toxicity, and have higher clinical application and transformation values.
Description
Technical Field
The invention belongs to the field of pharmaceutical preparations, relates to a brain targeting nano system, and particularly relates to a low-toxicity hydrogen sulfide controlled-release brain targeting nano system for protecting a nervous system after cardiac arrest and a preparation method thereof.
Background
With the disclosure of hydrogen sulfide as an endogenous metabolite, in recent 10 years, researchers at home and abroad have conducted a great deal of research on the physiological significance of hydrogen sulfide and its important role in nervous system injury. A number of studies have shown that: the hydrogen sulfide can inhibit the apoptosis of the neurons, reduce the generation of free radicals and play an effective role in protecting the excitatory damage of the neurons caused by the rise of the glutamic acid. Meanwhile, hydrogen sulfide can regulate cerebral blood supply by affecting vascular endothelial cells and smooth muscle cells. Of particular note, hydrogen sulfide exhibits excellent protective effects in a variety of related models of nervous system injury. However, current hydrogen sulfide research still lacks an ideal sulfur donor. In research practice, the most commonly used hydrogen sulfide donor is a saturated solution of hydrogen sulfide or a sodium hydrosulfide (NaHS) solution, both of which can effectively provide hydrogen sulfide, but are very unstable, toxic and smelly. It is disclosed in the literature that the use of NaHS often results in inconsistent or hard to repeat experimental phenomena, possibly due to its unstable nature. Another hydrogen sulfide donor, GYY4137, can slowly release hydrogen sulfide in vivo and in vitro, however, the hydrogen sulfide concentration is low, and the release rate cannot be controlled, so that the application is limited. Other hydrogen sulfide donors all have more or less of the above disadvantages, making the search for new donors of hydrogen sulfide even more urgent.
Diallyl trisulfide (DATS) is an allicin extract which can effectively release hydrogen sulfide in the presence of Glutathione (GSH), but has high release speed and high concentration and cannot be regulated; and DATS is insoluble in water, these drawbacks limit its application as a hydrogen sulfide donor. gamma-Fe2O3The nano particle is a superparamagnetic iron oxide material widely applied to the field of biomedicine, the metabolism and excretion processes of the nano particle are very similar to those of physiological iron, and related researches also prove that the iron oxide particle can be effectively degraded in a living body, and the safety of the nano particle enables Mesoporous Iron Oxide (MIONs) to be an ideal choice for constructing an in-vivo donor system. Meanwhile, MIONs can react with various functional groups under mild conditions, so that targeting and long-circulating transport of a carrier system can be realized through surface modification of appropriate groups.
Based on the foundation and the current situation of the prior art, the inventor of the application intends to provide a novel brain targeting nano system, and particularly relates to a low-toxicity hydrogen sulfide controlled-release brain targeting nano system for protecting the nervous system after cardiac arrest.
Disclosure of Invention
The invention aims to provide a hydrogen sulfide donor drug DATS nano delivery system based on the foundation and the current situation of the prior art, and particularly relates to a hydrogen sulfide controlled release brain targeting nano system for protecting a nervous system after sudden cardiac arrest and a preparation method thereof.
Specifically, the nano system takes mesoporous iron oxide nano particles as an inner core to entrap DATS, and the outer layer is sequentially modified by bifunctional PEG and LF through a chemical combination way to construct a low-toxicity hydrogen sulfide controlled-release brain targeting nano system (DATS @ MION-PEG-LF) for targeting delivery of drugs to the brain, and DATS is slowly released in a longer time to maintain the treatment effect.
