CN114917352B - Drug-loaded disc particles with functions of adhering and regulating macrophages, and preparation method and application thereof - Google Patents
Drug-loaded disc particles with functions of adhering and regulating macrophages, and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a medicine-carrying disc particle with functions of adhering and regulating macrophages, and a preparation method and application thereof, wherein the preparation method comprises the following steps: and sequentially layering catechol modified hyaluronic acid and catechol modified chitosan on a PDMS template, obtaining disc particles by a micro-contact printing method, and co-culturing with SPM. The invention prepares discoid particles with diameters and thicknesses of 10 mu m and 200-300nm respectively by a photoetching technology, a layer-by-layer self-assembly technology and a micro-contact printing technology, the discoid particles can adhere to the surface of macrophages through hyaluronic acid and macrophage surface receptor CD44, release SPM and regulate and control macrophage phenotypes, and the macrophages at the vascular lesion part are reasonably regulated and controlled to control the process of atherosclerosis, thereby treating atherosclerosis and promoting the repair and remodeling of vascular tissues.
Description
Technical Field
The invention belongs to the technical field of cardiovascular disease treatment, and particularly relates to a medicine-carrying disc particle with functions of adhering and regulating macrophages, and a preparation method and application thereof.
Background
Immune cell therapy is one of the emerging technologies for treating tumors, which has also been trended in the treatment of cardiovascular diseases. Atherosclerosis is the root cause of cardiovascular disease, and is a complex chronic inflammatory reaction in which macrophages play a central role in the formation, development and rupture of the entire atheromatous plaque, and is considered as a key target for treating atherosclerosis and promoting vascular tissue repair.
Hyaluronic Acid (HA) is polyanionic acid mucopolysaccharide, HAs better biocompatibility, and HAs the functions of treating joint inflammation, promoting wound healing and the like in the field of medicines. Chitosan (CS) is a natural cationic polysaccharide with good antibacterial property and biocompatibility, and has been widely used in biomedical fields such as surgical sutures, drug controlled release carriers, etc. However, natural chitosan has poor solubility in neutral aqueous solution, and can be completely dissolved only in an acidic environment, and in addition, the stability of hyaluronic acid and chitosan after being combined needs to be improved.
The development of micropatterning technology in the fields of optics, biomedicine, microelectronics and the like plays a key role, and photoetching is a method commonly used in micropatterning technology, and the pattern precision of construction can reach the nanometer level. However, photolithography has drawbacks such as high cost, inability to construct patterns on non-planar surfaces, and dependence on photoresist of a photosensitive material, and thus a technique using the principle of photolithography is required for constructing patterns of a micrometer scale that are not particularly high in accuracy.
Disclosure of Invention
Aiming at the prior art, the invention provides a drug-loaded disc particle with functions of adhering and regulating macrophages, and a preparation method and application thereof, so as to provide a drug-loaded material which has strong adhesiveness, oxidation resistance and stability, can reasonably regulate and control the macrophages at a vascular lesion part to control the process of atherosclerosis, further treat atherosclerosis and promote the repair and remodeling of vascular tissues.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows: the preparation method of the drug-loaded disc particles with the functions of adhering and regulating macrophages is provided, and comprises the following steps: and sequentially layering catechol modified hyaluronic acid and catechol modified chitosan on a PDMS template, obtaining disc particles by a micro-contact printing method, and co-culturing with SPM.
The natural chitosan has poor solubility in neutral aqueous solution, can be completely dissolved only in an acidic environment, and the catechol group grafted on the chitosan can obviously improve the water solubility of the chitosan at neutral pH. The adhesiveness and oxidation resistance of the material after catechol group grafting are improved, so that the bonding stability of the material is improved.
Hyaluronic acid is an anionic polymer, chitosan is a cationic polymer, and the hyaluronic acid and the chitosan can be combined through intermolecular electrostatic interaction, and the hyaluronic acid and the chitosan have good biocompatibility, so that the hyaluronic acid can be used as a material for constructing disc particles on a PDMS template in a layer-by-layer self-assembly mode. The catechol group grafting modification is carried out on the hyaluronic acid and the chitosan, so that the adhesiveness and the oxidation resistance of the grafted material can be improved. The modified hyaluronic acid and chitosan are prepared into disc particles by a photoetching technology, a layer-by-layer self-assembly technology and a microcontact printing technology, and after drug-loaded SPM (pro-inflammatory fading medium) is carried, macrophages at the vascular lesion part can be reasonably regulated and controlled to control the process of atherosclerosis, so that atherosclerosis is treated, and the repair and remodeling of vascular tissues are promoted.
On the basis of the technical scheme, the invention can be improved as follows.
