CN113769112A - GOQDs-based pH response type bionic nano preparation and preparation method and application thereof - Google Patents
GOQDs-based pH response type bionic nano preparation and preparation method and application thereof Download PDFInfo
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- CN113769112A CN113769112A CN202111137219.2A CN202111137219A CN113769112A CN 113769112 A CN113769112 A CN 113769112A CN 202111137219 A CN202111137219 A CN 202111137219A CN 113769112 A CN113769112 A CN 113769112A
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Abstract
The invention belongs to the technical field of biological medicines, and particularly relates to a pH response type bionic nano preparation based on GOQDs, and a preparation method and application thereof. The pH response type bionic nano preparation is composed of PEG (polyethylene glycol) oxidized graphene quantum dots, a hydrophobic drug, a bionic hybrid membrane and phosphatide hyaluronic acid, wherein the hydrophobic drug is loaded on the PEG oxidized graphene quantum dots, the bionic hybrid membrane is disguised on the outer layer of the PEG oxidized graphene quantum dots loaded with the hydrophobic drug, and the phosphatide hyaluronic acid is modified on the surface of the bionic hybrid membrane. The pH response type bionic nano preparation can effectively prolong the circulation half-life period of the bionic nano preparation in blood, enhance the targeting capability and realize the controllable release at a pathological change part.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a pH response type bionic nano preparation based on GOQDs, and a preparation method and application thereof.
Background
The initial pathomechanism of Atherosclerosis (As), the disease with the highest global morbidity and mortality, is the damage of vascular endothelial cells by oxidized low-density lipoprotein (ox-LDL) resulting in a local inflammatory response. Cytokines are secreted at the injury site to recruit circulating mononuclear cells and differentiate into macrophages to phagocytose ox-LDL, and finally form As plaques. As the disease progresses, a large amount of cholesterol accumulates in late As plaques, impairing macrophage autophagy flow leading to decreased plaque stability and induction of myocardial infarction. AT present, statins such As Atorvastatin (AT) are mainly used for treating As, and lipid and blood pressure are reduced. Although the existing treatment method effectively reduces the incidence rate of cardiovascular diseases, As has higher residual risk due to the defects of low targeting property, poor water solubility, quick clearance and the like of oral administration. Oral AT is difficult to achieve effective blood levels due to the "first pass effect". In addition, As patients are mostly asymptomatic before complications such As myocardial infarction occur, resulting in patients treated mostly in the late stage of As. Although oral AT high dose is effective in ablating advanced As plaques in mice, the use of high dose AT in humans is limited due to side effects such As liver, kidney toxicity and muscle damage. Therefore, how to solve the defects of low drug targeting property, poor water solubility, quick removal, strong toxic and side effects and the like of oral administration is the key of the current As prevention and treatment.
In recent years, with the development of nanotechnology, the nanometer material shows great advantages in the aspect of drug delivery in the form of nanometer preparations, can effectively improve the targeting property, water solubility and biological safety of the drugs, can realize the controllable release of the drugs at the disease parts, and provides a potential alternative scheme for preventing and treating As. However, the nano-formulation has a half-life of several hours or less due to its rapid elimination as recognized by the immune system of the human body as a foreign substance. Therefore, how to prolong the blood circulation time of the nano preparation and increase the aggregation capability of the nano preparation at the As plaque part is a main challenge to prevent and treat the As nano preparation in the delivery process at present.
The cells are the most basic unit of the body, and the characteristic proteins exist on the membrane of the cells to help the cells adapt to the complex internal environment of the body and move to specific positions. Therefore, by utilizing the characteristic of cell membranes, the bionic nano preparation is constructed to avoid the elimination of the immune system of the body, and the aggregation of the nano preparation at the disease part is increased through the ligand-receptor interaction in the inflammation microenvironment, thereby attracting wide attention. The 'top-down' technology utilizes complex physiological functions of cell membranes while keeping complete physicochemical properties of nanoparticle cores, and the functions are difficult to replicate by using single nano materials. For example, the macrophage membrane coated nano-drug can effectively increase the blood circulation half-life and the targeting capability of the As plaque position. However, because the content of macrophages in human bodies is small, and primary macrophages are difficult to culture and proliferate in vitro, the large-scale clinical transformation application of the macrophages is limited. Except for macrophage membrane, erythrocyte membrane has good biocompatibility and wide availability and is widely applied to the construction of bionic nano preparation.