More specifically, the invention provides a hydrogen sulfide controlled-release brain-targeted nano system for protecting a nervous system after cardiac arrest, wherein mesoporous iron oxide nanoparticles are prepared by a pyrolysis method, bifunctional PEG Mal-PEG-NHS and lactoferrin are used for surface modification of the nanoparticles, DATS is used as a hydrogen sulfide donor drug, and the hydrogen sulfide controlled-release brain-targeted nano system DATS @ MION-PEG-LF is constructed; in the nano system, the hydrogen sulfide donor medicament DATS encapsulated in the nano kernel constructed by the mesoporous ferric oxide has higher stability and encapsulation efficiency, and the nano system has the effect of improving brain targeted transport.
In the invention, the adopted model medicine is garlic extract propyl trisulfide Diallyl (DATS); after the drug-loaded particles cross the blood brain barrier, the DATS slowly released in the mesopores is combined with the GSH in the tissue environment, so that the hydrogen sulfide can be slowly released in a longer time.
The DATS @ MION-PEG-LF provided by the invention is used as a novel drug carrier and is proved to have good biological safety.
The inner layer surface modification group adopted by the invention is bifunctional PEG maleimide-polyethylene glycol-aminosuccinimide succinate (Mal-PEG-NHS), wherein the iminosuccinate group is condensed with the surface amino group of the aminated mesoporous iron oxide nanoparticle, and the maleimide at the other end provides a reaction site for the connection of the outer layer surface modification group on the surface of the nanoparticle;
after the surface of the ferric oxide nano-ions is modified by the PEG, the ferric oxide nano-ions can be prevented from being identified by macrophages, so that the ferric oxide nano-ions can avoid the cleaning effect of a reticuloendothelial system (RES) in the liver and the spleen, and the in vivo circulation time of the nano-particles is prolonged; in addition, the mutual repulsion force between the long chains of PEG helps to prevent the agglomeration of the iron oxide nanoparticles and maintain the stability of the iron oxide nanoparticles in the solution.
The second surface modification group adopted by the invention is lactoferrin, the lactoferrin can be combined with lactoferrin receptors distributed on the surface of Brain Capillary Endothelial Cells (BCECs) in a large quantity, the nano system is delivered to a brain part in a targeted mode, and efficient blood brain barrier crossing transportation of the nano system is realized based on a receptor-mediated endocytosis path.
The construction method of the nanoparticles adopted by the invention is as follows: mesoporous iron oxide nanoparticles are prepared by a pyrolysis method, surface modification of the nanoparticles is carried out by bifunctional PEG Mal-PEG-NHS and lactoferrin, and DATS is used as a hydrogen sulfide donor drug entrapped in the mesoporous iron oxide nanoparticle inner core.
The neuron cells adopted by the invention are primary rat cortical neuron cells, and are obtained by self-culture of SD (Sprague Dawley) suckling rats after material collection.
The carrier system is proved to have good biological safety by immunohistochemical experiments; hydrogen sulfide release experiments prove that the drug delivery system can slowly release hydrogen sulfide for a long time; brain targeting experiments prove that the drug delivery system can specifically target the brain part, and in vivo pharmacodynamics experiments prove that the drug delivery system can practically play a role in protecting neurons.
The invention designs a surface-modified mesoporous iron oxide nano delivery system based on the research result of the prior art, uses mesoporous iron oxide nano particles as cores to entrap hydrogen sulfide donor drugs, modifies the surface of the particles with a brain-targeting group lactoferrin in a bifunctional PEG bridging manner, constructs a low-toxicity hydrogen sulfide controlled-release brain-targeting nano system (DATS @ MION-PEG-LF), targets the delivery drugs at the brain part, and slowly releases the delivery drugs in a longer time to maintain the treatment effect. Compared with the traditional hydrogen sulfide drug delivery system, the nano delivery system based on the mesoporous iron oxide core has good safety, and the cell level shows that the drug delivery system has low cytotoxicity; on the animal level, no obvious pathological changes are found in important organs such as heart, liver, kidney and brain of a nude mouse after the drug-loaded nanoparticles are repeatedly administered, which shows that the drug-loaded nanoparticles can efficiently and lowly exert the effect of a nervous system and have higher clinical application prospect and conversion value.