Further, catechol-modified hyaluronic acid is prepared by the following steps:
(1) Dissolving morpholine ethanesulfonic acid monohydrate in water, adding hyaluronic acid, and regulating the pH value to 4-6 to obtain a first mixed solution;
(2) Adding carbodiimide into a mixed solution of water and absolute ethyl alcohol, and adding the obtained solution and N-hydroxysuccinimide into the mixed solution I for reaction for 25-35 min to obtain a mixed solution II;
(3) Adding dopamine into the mixed solution II, adjusting the pH value of the solution to 4-6, and stirring for reaction for more than 4 hours to obtain a mixed solution III;
(4) And (3) putting the mixed solution III into a dialysis bag with the molecular weight of 12000Da, dialyzing in water with the pH of 5-6 for 70-75 h, and then freeze-drying to obtain the product.
Further, catechol-modified chitosan is prepared by the following steps:
(1) Adding chitosan into water, adding concentrated hydrochloric acid, stirring until the chitosan is completely dissolved, adjusting the pH to 4-6, and adding an aqueous solution of dihydroxypropionyl glycoside to obtain a mixed solution I;
(2) Mixing water and absolute ethyl alcohol according to a volume ratio of 1:1, adding carbodiimide into the mixture, adding the obtained solution into the first mixed solution, stirring the mixture for more than 4 hours at room temperature, and adjusting pH to 4-6 during the stirring to obtain a second mixed solution;
(3) And (3) putting the mixed solution II into a dialysis bag with the molecular weight of 12000Da, dialyzing in water with the pH of 5-6 for 70-75 hours, and then freeze-drying to obtain the product.
Further, the layer-by-layer assembly comprises the following steps: cutting a PDMS template into small blocks with the length of 1cm multiplied by 1cm, putting the small blocks into an aqueous solution of catechol modified hyaluronic acid, taking out the small blocks and putting the small blocks into water for soaking, putting the small blocks into an aqueous solution of catechol modified chitosan, taking out the small blocks and putting the small blocks into water for soaking, repeating the operation flow 5 times, obtaining the PDMS template assembled with 5 layers of catechol modified hyaluronic acid/catechol modified chitosan structure, putting the small blocks into a stable airflow for 8-12 min, and drying the small blocks at the temperature of 35-40 ℃ for 08-1.2 h.
Further, the PDMS template is prepared by the following steps:
(1) Spin coating photoresist on the wafer, placing a mask plate above the wafer, exposing for 10s, and developing with NaOH solution to obtain a patterned wafer;
(2) And mixing PDMS and a cross-linking agent, pouring the mixture into a patterned wafer, curing for 1.5-2.5 hours, peeling the PDMS template when the temperature is restored to room temperature, placing the PDMS template into an oxygen plasma system, performing hydrophilic-hydrophobic modification, adjusting the power to 300-400W, adjusting the vacuum degree to 150-250 MPa, and adjusting the modification time to 4-6 minutes.
Further, the micro-contact printing method includes the steps of: placing one surface of cover glass with PVA film at 2 cm above RO water at 45-65deg.C, then taking down and quickly placing PDMS template pattern surface on the PVA film, applying 10-40 g/cm 2 And after the pressure is 10-40 s, the PDMS template is removed from the upper part of the PVA film to obtain the disc particles.
Further, the SPM concentration is 50 to 100nmol/L.
The invention also provides the medicine carrying disc particles with the functions of adhering and regulating and controlling macrophages, which are prepared by the preparation method.
Further, the particle diameter and thickness of the drug-loaded disc were 10 μm and 200-300nm, respectively.
The invention also provides application of the drug-loaded disc particles with the functions of adhering and regulating macrophages in preparing drugs for promoting vascular tissue repair and remodeling.
The beneficial effects of the invention are as follows:
1. the Polydimethylsiloxane (PDMS) selected by the invention has the characteristics of good biocompatibility, mechanical flexibility and durability. Catechol groups are introduced into the material, so that the adhesiveness and oxidation resistance of hyaluronic acid and chitosan are improved, and an important basis is provided for the layer-by-layer self-assembly of disc particles on a PDMS template and the structural stability of the disc particles.
2. The catechol modified hyaluronic acid (C-HA) and catechol modified chitosan (C-CS) layer-by-layer self-assembly technology utilizes static electricity, hydrogen bonds and covalent bonds as intermolecular forces to assemble the polymer layer by layer, and HAs the advantages of simple operation steps, low experimental requirements and the like.
3. The atherosclerotic plaque presents an acidic environment, whereas chitosan has ionizable amino groups and a pKa value of 6.2, H when the disc particles are in a locally acidic environment + Can attract a great deal of negative charges in the disc particles and cause the change of the structural molecular chain conformation of the disc particles and the water absorption swelling of the disc particles, promote the release of SPM in the disc particles and play a role of pH responsiveness.