Although the bionic nano preparation can effectively reduce the removal of the reticuloendothelial system to the nano medicine and enhance the targeting property of the plaque part, only by further targeting the nano medicine to target cells and enabling the nano medicine to respond to microenvironment stimulation to release the medicine at the disease part, the target cells can be effectively killed and killed so as to improve the curative effect of the medicine.
Disclosure of Invention
In view of the above, the invention provides a pH-responsive nano preparation based on GOQDs, and a preparation method and an application thereof, and the pH-responsive bionic nano preparation can effectively prolong the circulatory half-life period of the bionic nano preparation in blood, enhance the targeting capability and realize controllable release at a diseased part.
The pH response type bionic nano preparation based on GOQDs comprises PEG (polyethylene glycol) oxidized graphene quantum dots (GP), a hydrophobic drug, a bionic hybrid membrane and phosphatide hyaluronic acid, wherein the hydrophobic drug is loaded on the PEG oxidized graphene quantum dots, the bionic hybrid membrane is camouflaged on the outer layer of the PEG oxidized graphene quantum dots loaded with the hydrophobic drug, and the phosphatide hyaluronic acid is modified on the surface of the bionic hybrid membrane;
the phosphatide hyaluronic acid is obtained by activating carboxyl on the surface of hyaluronic acid, adding phosphatide polyethylene glycol amino and stirring;
the PEG graphene oxide quantum dot is obtained by activating carboxyl on the surface of the graphene oxide quantum dot, adding amino polyethylene glycol amino and stirring;
the particle size of the pH response type bionic nano preparation is 80nm-100 nm.
Preferably, the hydrophobic drug is Atorvastatin (AT).
The invention also provides application of the pH response type bionic nano preparation loaded with AT in preparation of a medicament for preventing and/or treating atherosclerosis.
The invention also provides a preparation method of the pH response type bionic nano preparation based on GOQDs, which comprises the following steps:
s1, crushing the macrophage membrane and the erythrocyte membrane, and stirring in a PBS solution to prepare the bionic hybrid membrane;
stirring hyaluronic acid water solution for 30min-1h under the action of activator, adding phospholipid polyethylene glycol amino (DSPE-PEG)2000-NH2) Then stirring, and dialyzing and freeze-drying the mixed solution in sequence to obtain the phosphatide hyaluronic acid;
stirring graphene oxide quantum dot aqueous solution for 30min-1h under the action of an activating agent, and adding amino polyethylene glycol amino (NH)2-PEG2000-NH2) Stirring, and sequentially dialyzing and freeze-drying the mixed solution to obtain PEG graphene oxide quantum dots (GP);
s2, dissolving a hydrophobic drug, adding the dissolved hydrophobic drug into the PEGylated graphene oxide quantum dot solution, stirring, and dialyzing and freeze-drying the obtained solution in sequence to obtain a hydrophobic drug-loaded nano preparation (D @ GP);
s3, mixing the nano preparation loaded with the hydrophobic drug with the PBS solution of the bionic hybrid membrane, and repeatedly extruding to obtain a bionic nano preparation (M @ D @ GP);
s4, adding the phosphatide hyaluronic acid solution into the bionic nano preparation (M @ D @ GP) solution, and stirring for 30min-1h to obtain the pH response type bionic nano preparation (HA-M @ D @ GP).
Preferably, in S1, the mass ratio of the macrophage membrane to the erythrocyte membrane is 1: 0.2-5;
the mass ratio of the hyaluronic acid to the phospholipid polyethylene glycol amino is 1: 1-5;
the mass ratio of the graphene oxide quantum dots to the amino polyethylene glycol amino groups is 1: 5-10.
Preferably, in S1, the activating agents are 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), and the mass ratio of the hyaluronic acid, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide is 1:2-8: 4-16; the mass ratio of the graphene oxide quantum dots to the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride to the N-hydroxysuccinimide is 1:20-60: 5-20.
Preferably, the mass ratio of the graphene oxide quantum dots to the hydrophobic drug is 1-5: 1;
the mass ratio of the graphene oxide quantum dots to the phosphatide hyaluronic acid is 2-4: 1;
in S3, the mass ratio of the bionic hybrid membrane to the nano preparation loaded with the hydrophobic drug is 1-2: 1.
Preferably, in S1 and S2, the dialysis is performed for 24-72h using dialysis bags with a molecular weight cut-off of 3.5 kDa.
Preferably, in S1, the specific preparation process of the biomimetic hybrid membrane is as follows: breaking macrophage membrane and erythrocyte membrane at 4 deg.C under 100W water bath ultrasound for 1-2min, and stirring in PBS solution at 37 deg.C and 500-600 rpm for 1-2 hr.
