CN111632038B - Platelet drug-loaded micro-nano motor and preparation method and application thereof - Google Patents
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Abstract
The invention discloses a platelet drug-loading micro-nano motor and a preparation method and application thereof. The invention utilizes the characteristic that dopamine is easy to self-polymerize under the alkalescent condition to form a polydopamine coating by in-situ polymerization on the surface of a platelet, and then utilizes the polydopamine and drug molecules to combine to load drugs to form the platelet drug-loaded micro-nano motor. According to the invention, by utilizing the asymmetry of the platelet, under the irradiation of near infrared light, the polydopamine coating with the photothermal effect can form an uneven thermophoresis phenomenon, so that the movement of the platelet drug-loaded micro-nano motor is promoted. The preparation method has the advantages of simple raw materials, mild reaction conditions and simple and convenient operation, and the prepared material has good biocompatibility and can autonomously tend to inflammation or wound parts, and platelet-derived particles are generated by activating platelet cells so as to release the medicine.
Description
Technical Field
The invention belongs to biomedical materials, and particularly relates to a platelet drug-loaded micro-nano motor and a preparation method and application thereof.
Background
In recent years, the multifunctional artificially synthesized micro-nano motor has good application potential in the fields of catalytic degradation, biosensing, toxin adsorption, drug transportation and the like. However, in order to realize the practical application of the micro-nano motor in the living body, a completely biocompatible smart material needs to be designed and developed, so that the synthesized micro-nano motor can play a role in a complex biological environment without generating harmful effects.
Some natural cells and microorganisms have inherent chemotactic properties and biological functions. The combination of these biological components and nanomaterials to produce cell-based micro-nano motors for use in biological environments for operation and performance of various biological functions has been investigated. The cell-based micro-nano motor can fully exert the inherent chemotactic characteristic of the biological component and promote the effective movement of the micro-nano motor in a complex biological environment; and functions in specific locations by mimicking the biological function of natural cells. The cell-based micron motor has wide application prospect in the biomedical field because no additional fuel or power device is needed.
Despite the unique advantages of such cell-based micromotors, their potential for in vivo applications remains a significant challenge. Including control of speed and direction of motion and size limitations of micron-scale motors. For example: macrophage motor sizes of tens of microns make it difficult to deliver drugs efficiently to the interior of target cells, greatly limiting their efficacy as drug delivery vehicles.
Disclosure of Invention
The purpose of the invention is as follows: aiming at the problems in the prior art, particularly the problem that most of the existing micro-nano motors are poor in biocompatibility, the invention provides a platelet drug-loaded micro-nano motor and a preparation method thereof. The platelet drug-loading micro-nano motor provided by the invention takes natural cell platelets as a base material, adopts a mild surface modification technology to coat a polydopamine coating on the surface, and then utilizes the o-phenylenediquinone group of polydopamine to carry out Schiff base reaction with the amino group of amine-containing drugs, or utilizes the pi-pi accumulation between the polydopamine and the amine-containing or aromatic drugs to load the drugs. The preparation method is simple in preparation process and good in material biocompatibility, the prepared platelet drug-loaded micro-nano motor can autonomously tend to tissues and cells in pathological environments such as inflammation, thrombus, operation wounds and the like by utilizing inherent chemotaxis of platelets, and cell uptake and tissue penetration can be enhanced by the movement of the micro-nano motor, so that the drug delivery efficiency is improved, and the platelet drug-loaded micro-nano motor has a wide application prospect in the field of biomedicine.
The invention provides a platelet micro-nano motor with stimulus-responsive morphology change and good biocompatibility, which takes natural cell platelets as a base material, and adopts a mild surface modification technology to wrap a polydopamine coating on the surface part of the platelet, so that on one hand, the natural chemotaxis of the platelets can be utilized to autonomously tend to a surgical wound or inflammatory environment; on the other hand, the photo-thermal effect of polydopamine can be utilized to generate an uneven thermophoresis phenomenon under the irradiation of near-infrared laser so as to drive the movement of the platelet and the derivative particle drug-loaded micro-nano motor.