Drawings
FIG. 1 is a view of PVA film mirrors of microcontact printing in different processes;
FIG. 2 shows the catechol-modified chitosan at D 2 Nuclear magnetic resonance hydrogen spectrogram in O;
FIG. 3 shows the catechol-modified hyaluronic acid at D 2 Nuclear magnetic resonance hydrogen spectrogram in O;
FIG. 4 is an SEM observation of disc particles;
FIG. 5 shows the optical microscopic observation of disc particles dispersed in RO water (A) and Zeta potential (B);
FIG. 6 shows the change in F value (A) and D value (B) of the self-assembly process of C-HA and C-CS on QCM-D;
FIG. 7 shows the CD86 expression of macrophages loaded with disc particles;
FIG. 8 shows the expression of CD206 by macrophages loaded with disc particles.
Detailed Description
The following describes the present invention in detail with reference to examples.
Example 1
The preparation method of the drug-loaded disc particles with the functions of adhering and regulating macrophages comprises the following steps:
(1) Sequentially ultrasonically cleaning a wafer with acetone, absolute ethyl alcohol and RO water for 5min, drying at 120 ℃ for 2h, placing on a spin coater, vacuumizing, and opening an adsorption button to fix the wafer on a table top; sucking photoresist by a suction pipe, uniformly dripping the photoresist at the center of a wafer, opening a photoresist homogenizing machine, regulating the rotating speed to 500r/min, regulating the rotating speed of a desk type photoresist homogenizing machine to 2000r/min after the operation for 18s at 500r/min, operating for 60s at the rotating speed, drying for 20min at 90 ℃, opening a mercury lamp of a photoetching machine, preheating for 10min, placing the wafer at a tray of the photoetching machine, opening a suction piece function, placing a mask plate above the wafer, setting the exposure time of the photoetching machine to 10s, developing by using a NaOH solution of 5g/L after the exposure, cleaning by using RO water, and drying for 20min at 120 ℃ to carry out hardening to obtain a patterned wafer;
(2) Stirring and mixing PDMS and a cross-linking agent 2- (dimethylaminopropyl) -ethylcarbodiimide hydrochloride according to the volume ratio of 1:1, pouring the mixture into a culture dish with a patterned wafer at the bottom for curing for 2 hours, peeling off the PDMS template by a knife when the temperature is restored to room temperature, and then placing the PDMS template into an oxygen plasma system for hydrophilic and hydrophobic modification, wherein the power is regulated to 350W, the vacuum degree is regulated to 200MPa, and the modification time is 5 minutes;
(3) Weighing 100mg of catechol modified chitosan (C-CS) and 100mg of catechol modified hyaluronic acid (C-HA), dissolving with 50ml of RO water respectively, and adding the C-HA solution, the C-CS solution and the RO water into a 24-hole cell pore plate respectively; cutting PDMS (polydimethylsiloxane) templates into small blocks with the length of 1cm multiplied by 1cm, putting 1 PDMS template into each hole filled with C-HA solution, taking out after 15min, putting into RO water for 1min to remove unstable deposited C-HA, putting the PDMS template into C-CS solution for 15min, soaking in RO water for 1min, repeating the above operation flow for 5 times to obtain a PDMS template with a 5-layer C-HA/C-CS structure, putting into a stable air flow for 10min, and putting into a baking oven at 37 ℃ for 1h to remove surface moisture;
(4) Coating a layer of 3wt% PVA aqueous solution on the surface of the cover glass, drying at 70 ℃ for 15min to obtain the cover glass with the PVA film, placing one surface of the cover glass with the PVA film at 2 cm above RO water at 55 ℃, then taking down and rapidly placing the PDMS template pattern surface on the PVA film, and applying 20g/cm 2 After 20s of pressure, removing the PDMS template from the upper side of the PVA film to obtain disc particles, and then co-culturing with 75nmol/L SPM to obtain the membrane;
wherein, the C-HA is prepared by the following steps:
(1) Dissolving 0.97g of morpholine ethanesulfonic acid Monohydrate (MES) in RO water, adding 1g of Hyaluronic Acid (HA), and adjusting the pH value to 5 to obtain a first mixed solution;
(2) Mixing 10ml of RO water and 10ml of absolute ethyl alcohol, adding 0.39g of carbodiimide (EDC) into the mixture, and adding the obtained solution and 0.29g N-hydroxysuccinimide (NHS) into the mixed solution I for reaction for 30min to obtain a mixed solution II;
(3) Adding 0.47g of Dopamine (DOPA) into the mixed solution II, adjusting the pH value of the solution to 5, and stirring and reacting for 4 hours to obtain a mixed solution III;