Preferably, the stirring in S1 is at a stirring speed of 800rpm to 1000 rpm;
the stirring in S2 and S4 is at a stirring speed of 600rpm-800 rpm;
the repeated pressing in S3 means repeated pressing at least 10 times with a micro-squeezer with a pore size of 100 nm.
Compared with the prior art, the invention has the beneficial effects that:
1. the invention designs a green and biodegradable bionic nano preparation, and realizes specific aggregation, permeation and controllable release of a medicament at a pathological change part by utilizing the design characteristics of a bionic hybrid membrane and pH response. According to the invention, the PEG graphene oxide quantum dots are loaded with the hydrophobic drugs by utilizing pi-pi conjugation and/or electrostatic adsorption, and the PEG graphene oxide quantum dots have high dispersity and hydrophilicity, so that a compound formed after the hydrophobic drugs are loaded can exist in a microcrystalline state or a molecular dispersion state, and has good wettability, and the solubility of the hydrophobic drugs in water is increased. The bionic hybrid membrane formed by fusing a macrophage membrane modified by phosphatide hyaluronic acid and an erythrocyte membrane is utilized to carry out camouflage (HA-M @ D @ GP), so that the long blood circulation of the bionic nano preparation and the targeting capability (from the macrophage membrane) of a pathological change part are realized, and meanwhile, the consumption of the macrophage membrane can be reduced on the premise of not influencing the specific function of the macrophage membrane. When the pH response type bionic nano preparation reaches a pathological change part, the interaction of HA and activated macrophage surface receptor CD44 is utilized to successfully realize that the pH response type bionic nano preparation enters activated macrophages in a targeted manner. Meanwhile, the release of the hydrophobic drug from the pH response type bionic nano preparation is promoted by using the micro acid (pH6.4-6.8) environment in the microenvironment, so that the controllable release of the drug at the pathological change part is realized.
2. In addition to loading hydrophobic drugs for treating and/or preventing atherosclerosis, the bionic nano preparation with the micro-environment response characteristic provided by the invention can be used as a substitute platform for treating other diseases, such as tumors, rheumatoid arthritis and the like, by substituting different cell membrane types and hydrophobic drugs and utilizing the 'homing effect' of cell membranes and the action mechanisms of different drugs, so that the bionic nano preparation combines a hybrid membrane coating and stimulus-response type nanoparticles and can provide multiple functions and advantages for treating various diseases.
3. The pH response type bionic nano preparation based on GOQDs provided by the invention utilizes anti-atherosclerosis effects of AT on anti-inflammation, oxidative stress removal, autophagy promotion and the like, and is assisted by a nano carrier and a bionic system, so that the maximization of atherosclerosis prevention and treatment effects is realized.
4. The invention solves the problems of untimely early prevention, difficult reversion of late treatment and large toxic and side effects of atherosclerosis, can improve the blood half-life period, the target effect of plaque and activated macrophage and the controllable release capacity of pH response of the medicament, provides a new theoretical support for developing novel medicaments for preventing and treating atherosclerosis and relevant clinical prevention and treatment, and has important scientific significance, use value and economic value.
Drawings
FIG. 1 is a fluorescence resonance energy transfer spectrum and ratio of the bionic hybrid membrane: A. a fluorescence resonance energy transfer profile; B. a ratio;
FIG. 2 is a Western blot of a biomimetic hybrid membrane;
FIG. 3 is a transmission electron micrograph and particle size of GOQDs, AT @ GP and HA-M @ AT @ GP: A. GOQDs; B. AT @ GP; C. HA-M @ AT @ GP;
FIG. 4 is an IR spectrum of GOQDs, PEG-GOQDs, AT and AT @ GP;
FIG. 5 is a graph of the cumulative AT release from AT @ GP and HA-M @ AT @ GP AT different pH values;
FIG. 6 is a graph of AT, AT @ GP and HA-M @ AT @ GP for scavenging ROS, NO and inflammatory cell performance; A. scavenging ROS levels; B. ROS clearance statistical plots; C. scavenging NO levels; D. ability to clear inflammatory macrophages;
FIG. 7 is the blood half-life of HA-M @ AT @ GP in mice; A. in vitro fluorescence plots of mouse blood at different time points; B. a fluorescence intensity histogram;
FIG. 8 shows targeting of HA-M @ AT @ GP to atherosclerotic plaque sites in mice; A. mouse blood vessel fluorescence mapping; B. a fluorescence intensity histogram;
FIG. 9 is a graph and statistics of vascular gross oil red O staining for treatment of early atherosclerosis in different treatment groups; A. oil red O staining pattern; B. a statistical chart;
FIG. 10 is a graph and statistics of vascular gross oil red O staining for treatment of advanced atherosclerosis in different treatment groups; A. oil red O staining pattern; B. a statistical chart;
FIG. 11 is a graph of H & E staining of heart, liver, spleen, lung and kidney of different treatment groups of mice treated for early and late stage atherosclerosis.