The technical scheme is as follows: in order to achieve the above object, the platelet drug-loaded micro-nano motor according to the present invention includes a polydopamine coating layer wrapped on the outer side of platelets and amine-containing compound or aromatic compound drug molecules physically or chemically combined with the polydopamine, and can move under near-infrared irradiation to form a drug-loaded micro-nano motor. Preferably, the thickness of the polydopamine coating is 0.1-1 μm.
The preparation method of the platelet drug-loaded micro-nano motor is characterized by comprising the following steps of:
(1) dispersing the extracted Platelets (PLTs) in a PBS buffer solution to obtain a platelet suspension to prevent unnecessary platelet activation, and storing at room temperature for later use;
(2) dissolving dopamine hydrochloride in Tris-HCl buffer solution (pH 8.5), mixing the dopamine hydrochloride with the platelet suspension according to a certain volume ratio, and polymerizing under illumination to obtain platelet micro-nano motor suspension containing a polydopamine coating; centrifugally washing, dispersing in a PBS buffer solution, and storing for later use;
(3) dissolving an amine-containing compound or aromatic compound medicine in a PBS buffer solution, mixing and oscillating the PBS buffer solution with the platelet micro-nano motor suspension containing the polydopamine coating at room temperature to obtain a platelet drug-loaded micro-nano motor suspension; after centrifugal washing, the mixture is dispersed in PBS buffer solution for storage and standby.
Wherein the thickness of the polydopamine coating is 0.1-1 μm.
Preferably, the step (1) of extracting platelets is to separate and extract platelets from fresh whole blood by using a gradient centrifugation method, wherein the gradient centrifugation method comprises the steps of firstly centrifuging at 1500rpm for 15-20 minutes at 1000-.
Preferably, the concentration of the Tris-HCl solution of dopamine hydrochloride in the step (2) is 0.1-2mg/mL, the volume ratio of the solution to the platelet suspension is 1:1-1:10, and the density of the platelets in the platelet suspension is 1 x 107-8one/mL, and the polymerization reaction time is 2-6 h.
Wherein, the medicine in the step (4) is amine-containing compound or aromatic compound, such as adriamycin, nimustine, mitomycin, etc.
Preferably, the drug concentration in the step (3) is 0.1-2mg/mL, the volume ratio of the drug concentration to the platelet micro-nano motor suspension containing the polydopamine coating is 1:1-5:1, and the platelet density is 1 × 107-8Mixing the powder per mL with platelet micro-nano motor suspension containing polydopamine coating for 2-12 hours at room temperature.
The platelet micro-nano motor disclosed by the invention is applied to the preparation of medicines for preventing the metastasis and recurrence of cancers.
The platelet drug-carrying micro-nano motor can automatically approach a wound after surgical excision, is stimulated by an inflammatory environment or a tumor microenvironment, activates and releases platelet-derived particles with nano sizes, and promotes the release of chemotherapeutic drugs such as adriamycin. The applied near-infrared laser irradiation can promote the platelet-derived microparticles to be taken up by cancer cells, and better exert the drug effect of the adriamycin.
Specifically, the characteristic that dopamine is easy to self-polymerize under a weak alkaline condition is utilized, a polydopamine coating is formed by in-situ polymerization on the surface of a platelet, and then Schiff base reaction is carried out on rich o-phenylenediquinone groups of polydopamine and amine-containing drugs or drug loading is carried out by pi-pi accumulation between polydopamine and amine-containing compounds or aromatic drugs, so that the platelet drug loading micro-nano motor is formed. By utilizing the asymmetry of the platelet, under the irradiation of near infrared light, the polydopamine coating with the photothermal effect can form an uneven thermophoresis phenomenon, thereby promoting the movement of the platelet drug-loaded micro-nano motor.