(4) Putting the mixed solution III into a dialysis bag with the molecular weight of 12000Da, dialyzing in RO water with the pH of 5.5 for 72 hours, changing the water solution once every 8 hours, and freeze-drying to obtain the product;
wherein, the C-CS is prepared by the following steps:
(1) Adding 0.5g of chitosan into 25mL of RO water, adding concentrated hydrochloric acid at a dosage of 20 mu L each time, stirring until the chitosan is completely dissolved, dropwise adding 1mol/L NaOH solution until the pH value is 5, and adding an aqueous solution of dihydroxypropionyl glucoside (DHPA) (0.59 g of DHPA is dissolved in 3mL of RO water) to obtain a first mixed solution;
(2) Mixing 10ml of water with 10ml of absolute ethyl alcohol, adding 0.623g of carbodiimide (EDC) into the mixture, dropwise adding the obtained solution into the first mixed solution after 12min, stirring the solution at room temperature for 4h, and adjusting the pH to 5 by using 1mol/L of HCL and 1mol/L of NaOH during the stirring to obtain a second mixed solution;
(3) Putting the mixed solution II into a dialysis bag with the molecular weight of 12000Da, dialyzing in RO water with the pH of 5.5 for 72 hours, replacing the water solution once every 8 hours, and freeze-drying to obtain the product;
wherein, the PVA aqueous solution with 3 weight percent is prepared by the following steps: adding 3g of PVA particles into 100ml of RO water, then placing the mixture into a 70 ℃ oil bath heating magnetic stirrer for dissolution, and reacting for 2 hours after the temperature reaches 70 ℃ to completely dissolve the PVA, thus obtaining the PVA.
Example 2
The preparation method of the drug-loaded disc particles with the functions of adhering and regulating macrophages comprises the following steps:
(1) Sequentially ultrasonically cleaning a wafer with acetone, absolute ethyl alcohol and RO water for 5min, drying at 120 ℃ for 2h, placing on a spin coater, vacuumizing, and opening an adsorption button to fix the wafer on a table top; sucking photoresist by a suction pipe, uniformly dripping the photoresist at the center of a wafer, opening a photoresist homogenizing machine, regulating the rotating speed to 500r/min, regulating the rotating speed of a desk type photoresist homogenizing machine to 2000r/min after the operation for 18s at 500r/min, operating for 60s at the rotating speed, drying for 20min at 90 ℃, opening a mercury lamp of a photoetching machine, preheating for 10min, placing the wafer at a tray of the photoetching machine, opening a suction piece function, placing a mask plate above the wafer, setting the exposure time of the photoetching machine to 10s, developing by using a NaOH solution of 5g/L after the exposure, cleaning by using RO water, and drying for 20min at 120 ℃ to carry out hardening to obtain a patterned wafer;
(2) Stirring and mixing PDMS and a cross-linking agent 2- (dimethylaminopropyl) -ethylcarbodiimide hydrochloride according to the volume ratio of 1:1, pouring the mixture into a culture dish with a patterned wafer at the bottom for curing for 1.5 hours, peeling off a PDMS template by a knife when the temperature is restored to room temperature, and then placing the PDMS template into an oxygen plasma system for hydrophilic and hydrophobic modification, wherein the power is regulated to 300W, the vacuum degree is regulated to 150MPa, and the modification time is 6 minutes;
(3) Weighing 100mg of catechol modified chitosan (C-CS) and 100mg of catechol modified hyaluronic acid (C-HA), dissolving with 50ml of RO water respectively, and adding the C-HA solution, the C-CS solution and the RO water into a 24-hole cell pore plate respectively; cutting PDMS (polydimethylsiloxane) templates into small blocks with the length of 1cm multiplied by 1cm, putting 1 PDMS template into each hole filled with C-HA solution, taking out after 15min, putting into RO water for 1min to remove unstable deposited C-HA, putting the PDMS template into C-CS solution for 15min, soaking in RO water for 1min, repeating the above operation flow for 5 times to obtain a PDMS template with a 5-layer C-HA/C-CS structure, putting into a stable air flow for 8min, and putting into a baking oven at 35 ℃ for 1.2h to remove surface moisture;
(4) Coating a layer of 3wt% PVA aqueous solution on the surface of the cover glass, drying at 70 ℃ for 15min to obtain the cover glass with the PVA film, placing one surface of the cover glass with the PVA film at 2 cm above RO water at 45 ℃, then taking down and rapidly placing the PDMS template pattern surface on the PVA film, and applying 10g/cm 2 After the pressure is 40s, the PDMS template is removed from the upper side of the PVA film to obtain disc particles, and then the disc particles are co-cultured with 50nmol/L SPM to obtain the disc particles;