Drawings
FIG. 1 is a DATS @ MION-PEG-LF model diagram.
FIG. 2 is a scheme showing the synthesis of DATS @ MION-PEG-LF.
FIG. 3 is a representation of a surface-modified mesoporous iron oxide nano delivery system, wherein,
(a) (b) DATS @ MION-PEG-LF transmission electron microscopy picture,
(c) particle size distribution of DATS @ MION-PEG-LF,
(d) drug loading of DATS @ MION-PEG-LF.
(e) (f) (g) (h) Infrared Spectroscopy of MION-PEG-LF.
FIG. 4. safety experiments with DATS @ MION-PEG-LF, in which,
(a) in vitro safety tests were carried out on the samples,
(b) in vivo safety experiments.
FIG. 5. evaluation of brain targeting properties of DATS @ MION-PEG-LF, wherein,
(a) (b) in vitro fluorescence experiment picture,
(c) the fluorescence density distribution.
FIG. 6 evaluation of hydrogen sulfide release of DATS @ MION-PEG-LF, wherein,
(a) the concentration of hydrogen sulfide in the in vitro environment changes along with the time,
(b) the concentration of hydrogen sulfide in the plasma varies with time,
(c) (d) hydrogen sulfide concentration changes in brain tissue 24 hours, 3 days after injection of different drugs.
FIG. 7. evaluation of nervous system protection by DATS @ MION-PEG-LF, wherein,
(a) the survival rate of the neuron cells changes after the action of DATS @ MION-PEG-LF with different concentrations,
(b) the concentration of LDH in neuron cells is changed after the action of DATS @ MION-PEG-LF with different concentrations,
(c) H.E. staining pattern of brain tissue section after effect of different concentrations of DATS @ MION-PEG-LF,
(d) (e) (f) (g) changes in MPO, MDA, SOD and CAT contents in brain tissue after DATS @ MION-PEG-LF action.
Detailed Description
Example 1
Preparing mesoporous iron oxide nanoparticle Materials (MIONs):
adding 36ml of polystyrene, 4ml of methacrylic acid and 140ml of deionized water into a three-neck flask, performing ultrasonic dispersion, heating and stirring for 30 minutes at 75 ℃, dissolving 0.4g of potassium persulfate into 40ml of deionized water, slowly and dropwise adding the potassium persulfate into the three-neck flask, continuously reacting for 24 hours at 75 ℃ to obtain polystyrene nano microspheres, cooling, adding 1g of polystyrene nano microspheres, 170ml of deionized water and 30ml of ethylene glycol into a new flask, and performing ultrasonic dispersion; respectively adding 0.446g of ferrous chloride, 0.111g of potassium nitrate and 2g of hexamethylenetetramine into the polypropylene nano microsphere solution, heating the solution to 80 ℃ under the protection of nitrogen, reacting for 3 hours, gradually cooling the solution to room temperature, and centrifuging at 12000r for 10 minutes; and (3) fully washing the collected product with deionized water, adding the product into a ceramic crucible, slowly heating to 500 ℃, fully burning for 3 hours, and cooling to room temperature to obtain the MIONS.