Detailed Description
The invention is further described below with reference to the drawings and specific preferred embodiments of the description, without thereby limiting the scope of protection of the invention.
In the following examples, unless otherwise specified, the starting materials and equipment used are commercially available and the methods used are conventional in the art.
Example 1
(1) Synthesis of HA-M @ AT @ GP bionic nano preparation
a. Preparation of Bionical hybrid Membrane (M)
Whole blood was taken from C57BL/6 mice, washed with PBS (pH7.4), and centrifuged at 3000rpm for 5min to remove plasma. The red blood cells were resuspended in 0.25 XPBS (pH7.4) and lysed at 4 ℃ for 6-8 h. The lysate is then centrifuged at 12000rpm for 10min to obtain RBCm and washed with 0.25 XPBS (pH7.4) until the supernatant becomes colorless. Collecting the cleaned RBCm. The preparation of macrophage (RAW264.7 cell) membranes was performed as described in the membrane protein extraction kit. When the RAW264.7 cells in the cell culture dish were overgrown, the cells were scraped off with a cell scraper and centrifuged at 800rpm for 5 min. The collected cells were washed 2 times with PBS (pH7.4) at 4 ℃ and then suspended in membrane protein extractant A containing PMSF (1 mM). The mixture was lysed in an ice bath for 30min and sonicated in a water bath at 4 ℃ for 10min (80W, 0.3s on, 0.5s off). Repeatedly freezing and thawing at 37 deg.C and-80 deg.C for 4 times, each for 20-30 min. Then centrifuged at 800rpm for 10min at 4 ℃. Collecting supernatant, and centrifuging at 13000rpm at 4 deg.C for 30min to obtain macrophagusCell membrane
Measurement of erythrocyte Membrane (RBCm) and macrophage Membrane with BCA protein preparation kit
The concentration of protein, the weight of membrane, is twice the weight of membrane protein. Combining RBCm with
Mixing according to the total protein mass ratio of 1:1, performing ultrasonic treatment in water bath at 4 ℃ for 1min (100W, 0.5s on and 0.5s off), stirring in water bath at 37 ℃ and 600rpm for 1h to complete membrane fusion;
b. preparation of PEG oxidized graphene Quantum dot (GP)
Synthesis of pegylated GOQDs: by means of NH2-PEG2000-NH2The surface of GOQDs is modified. The specific operation method comprises the following steps: to 1mL of GOQDs solution (1mg/mL) were added 20mg of EDC and 5mg of NHS crosslinking agent, and stirred at 800rpm for 30min at room temperature for activating carboxyl groups on the surface of GOQDs. Then, 10mg of NH was added2-PEG2000-NH2Stirring the solution at room temperature of 800-. The above mixed solution was dialyzed for 3d against 3.5kDa dialysis bag to remove free NH2-PEG2000-NH2And a crosslinking agent. Finally, freeze-drying the purified material by a freeze-drying method, and placing the freeze-dried sample in a refrigerator at the temperature of-20 ℃ for later use;
c. preparation of phosphatidized hyaluronic acid
8mg EDC, 16mg NHS and 1mg HA were dissolved in PBS and stirred at 800rmp for 30min at room temperature for activating carboxyl groups on the HA surface. Then, 1mg of DSPE-PEG was added2000-NH2Stirring the solution at room temperature of 800-. Dialyzing the above mixed solution in 3.5kDa dialysis bag for 24 hr to remove free EDC, NHS and DSPE-PEG2000-NH2. Finally, freeze-drying the purified material by a freeze-drying method, and placing the freeze-dried phosphatide hyaluronic acid in a refrigerator at the temperature of-20 ℃ for later use;
synthesis of AT @ GP
AT is loaded on the GP surface through pi-pi conjugation and electrostatic adsorption. The specific operation process is as follows: 20mg of AT powder was weighed out and dissolved in 1ml of DMSO solution to form a 20mg/ml AT stock solution. Dissolving the synthesized GP lyophilized powder in ddH2O to form a 1mg/mL GP solution in water (1mL), then, 10 μ lAT solution was added dropwise to the GP solution and stirred at 600-800rpm for 24h at room temperature. Putting the mixed solution into a 3.5kDa dialysis bag, dialyzing with deionized water for 24h, finally, freeze-drying the purified material (AT @ GP) by using a freeze-drying method, and putting the freeze-dried sample into a refrigerator AT-20 ℃ for later use;
e. preparing M @ AT @ GP bionic nano preparation
Dissolving AT @ GP again in a PBS solution, mixing the dissolved solution with a bionic hybrid membrane (M) (1mL, 1mg/mL) subjected to water bath ultrasonic treatment according to a mass ratio of 1:2, and repeatedly extruding the mixture for AT least 10 times by a miniature extruder with the aperture of 100nm to obtain an M @ AT @ GP bionic nano preparation;
f. preparing HA-M @ AT @ GP bionic nano preparation
And (2) redissolving the phosphatide hyaluronic acid in a PBS solution, adding 250-.