wherein, the C-HA is prepared by the following steps:
(1) Dissolving 0.97g of morpholine ethanesulfonic acid Monohydrate (MES) in RO water, adding 1g of Hyaluronic Acid (HA), and adjusting the pH value to 4 to obtain a first mixed solution;
(2) Mixing 10ml of RO water and 10ml of absolute ethyl alcohol, adding 0.39g of carbodiimide (EDC) into the mixture, and adding the obtained solution and 0.29g N-hydroxysuccinimide (NHS) into the mixed solution I for reaction for 25min to obtain a mixed solution II;
(3) Adding 0.47g of Dopamine (DOPA) into the second mixed solution, adjusting the pH value of the solution to 4, and stirring and reacting for 4.2 hours to obtain a third mixed solution;
(4) Putting the mixed solution III into a dialysis bag with the molecular weight of 12000Da, dialyzing in RO water with the pH of 5 for 75 hours, changing the water solution once every 8 hours, and freeze-drying to obtain the product;
wherein, the C-CS is prepared by the following steps:
(1) Adding 0.5g of chitosan into 25mL of RO water, adding concentrated hydrochloric acid at a dosage of 20 mu L each time, stirring until the chitosan is completely dissolved, dropwise adding 1mol/L NaOH solution until the pH value is 4, and adding an aqueous solution of dihydroxypropionyl glucoside (DHPA) (0.59 g of DHPA is dissolved in 3mL of RO water) to obtain a first mixed solution;
(2) Mixing 10ml of water with 10ml of absolute ethyl alcohol, adding 0.623g of carbodiimide (EDC) into the mixture, dropwise adding the obtained solution into the first mixed solution after 10min, stirring the solution at room temperature for 4.2h, and adjusting the pH to 4 by using 1mol/L of HCL and 1mol/L of NaOH during the stirring to obtain a second mixed solution;
(3) Putting the mixed solution II into a dialysis bag with the molecular weight of 12000Da, dialyzing in RO water with the pH of 5 for 75 hours, replacing the water solution once every 8 hours during the dialysis, and freeze-drying to obtain the product;
wherein, the PVA aqueous solution with 3 weight percent is prepared by the following steps: adding 3g of PVA particles into 100ml of RO water, then placing the mixture into a 70 ℃ oil bath heating magnetic stirrer for dissolution, and reacting for 2 hours after the temperature reaches 70 ℃ to completely dissolve the PVA, thus obtaining the PVA.
Example 3
The preparation method of the drug-loaded disc particles with the functions of adhering and regulating macrophages comprises the following steps:
(1) Sequentially ultrasonically cleaning a wafer with acetone, absolute ethyl alcohol and RO water for 5min, drying at 120 ℃ for 2h, placing on a spin coater, vacuumizing, and opening an adsorption button to fix the wafer on a table top; sucking photoresist by a suction pipe, uniformly dripping the photoresist at the center of a wafer, opening a photoresist homogenizing machine, regulating the rotating speed to 500r/min, regulating the rotating speed of a desk type photoresist homogenizing machine to 2000r/min after the operation for 18s at 500r/min, operating for 60s at the rotating speed, drying for 20min at 90 ℃, opening a mercury lamp of a photoetching machine, preheating for 10min, placing the wafer at a tray of the photoetching machine, opening a suction piece function, placing a mask plate above the wafer, setting the exposure time of the photoetching machine to 10s, developing by using a NaOH solution of 5g/L after the exposure, cleaning by using RO water, and drying for 20min at 120 ℃ to carry out hardening to obtain a patterned wafer;
(2) Stirring and mixing PDMS and a cross-linking agent 2- (dimethylaminopropyl) -ethylcarbodiimide hydrochloride according to the volume ratio of 1:1, pouring the mixture into a culture dish with a patterned wafer at the bottom for curing for 2.5 hours, peeling off a PDMS template by a knife when the temperature is restored to room temperature, and then placing the PDMS template into an oxygen plasma system for hydrophilic and hydrophobic modification, wherein the power is regulated to 400W, the vacuum degree is regulated to 250MPa, and the modification time is 4min;
(3) Weighing 100mg of catechol modified chitosan (C-CS) and 100mg of catechol modified hyaluronic acid (C-HA), dissolving with 50ml of RO water respectively, and adding the C-HA solution, the C-CS solution and the RO water into a 24-hole cell pore plate respectively; cutting PDMS (polydimethylsiloxane) templates into small blocks with the length of 1cm multiplied by 1cm, putting 1 PDMS template into each hole filled with C-HA solution, taking out after 15min, putting into RO water for 1min to remove unstable deposited C-HA, putting the PDMS template into C-CS solution for 15min, soaking in RO water for 1min, repeating the above operation flow for 5 times to obtain a PDMS template with a 5-layer C-HA/C-CS structure, putting into a stable air flow for 12min, and putting into a baking oven at 40 ℃ for 0.8h to remove surface moisture;