The surface modification process of the mesoporous iron oxide nano particle Material (MIONs) comprises the following steps:
adding 10mg of MIONs and 30ml of deionized water into the flask, and performing ultrasonic dispersion; adding 5ml of APTES into hydrochloric acid with the pH value of 4.0 and the amount of a catalyst, adding the mixture into the flask after the reaction is stopped, heating and stirring the mixture for 4 hours at 65 ℃ under the protection of nitrogen, cooling the mixture, adding ethanol and deionized water into the mixture for alternate elution, adding the mesoporous iron oxide nano solution into a new flask after the elution is finished, blowing nitrogen into the flask for 30 minutes, adding 20mg of Mal-PEG-NHS, and stirring the mixture for reaction for 5 hours at room temperature; after the reaction is finished, the mixture is dialyzed for 24 hours to obtain Mal-PEG-MION, and the molar ratio of Mal-PEG-MION is 1: 8 molar ratio of lactoferrin to SATA in pH7.0HBS solution, continuously shaking for 1 hour at room temperature, ultrafiltering in a centrifugal tube with 30Kd, centrifuging for 15 minutes at 4000g and 4 ℃, collecting all supernatants, and diluting with HBS; preparing 0.1M hydroxylamine hydrochloride solution, reacting with the lactoferrin solution for 45 minutes at room temperature, finally adding the activated lactoferrin solution into the Mal-PEG-MION solution according to a certain proportion, and continuously shaking for 2 hours at room temperature; after the reaction is finished, deionized water is dialyzed for 48 hours, then impurities are removed by filtration through a 0.45-micron filter membrane, and the filtrate is freeze-dried and weighed in a vacuum environment.
The drug loading process of diallyl trisulfide (DATS), namely the synthesis process of DATS @ MION-PEG-LF:
ultrasonically dispersing 10mg of MION-PEG-LF in 2mL of water, adding 5 mu L of DATS, magnetically stirring, centrifuging the product after 12 hours, and removing the supernatant to obtain drug-loaded particles;
the results are shown in the figure: wherein, FIG. 2 shows the synthesis process of the present low toxicity nano system; FIGS. 3(a) and (b) are scanning electron micrographs of DATS @ MION-PEG-LF, which show that mesopores on the surfaces of the prepared iron oxide particles are clearly visible, the particles have similar shapes and sizes, and show obvious monodispersity; FIG. 3(c) shows the particle size distribution of DATS @ MION-PEG-LF, at 225.5 nm. + -. 25 nm; FIG. 3(d) shows the drug loading rate of DATS @ MION-PEG-LF 40%; FIG. 3(e) (f) (g) (h) shows the IR spectrum of MION-PEG-LF, the characteristic carbon-sulfur bond reflects the successful modification of lactoferrin and bifunctional PEG.
Example 2
In vitro safety experiments:
obtaining primary rat cortical neuron cells by a conventional method; neuronal cells (10)5/mL) was routinely cultured in a carbon dioxide incubator (37 ℃, 5% CO) using DMEM/F12 and 10% calf serum2) Culturing for 12 h, adding DATS @ MION-PEG-LF (0-100 μ g/mL) into 96-well plate for neuron cell planting, changing culture solution into CCK-8 solution, reacting for 24 h, and measuring cell activity (450nm) with enzyme labeling instrument.
In vivo safety experiments:
conventionally selecting 30 male Balb/c nude mice with similar age and weight, dividing into A, B groups, injecting 0.1ml of DATS-MION solution into each nude mouse of group A by tail vein injection, injecting 0.9% NaCl solution into each nude mouse of group B by same amount, feeding A, B two groups of nude mice under same ventilation, illumination, temperature, humidity and feeding conditions, selecting 5 nude mice from A, B groups at morning of 7, 14 and 21 days after injection, killing after anesthesia, taking the whole brain, heart, liver and kidney of each nude mouse, fixing in prepared 4% formaldehyde solution, slicing, staining, and microscopic examination.
The results show that: in vitro experiments, FIG. 4(a) shows that the survival rate of cortical neuron cells gradually decreases with increasing addition of DATS @ MION-PEG-LF, but even if the concentration of DATS @ MION-PEG-LF approaches 100. mu.g/mL, the survival rate is not lower than 90%, which indicates that the DATS @ MION-PEG-LF hydrogen sulfide donor system has certain toxicity to cortical neuron cells, but still has in vitro safety required by further experiments; in vivo experiments, FIG. 4(b) shows that the glomerular and tubular systems in kidney sections do not show pathologically significant structural changes as the time between DATS @ MION-PEG-LF injection and specimen preparation is extended; in the liver section, liver lobules in samples with different material taking time basically keep normal tissue structures; similarly, no significant tissue damage was observed in both cardiac and cerebral sections, whether cardiomyocytes or neuronal cells, due to injection of DATS @ MION-PEG-LF; the result shows that DATS @ MION-PEG-LF has excellent biological safety.