(2) Characterization of HA-M @ AT @ GP biomimetic Nanoparticulation
As shown in FIG. 1 and FIG. 2, the fluorescence energy resonance transfer and membrane characteristic protein analysis were performed on the biomimetic hybrid membrane prepared in this example, and the results show that the biomimetic hybrid membrane was successfully fused and prepared.
As shown in FIG. 3, transmission electron microscope images and DLS particle size analysis were performed on the bionic nano-preparations GOQDs, AT @ GP and HA-M @ AT @ GP prepared in the present example. The result shows that the uniformly dispersed spherical GOQDs material is successfully prepared, and the grain diameter is about 8 nm; the AT-loaded nano-particles (AT @ GP) are spherical, and the particle size is about 32 nm; the HA modified bionic hybrid membrane is wrapped on the outer layer of AT @ GP, an obvious shell-core structure is shown, the particle size is about 80nm-100nm, and 3-4 AT @ GP nano particles are wrapped in one bionic nano preparation.
As shown in FIG. 4, the infrared spectrum (FT-IR) detection showsIt shows that AT @ GP nanoparticles are 1216cm-1And 843cm-1The characteristic peak of AT appears, indicating that AT is successfully loaded to GP surface.
As shown in FIG. 5, the cumulative release of AT increased with decreasing pH. Compared with AT @ GP, the bionic hybrid membrane has reduced AT accumulated release amount after being wrapped. The prepared bionic nano preparation realizes controllable and sustained release.
Example 2
The difference of the pH response type bionic nano preparation based on GOQDs from example 1 is that EDC is added in 60mg and NHS cross-linking agent is added in 20mg during preparation of the PEGylated graphene oxide quantum dots.
Example 3
A pH response type bionic nano preparation based on GOQDs, which is different from the pH response type bionic nano preparation in example 1 in NH (hydrogen sulfide) during preparation of PEG (polyethylene glycol) oxidized graphene quantum dots2-PEG2000-NH2Was added in an amount of 5 mg.
Example 4
A pH-responsive biomimetic nano-formulation based on GOQDs, which is different from example 1 in that EDC is added in an amount of 2mg and NHS is added in an amount of 4mg at the time of preparation of the phosphatide hyaluronic acid.
Example 5
A pH responsive biomimetic nano-formulation based on GOQDs, which is different from the embodiment 1 in that the phospholipid hyaluronic acid DSPE-PEG is prepared2000-NH2Was added in an amount of 5 mg.
Example 6
A pH-responsive biomimetic nano-formulation based on GOQDs, differing from example 1 in that the addition amount of AT solution AT the synthesis of AT @ GP was 50 μ l.
Example 7
A pH-responsive biomimetic nano-formulation based on GOQDs, which is different from example 6 in that 10 μ lAT solution is dropwise added into GP solution, and stirred at 600rpm-800rpm for 24h at room temperature.
Example 8
A pH-responsive biomimetic nanopreparation based on GOQDs, which is different from example 1 in the preparation of M @ AT @ GP biomimetic nanopreparation: dissolving AT @ GP again in a PBS solution, mixing the dissolved solution with a bionic hybrid membrane (M) (1mL, 1mg/mL) subjected to water bath ultrasonic treatment according to the mass ratio of 1:1, and repeatedly extruding the mixture for AT least 10 times by a miniature extruder with the aperture of 100nm to obtain the M @ AT @ GP bionic nano preparation.