(4) Coating a layer of 3wt% PVA aqueous solution on the surface of the cover glass, drying at 70 ℃ for 15min to obtain the cover glass with the PVA film, placing one surface of the cover glass with the PVA film at 2 cm above RO water at 65 ℃, then taking down and rapidly placing the PDMS template pattern surface on the PVA film, and applying 40g/cm 2 After 10s of pressure, the PDMS template is removed from the upper part of the PVA film to obtain disc particles, and then the disc particles are co-cultured with 100nmol/L SPM to obtain the disc particles;
wherein, the C-HA is prepared by the following steps:
(1) Dissolving 0.97g of morpholine ethanesulfonic acid Monohydrate (MES) in RO water, adding 1g of Hyaluronic Acid (HA), and adjusting the pH value to 6 to obtain a mixed solution I;
(2) Mixing 10ml of RO water and 10ml of absolute ethyl alcohol, adding 0.39g of carbodiimide (EDC) into the mixture, and adding the obtained solution and 0.29g N-hydroxysuccinimide (NHS) into the mixed solution I for reaction for 35min to obtain a mixed solution II;
(3) Adding 0.47g of Dopamine (DOPA) into the second mixed solution, adjusting the pH value of the solution to 6, and stirring and reacting for 4.5 hours to obtain a third mixed solution;
(4) Putting the mixed solution III into a dialysis bag with the molecular weight of 12000Da, dialyzing in RO water with the pH of 6 for 70 hours, replacing the water solution once every 8 hours during the dialysis, and freeze-drying to obtain the product;
wherein, the C-CS is prepared by the following steps:
(1) Adding 0.5g of chitosan into 25mL of RO water, adding concentrated hydrochloric acid at a dosage of 20 mu L each time, stirring until the chitosan is completely dissolved, dropwise adding 1mol/L NaOH solution until the pH value is 6, and adding an aqueous solution of dihydroxypropionyl glucoside (DHPA) (0.59 g of DHPA is dissolved in 3mL of RO water) to obtain a first mixed solution;
(2) Mixing 10ml of water with 10ml of absolute ethyl alcohol, adding 0.623g of carbodiimide (EDC) into the mixture, dropwise adding the obtained solution into the first mixed solution after 10min, stirring the solution at room temperature for 4.5h, and adjusting the pH to 6 by using 1mol/L of HCL and 1mol/L of NaOH during the stirring to obtain a second mixed solution;
(3) Putting the mixed solution II into a dialysis bag with the molecular weight of 12000Da, dialyzing in RO water with the pH of 6 for 70 hours, replacing the water solution once every 8 hours during the dialysis, and freeze-drying to obtain the product;
wherein, the PVA aqueous solution with 3 weight percent is prepared by the following steps: adding 3g of PVA particles into 100ml of RO water, then placing the mixture into a 70 ℃ oil bath heating magnetic stirrer for dissolution, and reacting for 2 hours after the temperature reaches 70 ℃ to completely dissolve the PVA, thus obtaining the PVA.
The effect of microcontact printing is determined by the temperature of the water vapor passing through the PVA film, the pressure at the time of microcontact printing, and the time of imprinting, and therefore the optimum process for microcontact printing needs to be explored in order to ensure the integrity of the disc particles, and the specific exploration scheme is shown in table 1, and the effect is shown in fig. 1.
TABLE 1 optimal process exploration scheme for microcontact printing
FIG. 2 is a nuclear magnetic resonance hydrogen spectrum of modified chitosan, wherein the characteristic peak at 4.79ppm is solvent heavy water (D 2 O), the peak at point b (3.1 ppm) is a proton characteristic peak of chitosan C-2, the peak at point C (2.1 ppm) is a hydrogen proton characteristic peak of the incomplete deacetylated methyl group (-o=ch3), and the peak at point a (6.6-6.8 ppm) is a hydrogen proton characteristic peak on the benzene ring; the chitosan not subjected to catechol group grafting modification was free from characteristic peaks at 6.6 to 6.8 ppm.
FIG. 3 is a nuclear magnetic resonance hydrogen spectrum of modified hyaluronic acid, showing a characteristic peak at 4.79ppm as a solventHeavy water (D) 2 O), the peak at point C (3.3 ppm) is a proton characteristic peak of chitosan C-2, the peak located near 2.1 is a hydrogen proton characteristic peak of the unacetylated methyl group (-o=ch3), the peak at point a (6.6-6.8 ppm) is a hydrogen proton characteristic peak on the benzene ring, and the peak near 3.35ppm is a hydrogen proton characteristic peak on the alkyl C on dopamine.