Example 3 evaluation of Hydrogen sulfide Release of DATS @ MION-PEG-LF
In vitro release: hydrogen sulfide evolution was determined by high-performance liquid chromatography (HPLC): DATS @ MION-PEG-LF (10. mu.g/mL), NaHS (10. mu.M) and DATS (10. mu.g/mL) were added to 4mL of 100mM phosphate solution (PBS), followed by addition of 200. mu.M GSH, followed by measurement of hydrogen sulfide, by mixing 30. mu.L of the resulting sample with 70. mu.L of Tris-HCl (100mM, pH 9.5,0.1Mm DPTA), mixing with 50. mu.L of monobromobimane solution, after 30 minutes of reaction, adding 50. mu.L of glacial acetic acid (200mM) to terminate the reaction, and then taking 100. mu.L of the supernatant to a chromatographic column (XDB-C18column, 150 x 4.6mM, 5. mu.m) of an HPLC apparatus (1260) to measure the concentration of hydrogen sulfide (. lamda. ex:390nm and. lamda.: 475nm) by fluorescence method, and comparing with a standard curve, for 0 to 120 minutes.
And (3) in vivo release: SD rats (200-220 g) are respectively administered DATS @ MION-PEG-LF (10mg/kg), NaHS (100 mu mol/kg) and DATS (10mg/kg) for tail vein injection after anesthesia, blood is taken from a carotid artery catheter 0.2mL at a timing of 0-12 hours after injection, blood plasma is obtained after anticoagulation (heparin sodium 50U/mL) and centrifugation (3000 r/min, 15 min), the hydrogen sulfide determination method is the same as that of the SD rats, the SD rats need to be centrifuged at 4 ℃ for 10 min (12000 r/min) and then enter an instrument for analysis after glacial acetic acid is added, and the SD rats are treated in batches at 24 hours and 3 days after injection to prepare brain tissue homogenate, and the hydrogen sulfide determination method is the same as that of the SD rats;
the results show that: DATS @ MION-PEG-LF shows obvious hydrogen sulfide slow-release effect compared with DATS and NaHS, and FIGS. 6(a) and 6(b) show that the hydrogen sulfide concentration in the solution slowly rises and is maintained at a certain concentration level for a long time after the DATS @ MION-PEG-LF is added; FIGS. 6(c) and 6(d) show that the concentration of hydrogen sulfide in the brain homogenate after DATS @ MION-PEG-LF24 hours and 3 days was still significantly higher than that after NaHS and DATS injection, fully reflecting the sustained release properties.