Example 9
A pH response type bionic nano preparation based on GOQDs, which is different from the preparation in example 1 in that the mass ratio of a macrophage membrane to an erythrocyte membrane is 1: 0.2.
Example 10
A pH response type bionic nano preparation based on GOQDs, which is different from the preparation in example 1 in that the mass ratio of a macrophage membrane to an erythrocyte membrane is 1: 5.
Example 11
A pH response type bionic nanometer preparation based on GOQDs is different from the preparation method of the embodiment 1 in that the bionic hybrid membrane is prepared by stirring in a water bath at 500rpm for 2 hours to complete membrane fusion.
Example 12
A pH response type bionic nanometer preparation based on GOQDs is different from that in the embodiment 1, the activation process of carboxyl on the surface of the GOQDs is stirring at 1000rpm for 1h when the PEG oxidized graphene quantum dots are prepared.
Example 13
A pH-responsive biomimetic nano-formulation based on GOQDs, which is different from example 1 in that the process of activating carboxyl groups on the surface of HA is stirring at 1000rpm for 1h when preparing phosphatide hyaluronic acid.
Example 14
The application of the pH response type bionic nano preparation based on GOQDs in prevention and treatment of atherosclerosis is described by taking the HA-M @ AT @ GP bionic nano preparation prepared in the embodiment 1 as an example.
The scavenging capacity of the HA-M @ AT @ GP biomimetic nano-preparation prepared in example 1 on macrophage oxidative stress is determined by adopting fluorescence imaging and NO detection kit. AT concentration was 10. mu.M, and the results are shown in FIGS. 6A-C. The results show that the HA-M @ AT @ GP biomimetic nanopreparation was effective in scavenging ROS and NO levels in macrophages compared to the AT and AT @ GP groups alone.
The scavenging capacity of the HA-M @ AT @ GP biomimetic nano-preparation prepared in example 1 on inflammatory macrophages is determined by adopting an MTT method. The AT concentration was 10. mu.M, and the results are shown in FIG. 6D. The results show that the HA-M @ AT @ GP biomimetic nano-formulation can effectively eliminate inflammatory macrophages compared with the AT and AT @ GP groups alone.
The above results show that the HA-M @ AT @ GP biomimetic nano-preparation prepared in example 1 can effectively eliminate inflammation and oxidative stress.
Example 15
The blood half-life period and the in vivo target of the HA-M @ AT @ GP biomimetic nano preparation are measured by a semi-quantitative means for detecting the fluorescence intensity along with the HA-M @ AT @ GP biomimetic nano preparation prepared in the example 1.
Test one: after tail vein injection of 200. mu.L of Cy5 succinimidyl ester reactive dye (Cy5.5), Cy5.5@ GP and HA-M @ Cy5.5@ GP at a dose concentration of 10mg/kg into C57BL/6 mice, blood samples were taken at various time points for fluorescence intensity determination.
Wherein Cy5.5@ GP is prepared according to the following method: 50mg EDC, 100mg NHS and 5mg HA in ddH2Stirring at room temperature of 800 ℃ and 1000rmp for 30min-1h in O. Then, 10mg GP was added to the above solution and stirred at room temperature 800-1000rpm for 24 h. The above mixed solution was dialyzed in a 3.5kDa dialysis bag for 24h to remove free EDC and NHS. Finally, the purified material is freeze-dried by a freeze-drying method, and the freeze-dried Cy5.5@ GP is placed in a refrigerator at the temperature of-20 ℃ for standby.
HA-M @ Cy5.5@ GP was prepared according to the following method: re-dissolving the Cy5.5@ GP in a PBS solution, mixing the solution with a bionic hybrid membrane (M) (1mL, 1mg/mL) subjected to water bath ultrasonic treatment according to the mass ratio of 1:2, and repeatedly extruding the mixture for at least 10 times by using a miniature extruder with the aperture of 100nm to obtain a M @ Cy5.5@ GP bionic nano preparation; and (2) re-dissolving the phosphatidized hyaluronic acid in the PBS solution, adding 250 mu g of the phosphatidized hyaluronic acid solution into the M @ Cy5.5@ GP solution, stirring in a water bath at the speed of 600-.