Fig. 4 shows the results of SEM observation of the disc particles, which are relatively uneven in surface due to the influence of freeze-drying on the loss of moisture in the disc particle structure. When the disc particles are placed in the disc direction with the front face upwards in the freeze drying process, the shape of the disc particles is a relatively regular circle, as shown in fig. 4 (A); however, when the disc particles are placed in other directions during the freeze-drying process, the disc particles are folded and curled, and the SEM morphology is irregular and circular, as shown in fig. 4 (B).
Zeta potential is an important index for analyzing the dispersion stability of particles in a system, and charged particles have a certain attraction effect on particles with opposite charges in the dispersion system, and Zeta potential measures the mutual repulsion or attraction force; in this experiment, zeta potential of the disk particle dispersion solution was measured by Malvern ZEN3690 to evaluate dispersion stability of the disk particles in RO aqueous solution. FIG. 5 (A) is the observation result under a fluorescence microscope and an optical microscope of disk particles dispersed in RO water; from the figure, it can be observed that some disc particles have a size of less than 10 μm, presumably the first cause is due to partial folding, bending of the disc particles in the aqueous solution, which occurs due to the disc-like shape; the second reason is that the disc surface direction is different after the disc particles are dispersed in the solution, resulting in a difference in size as seen from the optical microscope viewing direction. FIG. 5 (B) shows the Zeta potential results of a dispersion of disc particles, the absolute value of the average Zeta potential being 13mV, indicating that the dispersion is relatively stable, and the Zeta potential is negative because the outer layer of the disc particles is the polyanionic polymer catechol modified hyaluronic acid.
The invention monitors the layer-by-layer self-assembly process of the C-CS and the C-HA in real time through QCM-D, and evaluates the stability of the assembled structure through the change of the F value and the D value. A dissipative quartz crystal microbalance analyzer (QCM-D) is a high-sensitivity instrument which can be used for analyzing surface molecular interactions in real time, and the resonant frequency delta F and the dissipation value delta D are obtained through a quartz matrix sensor by monitoring the change generated by the vibration frequency of the surface of a quartz gold plate in a module in real time, so that the mass and the structure of a sample are analyzed, wherein the delta F value is related to the mass of the sample, and the delta D value is related to the hardness of the sample. The specific experimental method is as follows:
(1) The quartz gold piece is required to be cleaned before being used, the cleaning liquid is prepared by ammonia water, hydrogen peroxide and RO water in a volume ratio of 1:1:5, the quartz gold piece is placed in the mixed solution and heated to 75 ℃, and after 20 minutes, the quartz gold piece is taken out and cleaned by RO water in an ultrasonic manner for 5 minutes.
(2) And (3) putting the cleaned quartz gold plate into a QCM-D module, mounting the module on a module object stage, connecting a sample inlet and outlet catheter, opening an instrument and a computer, and starting to level a base line by taking RO water as a solvent.
(3) After the baseline is flattened, placing the sample tube into a C-HA solution, placing the sample tube into RO water after the F value curve is stable, placing the sample tube into a C-CS solution after the F value curve is stable, and then placing the sample tube into RO water; this process was repeated 5 times to complete the self-assembly of BPs.
The F value is related to the quality of the adsorption layer, and when the quality of the adsorption layer on the quartz gold sheet is increased, the F value is correspondingly reduced; as can be seen from FIG. 6 (A), after the assembly of the BPs structure is completed, the F value is stabilized at about-80 Hz, and the F value is unchanged after the RO water is washed for 8 hours, so that the polyelectrolyte is not separated, and the assembled structure has stronger acting force between layers and higher stability.
The D value is related to the viscoelasticity of the adsorption layer, which is generally classified into rigid and flexible, and when the adsorption layer is a rigid film, it is coupled with the oscillating motion WM of the sensor surface, whereas when the adsorption layer is a flexible film, the viscoelasticity shear wave caused by the oscillation generates a delayed motion when propagating in the film. From the range of variation of D values in fig. 6 (B), the layer-by-layer assembled BPs structure is a flexible structure.
FIG. 7 shows the expression of CD86 by macrophages loaded with disc particles, using DAPI to stain the nuclei, which appear blue after excitation; the secondary antibody with FITC label is used, and is green under a fluorescence microscope after being specifically combined with the primary antibody. From the analysis in the figure, the disc particles resulted in a significant decrease in macrophage surface CD86 expression and a significant decrease in the positive rate relative to the blank, CD86 being a marker for M2 type macrophages, indicating that the polarization of macrophages towards M1 type was inhibited under the disc particle loading.