The invention utilizes the natural characteristic of the blood platelet, the blood platelet under the physiological condition is in a resting state and is in a disc shape, and the blood platelet circulates near the blood vessel wall. When stimulated by activation signals, platelets adhere, activate, and aggregate. The activated platelets extend pseudopodically and become dendritic, releasing platelet-derived microparticles (plasma membranes of platelets) approximately 0.1-1 micron in diameter. The invention extracts platelets from whole blood, modifies polydopamine coating (in resting state) on the membrane surface, then adds thrombin to activate the modified platelets in vitro to obtain activated platelets and platelet-derived microparticles, and the two products are both derived from original platelets, so that the surfaces of the platelets are also covered with the polydopamine coating. And the drug molecules combined with the polydopamine exist on the surfaces of the activated platelets and platelet-derived particles to form nanoscale platelet-derived particles carrying the drug, which is more favorable for tissue permeation of the drug than micron-sized cells.
Has the advantages that: compared with the prior art, the invention has the following advantages:
the platelet drug-carrying micro-nano motor is prepared by using natural cell platelets and dopamine with good biocompatibility as raw materials. The method has the advantages of simple raw materials, mild reaction conditions and simple and convenient operation, and the prepared material has good biocompatibility, can autonomously tend to inflammation or wound parts, and generates platelet-derived microparticles through platelet cell activation, thereby releasing the medicine. Compared with the existing cell-based micro-nano motor, the platelet drug-loaded micro-nano motor has the advantages that the appearance change of the platelet drug-loaded micro-nano motor is stimulated, nanoscale derivative particles can be generated after activation, the defect that the existing biological micro-motor drug delivery system is large in size is overcome compared with the existing living cell drug delivery system (such as macrophages, sperm cells, bacteria and the like), drug permeation in tissues is facilitated, meanwhile, the poly-dopamine coating with the photo-thermal effect can form an uneven thermophoresis phenomenon under the irradiation of near infrared light by utilizing the asymmetry of platelets, so that the movement of the platelet drug-loaded micro-nano motor is promoted, the cell uptake and the tissue permeation can be enhanced through the movement performance of the micro-nano motor, and the drug delivery efficiency is improved.
The platelet drug-loaded micro-nano motor prepared by the invention can autonomously tend to tissues and cells in pathological environments such as inflammation, thrombus, postoperative wound and the like by utilizing the inherent chemotaxis of platelets, and the cell uptake and tissue penetration can be enhanced by the movement of the micro-nano motor, so that the drug delivery efficiency is improved, the platelet drug-loaded micro-nano motor can be effectively used for preparing drugs for preventing the metastasis and recurrence of cancers, and has wide application prospect in the field of biomedicine.
Drawings
FIG. 1 is a transmission electron micrograph of platelets;
FIG. 2 is a transmission electron microscope image of a platelet micro-nano motor;
fig. 3 is an immunofluorescence image of a platelet drug-loaded micro-nano motor loaded with doxorubicin;
FIG. 4 shows a near-infrared laser (808nm, 2.5W/cm)2) Motion track of platelet drug-loaded micro-nano motor loaded with adriamycin under irradiation(5s)；
FIG. 5 shows the cell viability of different materials after 24 hours of co-incubation with MCF-7 cells.
Detailed Description
The technical solutions of the present invention are further described in detail by the following specific examples, but it should be noted that the following examples are only for describing the contents of the present invention and should not be construed as limiting the scope of the present invention.