As shown in FIG. 7, Cy5.5@ GP and HA-M @ Cy5.5@ GP have blood circulation half-lives of 0.82h, 2.52h and 4.51h, respectively. Compared with Cy5.5 and Cy5.5@ GP, the blood circulation period of HA-M @ Cy5.5@ GP is prolonged significantly, 5.5 times that of Cy5.5 (4.51h vs 0.82h) and 1.80 times that of Cy5.5@ GP (4.51h vs 2.52 h). The nano preparation disguised by the bionic hybrid membrane is beneficial to prolonging the blood circulation time.
And (2) test II: ApoE-/-After mice were raised on a high-fat diet for one month, 200. mu.L of Cy5.5, Cy5.5@ GP (test one) and HA-M @ Cy5.5@ GP (test one) were injected into the tail vein at a dose concentration of 10 mg/kg. After 12h, the aorta of the mice was taken for fluorescence imaging.
The results are shown in fig. 8, compared with the groups of Cy5.5 and Cy5.5@ GP, the group HA-M @ Cy5.5@ GP significantly aggregates AT the aortic arch and abdominal aorta, and the HA-M @ AT @ GP biomimetic nano-preparation prepared in example 1 can effectively target to the atherosclerotic plaque part.
Example 16
The HA-M @ AT @ GP biomimetic nano preparation prepared in example 1 is continuously used for early prevention and late treatment of atherosclerosis.
ApoE-/-After one month of high fat diet, mice are divided into two treatment modes: an early preventive administration group, wherein the administration dose is 2mg/kg twice a week, and the administration is continuously carried out for 2 months, and high fat diet is maintained in the period; the late treatment group is administered once daily at a dose of 10mg/kg for 1 week while maintaining a high fat diet. All the drugs are administered by tail vein injection, and the administration components are an independent AT group, an AT @ GP group and an HA-M @ AT @ GP group. 3 days after the end of the treatment, the aorta of the mice was dissected for gross oil red O staining to examine the therapeutic effect of the different treatment groups.
Results as shown in fig. 9 and fig. 10, the HA-M @ AT @ GP treatment group significantly reduced the formation of atherosclerotic plaques in both the early treatment regimen (fig. 9) and the late treatment regimen (fig. 10), indicating that the biomimetic nano-formulation constructed by the present invention can effectively prevent the progression of early atherosclerosis and treat late atherosclerosis.
Example 17
The HA-M @ AT @ GP biomimetic nano preparation prepared in example 1 is continuously used for in vivo biosafety inspection.
Using the treatment protocol adopted in example 16, in ApoE-/-After the treatment of the mouse is finished,collecting main organs (heart, liver, spleen, lung and kidney) of mice with different treatment components under different treatment schemes to prepare H&E staining to investigate the biological safety of the different treatment groups.
The results are shown in fig. 11, no obvious organic lesions are seen in the organs of mice of different treatment groups, which shows that the bionic nano preparation constructed by the invention has good biological safety.
The foregoing is merely a preferred embodiment of the invention and is not intended to limit the invention in any manner. Although the present invention has been described with reference to the preferred embodiments, it is not intended to be limited thereto. Those skilled in the art can make many possible variations and modifications to the disclosed embodiments, or equivalent modifications, without departing from the spirit and scope of the invention, using the methods and techniques disclosed above. Therefore, any simple modification, equivalent replacement, equivalent change and modification made to the above embodiments according to the technical essence of the present invention are still within the scope of the protection of the technical solution of the present invention.
Claims (10)
1. A pH response type bionic nano preparation based on GOQDs is characterized by comprising PEG (polyethylene glycol) oxidized graphene quantum dots, a hydrophobic drug, a bionic hybrid membrane and phosphatide hyaluronic acid, wherein the hydrophobic drug is loaded on the PEG oxidized graphene quantum dots, the bionic hybrid membrane is camouflaged on the outer layer of the PEG oxidized graphene quantum dots loaded with the hydrophobic drug, and the phosphatide hyaluronic acid is modified on the surface of the bionic hybrid membrane;
the phosphatide hyaluronic acid is obtained by activating carboxyl on the surface of hyaluronic acid, adding phosphatide polyethylene glycol amino and stirring;
the PEG graphene oxide quantum dot is obtained by activating carboxyl on the surface of the graphene oxide quantum dot, adding amino polyethylene glycol amino and stirring;
the particle size of the pH response type bionic nano preparation is 80nm-100 nm.
2. The GOQDs-based pH-responsive biomimetic nano-formulation according to claim 1, wherein the hydrophobic drug is atorvastatin.
3. Use of the pH-responsive biomimetic nano formulations based on GOQDs as claimed in claim 2 in the preparation of medicaments for preventing and/or treating atherosclerosis.