FIG. 8 shows the expression of macrophage CD206 loaded with disc particles, using a secondary antibody that appears red under a fluorescence microscope for differentiation from the CD86 fluorescent label. From the analysis in the figure, the disc particles resulted in a significant increase in macrophage surface CD206 expression, and a significant increase in the positive rate, relative to the blank, CD206 being a marker for M2 type macrophages, indicating that the macrophage polarization to M2 type was promoted under the disc particle loading.
Although specific embodiments of the invention have been described in detail with reference to the accompanying drawings, it should not be construed as limiting the scope of protection of the present patent. Various modifications and variations which may be made by those skilled in the art without the creative effort are within the scope of the patent described in the claims.
Claims (6)
1. A preparation method of drug-loaded disc particles with functions of adhering and regulating macrophages, which is characterized by comprising the following steps: sequentially layering catechol modified hyaluronic acid and catechol modified chitosan on a Polydimethylsiloxane (PDMS) template, obtaining disc particles by a microcontact printing method, and co-culturing with a pro-inflammatory fading medium (SPM) to obtain the compound;
wherein, the layer-by-layer assembly comprises the following steps: putting a PDMS template into an aqueous solution of catechol modified hyaluronic acid, taking out and putting into water for soaking, putting into an aqueous solution of catechol modified chitosan, taking out and putting into water for soaking, repeating the above operation flow 5 times to obtain a PDMS template assembled with 5 layers of catechol modified hyaluronic acid/catechol modified chitosan structures, putting into a stable air flow for 8-12 min, and drying at 35-40 ℃ for 08-1.2 h;
the PDMS template is prepared by hydrophilic-hydrophobic modification in an oxygen plasma system;
the micro-contact printing method comprises the following steps: placing one surface of the cover glass with the PVA film at a position 2 cm above RO water at 45-65 ℃, then taking down and rapidly placing the PDMS template pattern surface on the PVA film, and applying 10-40 g/cm 2 After the pressure is 10-40 s, removing the PDMS template from the upper side of the PVA film to obtain disc particles;
the catechol modified hyaluronic acid is prepared by the following steps:
(1) Dissolving morpholine ethanesulfonic acid monohydrate in water, adding hyaluronic acid, and adjusting the pH value to 4-6 to obtain a first mixed solution;
(2) Adding carbodiimide into a mixed solution of water and absolute ethyl alcohol, and adding the obtained solution and N-hydroxysuccinimide into the mixed solution I for reaction for 25-35 min to obtain a mixed solution II;
(3) Adding dopamine into the mixed solution II, adjusting the pH value of the solution to 4-6, and stirring for reaction for more than 4 hours to obtain a mixed solution III;
(4) Putting the mixed solution III into a dialysis bag with the molecular weight of 12000Da, dialyzing in water with the pH of 5-6 for 70-75 hours, and then freeze-drying to obtain the product;
the catechol modified chitosan is prepared by the following steps:
(1) Adding chitosan into water, adding concentrated hydrochloric acid, stirring until the chitosan is completely dissolved, adjusting the pH to 4-6, and adding an aqueous solution of dihydroxypropionyl glycoside to obtain a mixed solution I;
(2) Adding carbodiimide into a mixed solution of water and absolute ethyl alcohol, adding the obtained solution into the mixed solution I, stirring for more than 4 hours at room temperature, and adjusting the pH value to 4-6 during the stirring to obtain a mixed solution II;
(3) And (3) putting the mixed solution II into a dialysis bag with the molecular weight of 12000 and Da, dialyzing in water with the pH of 5-6 for 70-75 hours, and then freeze-drying to obtain the product.
2. The method of manufacturing according to claim 1, characterized in that: the PDMS template is prepared by the following steps:
(1) Spin coating photoresist on the wafer, placing a mask plate above the wafer, exposing for 10s, and developing with NaOH solution to obtain a patterned wafer;
(2) And mixing PDMS and a cross-linking agent, pouring the mixture into a patterned wafer, curing for 1.5-2.5 hours, peeling the PDMS template when the temperature is restored to room temperature, placing the PDMS template into an oxygen plasma system, performing hydrophilic-hydrophobic modification, adjusting the power to 300-400W, adjusting the vacuum degree to 150-250 MPa, and adjusting the modification time to 4-6 minutes.
3. The method of manufacturing according to claim 1, characterized in that: the concentration of the SPM is 50-100 nmol/L.
4. The drug-loaded disc particles with functions of adhering and regulating macrophages, which are prepared by the preparation method according to any one of claims 1-3.
5. The drug-loaded disc particle with functions of adhering and controlling macrophages of claim 4, wherein: the particle diameter and the thickness of the drug-loaded disc are respectively 10 mu m and 200-300nm.
6. The use of the drug-loaded disc particles with functions of adhering and controlling macrophages according to claim 4 or 5 for preparing a medicament for promoting vascular tissue repair and remodeling.
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