Example 4 brain-targeted transport experiments
Dissolving 1mg of MION-PEG-LF in 5ml of deionized water, and marking as a solution a; dissolving 1mg MION in 5ml deionized water, and marking as solution b; weighing 0.2mgDiR, dissolving in 2mlDMSO in dark condition, and marking as solution c; then, 1ml of the solution was mixed with solution a, and the mixture was stirred at room temperature in the dark for 9 hours, and the mixture was designated as solution A. Mixing the 1ml of the liquid with the liquid B, stirring the mixture at room temperature for 9 hours in a dark place, and marking the mixture as liquid B; selecting 12 Balb/c male healthy nude mice with approximate size and weight of three weeks, dividing into A, B groups (6 mice in each group) randomly, ligating the common carotid artery on the right side of each mouse to simulate the state of central nervous system ischemia and anoxia after sudden cardiac arrest, injecting 0.1ml of A solution into each nude mouse in the group A in a tail vein injection mode, injecting 0.1ml of B solution into each nude mouse in the group B in the same mode, recording the time of injection completion, anesthetizing the two groups of mice in an intraperitoneal injection mode 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours and 24 hours after the injection completion, and then carrying out image acquisition by using a Burker in vivo xvo Xtreme imaging system;
the results show that: after administration for half an hour through the tail vein, fluorescence signals are observed to appear at the position of the liver in A, B two groups of nude mice at the same time, the signal intensity between the two groups has no obvious difference, and the distribution of the obvious fluorescence signals is not seen in the other tissues except the tail and the liver; then, in B group of nude mice injected with the DiR-loaded mesoporous iron oxide nanoparticles without surface modification, the fluorescence signals in the liver are observed to be gradually enhanced and reach a peak value within about 2 hours, and then the signal intensity is gradually reduced, and the distribution of the fluorescence signals in other tissues is not found in the process; the result is basically consistent with the in vivo metabolic pathway of the iron oxide nanoparticles reported in the literature;
in the group A nude mice injected with the surface-modified DiR-loaded mesoporous iron oxide nanoparticles, the fluorescence signal change similar to that of the group B nude mice at the liver is observed; meanwhile, the fluorescent signal distribution of the brain of a nude mouse can be seen in an image 4 hours after injection, the fluorescent signal of the brain is gradually enhanced in an image 6 hours after injection and disappears in an image 24 hours after injection, the fluorescent signal distribution of the other tissues is not found, although the signal intensity is relatively weaker than that of the liver, the fluorescent signal of the brain is still obviously visible after background interference is eliminated by adjusting a filtering threshold value, the signal intensity change accords with a physiological metabolic process, and the result shows that the phenomenon is not an interference signal, but the DiR-loaded mesoporous iron oxide nanoparticles with the modified surfaces actually cross the blood brain barrier to enter the brain.
Example 5 nervous System protection experiments
Randomly dividing the same batch of rats into two groups, namely a DATS @ MION-PEG-LF group and a sudden cardiac arrest/resuscitation group; induction of cardiac arrest in the classical Utstein mode: anesthetizing a rat by a conventional method, then cutting an air moving tube, intubating the air moving tube, connecting a breathing machine, inserting a carotid artery and a jugular vein, connecting a body surface electrocardiogram, clamping the air tube at the end of expiration to cause respiratory arrest, observing the electrocardiogram, and starting timing after the occurrence of heartbeat arrest (the heartbeat arrest is defined as ventricular fibrillation, pulse arrest or electromechanical separation, and the average carotid artery pressure is less than 30 mmHg); performing precordial chest compression (the frequency is 200bpm, the depth is 1/3 of the front and back chest diameters) after 3 minutes of cardiac arrest, opening an airway to perform continuous mechanical ventilation (FiO2100 percent and 60 times/minute), simultaneously administering 20 mu g/kg of intravenous epinephrine, performing biphasic electrical defibrillation (the energy intensity is 2J) after 2 minutes of compression, repeatedly performing periodic cardiopulmonary resuscitation and electrical defibrillation (30 seconds) if the autonomic heart rate is not recovered, and declaring resuscitation failure if the autonomic heart rate is not recovered after 5 minutes of resuscitation time; after cardiac arrest is induced according to the steps, injecting 0.5ml of DATS @ MION-PEG-LF with different concentrations through tail