Example 1
A preparation method of a platelet drug-loaded micro-nano motor specifically comprises the following steps:
(1) preparing a platelet micro-nano motor:
collecting fresh rabbit whole blood, centrifuging at 1500rpm for 15 minutes, and carefully sucking and collecting platelet-rich plasma on the upper layer; the platelet suspension was obtained by centrifugation at 3000rpm for 10 minutes, the upper plasma layer was discarded, and the precipitated platelets were slowly resuspended in a PBS buffer solution (pH 7.4, the same applies hereinafter). The transmission electron micrograph of the platelet is shown in FIG. 1, and it can be seen from FIG. 1 that the platelet has a disk shape. 10mg of dopamine hydrochloride was weighed and dissolved in 10mL of 10mM Tris-HCl buffer (pH 8.5). Adjustment of platelet Density to 1X 10 in platelet suspension by cytometry8Taking 1mL and 1mL (1X 10) of the dopamine hydrochloride solution per mL8Respectively) mixing the platelet suspension, placing the platelet suspension in a silent mixer, polymerizing for 2 hours at room temperature to obtain platelet micro-nano motor suspension containing a polydopamine coating, centrifuging at 3000rpm for 10min to take a lower layer precipitate, adding PBS buffer solution, mixing uniformly, centrifuging, washing, and dispersing in the PBS buffer solution for later use. Fig. 2 is a transmission electron microscope image of the platelet micro-nano motor, and it can be seen from fig. 2 that the polydopamine is wrapped around the disc-shaped platelet, the thickness of the polydopamine is about 0.3-1 μm, and the outer ring with lighter color is the thickness of the polydopamine coating is about 0.1-1 μm.
(2) Preparing a platelet drug-loaded micro-nano motor:
10mg of doxorubicin hydrochloride was weighed and dissolved in 10mL of PBS buffer (pH 7.4, the same applies below) with sonication. Regulating the density of the platelets in the platelet micro-nano motor suspension containing the polydopamine coating in the step (1) to be 1 multiplied by 10 by a cell counting method8one/mL.Taking 4mL and 1mL of platelet micro-nano motor suspension (1X 10) containing polydopamine coating of the doxorubicin hydrochloride solution8Respectively) mixing, placing in a silent mixer, reacting for 2 hours in a dark place at room temperature to obtain an adriamycin-loaded platelet drug-loaded micro-nano motor suspension, centrifuging at 3000rpm for 10min to take a lower layer precipitate, adding a PBS buffer solution, mixing uniformly, centrifuging, washing, and dispersing in the PBS buffer solution for later use. The obtained adriamycin-loaded platelet drug-loaded micro-nano motor and DiO dye are incubated together, a platelet membrane is marked and placed under a confocal microscope for observation, and an immunofluorescence image is shown in figure 3. From figure 3 it can be seen that the red fluorescence (doxorubicin DOX, middle panel) almost coincides with the green fluorescence (platelet PLTs, left panel) (right panel), demonstrating successful loading of the drug. Subjecting the obtained adriamycin-loaded platelet drug-loaded micro-nano motor to near-infrared laser (808nm, 2.5W/cm)2) The shot of the motion video under irradiation is shown in fig. 4, and the black thin line represents the motion track of the platelet drug-loaded micro-nano motor in 5s, which proves that the platelet drug-loaded micro-nano motor can move under the irradiation of the near-infrared laser.
0.1mL of cells were cultured at a cell density of 5X 104The individual/mL human breast cancer cells (MCF-7) were seeded in 96-well plates and placed at 37 ℃ in 5% CO2Culturing in an incubator for 12 h. The medium was aspirated and 0.2mL (1X 10) was added to each well of the experimental group8individual/mL) Platelets (PLTs) or the platelet drug-loaded micro-nano motor or fresh medium, adding a near-infrared laser (808nm, 2.5W/cm) to each well of the group of the fresh medium2) Irradiating for 10min, adding an equal amount of fresh cell culture medium into each well of a blank group (Control) as a Control, and testing the cell survival rate by adopting an MTT method after incubating for 24 h. The results are shown in fig. 5, the cell survival rates of the experimental groups are all larger than 90%, which indicates that the platelet micro-nano motor prepared by the research has good biocompatibility.