4. A method for preparing pH-responsive biomimetic nano-formulations based on GOQDs according to claim 1, characterized in that it comprises the following steps:
s1, crushing the macrophage membrane and the erythrocyte membrane, and stirring in a PBS solution to prepare the bionic hybrid membrane;
stirring hyaluronic acid aqueous solution for 30min-1h under the action of an activating agent, adding phospholipid polyethylene glycol amino, stirring, and dialyzing and freeze-drying the mixed solution in sequence to obtain phosphatide hyaluronic acid;
stirring the graphene oxide quantum dot aqueous solution for 30min-1h under the action of an activating agent, adding amino polyethylene glycol amino, stirring, and dialyzing and freeze-drying the mixed solution in sequence to obtain the PEG graphene oxide quantum dot;
s2, dissolving a hydrophobic drug, adding the dissolved hydrophobic drug into the PEGylated graphene oxide quantum dot solution, stirring, and dialyzing and freeze-drying the obtained solution in sequence to obtain a hydrophobic drug-loaded nano preparation;
s3, mixing the nano preparation loaded with the hydrophobic drug with the PBS solution of the bionic hybrid membrane, and repeatedly extruding to obtain a bionic nano preparation;
and S4, adding the phosphatide hyaluronic acid solution into the bionic nano preparation solution, and stirring for 30min-1h to obtain the pH response type bionic nano preparation.
5. The method for preparing pH responsive biomimetic nano-formulations according to GOQDs as claimed in claim 4, wherein in S1, the mass ratio of the macrophage membrane to the erythrocyte membrane is 1: 0.2-5;
the mass ratio of the hyaluronic acid to the phospholipid polyethylene glycol amino is 1: 1-5;
the mass ratio of the graphene oxide quantum dots to the amino polyethylene glycol amino groups is 1: 5-10.
6. The method for preparing pH responsive biomimetic nano-formulations based on GOQDs as claimed in claim 4, wherein in S1,
the activating agents are 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide; and is
The mass ratio of the hyaluronic acid to the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride to the N-hydroxysuccinimide is 1:2-8: 4-16;
the mass ratio of the graphene oxide quantum dots to the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride to the N-hydroxysuccinimide is 1:20-60: 5-20.
7. The method for preparing pH-responsive biomimetic nano-formulations based on GOQDs according to claim 4,
the mass ratio of the graphene oxide quantum dots to the hydrophobic drug is 1-5: 1;
the mass ratio of the graphene oxide quantum dots to the phosphatide hyaluronic acid is 2-4: 1;
in S3, the mass ratio of the bionic hybrid membrane to the nano preparation loaded with the hydrophobic drug is 1-2: 1.
8. The method for preparing pH-responsive biomimetic nano-formulations based on GOQDs as claimed in claim 4, wherein in S1 and S2, the dialysis is performed for 24-72h using dialysis bag with molecular weight cut-off of 3.5 kDa.
9. The method for preparing pH-responsive biomimetic nano-formulations according to claim 4, wherein in S1, the specific process for preparing the biomimetic hybrid membrane is as follows: breaking macrophage membrane and erythrocyte membrane at 4 deg.C under 100W water bath ultrasound for 1-2min, and stirring in PBS solution at 37 deg.C and 500-600 rpm for 1-2 hr.
10. The method for preparing pH-responsive biomimetic nano-formulations based on GOQDs according to claim 4,
in the step S1, the stirring speed is 800-1000 rpm;
the stirring in S2 and S4 is at a stirring speed of 600rpm-800 rpm;
the repeated pressing in S3 means repeated pressing at least 10 times with a micro-squeezer with a pore size of 100 nm.
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| CN115137709A (en) * | 2022-06-30 | 2022-10-04 | 重庆医科大学 | Podocyte active targeting reinforced biological homogeneous composite membrane nano-drug delivery system and preparation method and application thereof |
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| US20170319699A1 (en) * | 2016-05-03 | 2017-11-09 | Korea University Research And Business Foundation | Activated macrophage targetable drug carrier for treatment of atherosclerosis and methods of preparing the same |
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| CN115137709A (en) * | 2022-06-30 | 2022-10-04 | 重庆医科大学 | Podocyte active targeting reinforced biological homogeneous composite membrane nano-drug delivery system and preparation method and application thereof |
| CN115137709B (en) * | 2022-06-30 | 2023-05-09 | 重庆医科大学 | Podocyte active targeting reinforced biological homogeneous composite membrane nano drug delivery system and preparation method and application thereof |
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