veins in a DATS @ MION-PEG-LF group 1 minute before the start of cardiopulmonary resuscitation, and injecting equal amount of physiological saline through the tail veins in a cardiac arrest/resuscitation group; taking another group of Sham operation groups (Sham), performing anesthesia, tracheal intubation and arteriovenous intubation only without the processes of cardiac arrest/cardiopulmonary resuscitation and tail vein administration, killing the rat after completing resuscitation for 2 hours, randomly selecting half of the groups to prepare brain tissue homogenate, respectively determining the contents of MDA and MPO and the activities of SOD and CAT according to the specification of an ELISA kit, preparing brain tissue slices of the other half, and observing the brain tissue slices under a microscope after H.E. staining;
the results show that: the DATS @ MION-PEG-LF hydrogen sulfide donor system shows a protection effect on the hypoxic damage of neurons in a certain concentration range, the protection effect on the neurons shows a trend of increasing and then decreasing with the increase of the concentration of DATS @ MION-PEG-LF, and the maximum value is reached when the concentration of the DATS @ MION-PEG-LF reaches 5 mu g/mL;
the results of the DATS @ MION-PEG-LF hydrogen sulfide donor system on cardiopulmonary arrest/resuscitation rat protection experiments are shown in the figure, wherein the results of h.e. staining of brain tissue sections are shown in figure 7 (c); no obvious damaged area is found in the sham operation group, and the number and the shape of the H.E. staining positive cells are not obviously changed; compared with the sham operation group, the damaged area can be obviously increased in the cardiopulmonary arrest/resuscitation group, a large number of neurons with swelling and necrosis can be observed, and the number of H.E. staining positive cells is obviously reduced, which indicates that the nervous system is seriously damaged; the damaged area and the number of necrotic neurons in the DATS @ MION-PEG-LF treatment group are reduced compared with those in the cardiopulmonary arrest/resuscitation group, and the number of H.E. staining positive cells is increased, which indicates the protective effect of the DATS @ MION-PEG-LF on the nervous system of the cardiopulmonary arrest/resuscitation rat;
FIG. 7(d) (e) (f) (g) shows the change in MPO, MDA, SOD, CAT levels in rat brain tissue after DATS @ MION-PEG-LF action, where a decrease in SOD, CAT activity and an increase in MDA, MPO levels indicates an increase in oxidative damage, and vice versa indicates that oxidative damage is inhibited and the nervous system is protected; compared with the control group, the DATS @ MION-PEG-LF action group shows statistical difference of each index, which indicates that the DATS @ MION-PEG-LF really plays a role in protecting the nervous system.
Claims (8)
1. The hydrogen sulfide controlled-release brain-targeted nano system for protecting the nervous system after cardiac arrest is characterized in that mesoporous iron oxide nanoparticles are used as a core to entrap hydrogen sulfide donor drugs, lactoferrin serving as a brain-targeted group is modified on the surfaces of the particles in a bifunctional PEG bridging mode, so that a low-toxicity hydrogen sulfide controlled-release brain-targeted nano system DATS @ MION-PEG-LF is constructed, and the drugs are delivered in a targeted manner to the brain;
the nanosystems are prepared by the following method:
mesoporous iron oxide nanoparticles are prepared by a pyrolysis method, surface modification of the nanoparticles is carried out by bifunctional PEG Mal-PEG-NHS and lactoferrin, and DATS is used as a hydrogen sulfide donor drug entrapped in the mesoporous iron oxide nanoparticle inner core.
2. The hydrogen sulfide controlled-release brain-targeting nanosystem for protecting the nervous system after cardiac arrest of claim 1, wherein the nanosystem is a nanoparticle with a core-shell like structure, the core composed of mesoporous iron oxide nanoparticles carries a drug, and bifunctional PEG and lactoferrin are used as surface modifying groups.
3. The controlled-release brain-targeting nanosystem of hydrogen sulfide for nervous system protection after cardiac arrest as claimed in claim 1, wherein the hydrogen sulfide donor drug entrapped in the mesoporous iron oxide nanoparticle core is garlic extract propyl trisulfide diallyl DATS.
4. The hydrogen sulfide controlled-release brain-targeting nanosystem for nervous system protection after cardiac arrest as claimed in claim 1, wherein the inner layer surface modification group is bifunctional PEG maleimide-polyethylene glycol-aminosuccinimide succinate (Mal-PEG-NHS), the aminosuccinimide succinate group at one end of the nanosystem is condensed with the surface amino group of the aminated mesoporous iron oxide nanoparticle, and the maleimide at the other end provides a reaction site for the connection of the outer layer surface modification group on the nanoparticle surface.
5. The controlled-release brain-targeting nanosystem of hydrogen sulfide for nervous system protection after cardiac arrest of claim 1, wherein the outer layer surface modifying group is lactoferrin LF.
6. The controlled-release brain-targeted nanosystem of hydrogen sulfide for nervous system protection after cardiac arrest as claimed in claim 5, wherein said lactoferrin LF mediates brain-targeted transport through receptor-mediated endocytosis, and surface modification of mesoporous iron oxide nanoparticles is accomplished by reaction with maleimide after thiol activation.
7. The controlled-release brain-targeting nanosystem of hydrogen sulfide for nervous system protection after cardiac arrest as claimed in claim 1, wherein the nanosystem construction method comprises:
preparing mesoporous iron oxide nanoparticles by a pyrolysis method: firstly, preparing polystyrene nano-microspheres, taking the polystyrene nano-microspheres as a core to wrap iron oxide particles, and removing the polystyrene nano-microspheres through a pyrolysis method to prepare the mesoporous iron oxide nanoparticles.
8. Use of the controlled release brain-targeted nanosystems of hydrogen sulfide for nervous system protection after cardiac arrest of claim 1 in the preparation of a low toxicity medicament for protecting the nervous system.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1632889A (en) * | 2003-12-23 | 2005-06-29 | 中国科学院理化技术研究所 | Magnetic iron oxide hollow microsphere particle with mesoporous structure and preparation method and application thereof |
| CN102614525A (en) * | 2011-02-01 | 2012-08-01 | 复旦大学 | Protein-mediated brain-targeting gene delivery system and application thereof |
| CN104288174A (en) * | 2014-10-17 | 2015-01-21 | 复旦大学附属中山医院 | Controlled-release hydrogen sulfide donor as well as preparation method and application thereof |
-
2020
- 2020-09-02 CN CN202010907153.XA patent/CN113350520A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1632889A (en) * | 2003-12-23 | 2005-06-29 | 中国科学院理化技术研究所 | Magnetic iron oxide hollow microsphere particle with mesoporous structure and preparation method and application thereof |
| CN102614525A (en) * | 2011-02-01 | 2012-08-01 | 复旦大学 | Protein-mediated brain-targeting gene delivery system and application thereof |
| CN104288174A (en) * | 2014-10-17 | 2015-01-21 | 复旦大学附属中山医院 | Controlled-release hydrogen sulfide donor as well as preparation method and application thereof |
Non-Patent Citations (6)
| Title |
|---|
| MAJID SHARIFI ET AL: "Antimetastatic Activity of Lactoferrin-Coated Mesoporous Maghemite Nanoparticles in Breast Cancer Enabled by Combination Therapy", 《ACS BIOMATER. SCI. ENG.》 * |
| WENSHUO WANG ET AL: "Controlled release hydrogen sulfide delivery system based on mesoporous silica nanoparticles protects graft endothelium from ischemia–reperfusion injury", 《INTERNATIONAL JOURNAL OF NANOMEDICINE》 * |
| XIAOTIAN SUN ET AL: "Mesoporous silica nanoparticles for glutathione-triggered long-range and stable release of hydrogen sulfide", 《J. MATER. CHEM. B》 * |
| XIAOTIAN SUN ET AL: "Novel controlled and targeted releasing hydrogen sulfide system exerts combinational cerebral and myocardial protection after cardiac arrest", 《JOURNAL OF NANOBIOTECHNOLOGY》 * |
| 姚慧敏 著: "《靶向定位给药系统及评价方法》", 31 October 2013, 吉林大学出版社 * |
| 陶恒沂 等主编: "《气体生物医学》", 31 January 2014, 第二军医大学出版社 * |
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