Example 2
A preparation method of a platelet drug-loaded micro-nano motor specifically comprises the following steps:
(1) preparing a platelet micro-nano motor:
platelets were separated and extracted by gradient centrifugation as described in example 1 to obtain a platelet suspensionAnd (4) liquid. Adjustment of platelet Density to 1X 10 in platelet suspension by cytometry7one/mL. 20mg of dopamine hydrochloride was weighed and dissolved in 10mL of 10mM Tris-HCl buffer (pH 8.5), and 1mL and 10mL (1X 10)7Respectively) mixing the platelet suspension, placing the platelet suspension in a silent mixer, polymerizing for 2 hours at room temperature to obtain platelet micro-nano motor suspension, centrifuging at 3000rpm for 10min to take down a lower layer precipitate, adding PBS buffer solution, mixing uniformly, centrifuging, washing, and dispersing in the PBS buffer solution for later use.
(2) Preparing a platelet drug-loaded micro-nano motor:
10mg of doxorubicin hydrochloride was weighed and dissolved in 10mL of PBS buffer by sonication. Regulating the density of the platelets in the platelet micro-nano motor suspension containing the polydopamine coating in the step (1) to be 1 multiplied by 10 by a cell counting method7one/mL. Transferring 1mL of the doxorubicin hydrochloride solution and mixing with 1mL of platelet micro-nano motor suspension containing a polydopamine coating (1X 10)7And (2) placing the mixture in a silent mixer, reacting for 2 hours at room temperature in a dark place to obtain a platelet drug-loaded micro-nano motor suspension loaded with adriamycin, centrifuging at 3000rpm for 10min to take a lower layer precipitate, adding PBS buffer solution, mixing uniformly, centrifuging, washing, and dispersing in the PBS buffer solution for later use.
Example 3
A preparation method of a platelet drug-loaded micro-nano motor specifically comprises the following steps:
(1) preparing a platelet micro-nano motor:
collecting fresh rabbit whole blood, centrifuging at 1000rpm for 20 minutes, and carefully sucking and collecting platelet-rich plasma on the upper layer; centrifugation was carried out at 2500rpm for 15 minutes, the upper plasma layer was discarded, and the precipitated platelets were slowly resuspended in a PBS buffer solution to obtain a platelet suspension. Adjustment of platelet Density to 1X 10 in platelet suspension by cytometry8one/mL. 1mg of dopamine hydrochloride was weighed, dissolved in 10mL of 10mM Tris-HCl buffer (pH 8.5), and taken out 1mL and 1mL (1X 10)8Respectively) mixing the platelet suspension, placing the platelet suspension in a silent mixer, polymerizing for 6 hours at room temperature to obtain platelet micro-nano motor suspension, centrifuging at 3000rpm for 10min to take down a lower layer precipitate, adding PBS buffer solution, mixing uniformly, centrifuging, washing, and dispersing in the PBS buffer solution for later use.
(2) Preparing a platelet drug-loaded micro-nano motor:
4mg of nimustine hydrochloride is weighed and dissolved in 4mL of PBS buffer. Regulating the density of the platelets in the platelet micro-nano motor suspension containing the polydopamine coating in the step (1) to be 1 multiplied by 10 by a cell counting method8And mixing 1mL of the suspension with the 4mL of the nimustine hydrochloride solution, carrying out a dark reaction for 2 hours at room temperature to obtain a nimustine-loaded platelet drug-loaded motor suspension, centrifuging at 3000rpm for 10min, taking out the lower-layer precipitate, adding a PBS buffer solution, mixing uniformly, centrifuging, washing, dispersing in the PBS buffer solution, and storing for later use.
Example 4
A preparation method of a platelet drug-loaded micro-nano motor specifically comprises the following steps:
(1) preparing a platelet micro-nano motor:
platelets were separated and extracted by gradient centrifugation as described in example 1 to obtain a platelet suspension. Adjustment of platelet Density to 1X 10 in platelet suspension by cytometry8one/mL. 5mg of dopamine hydrochloride was weighed, dissolved in 10mL of 10mM Tris-HCl buffer (pH 8.5), and taken out 1mL and 1mL (1X 10)8Respectively) mixing the platelet suspension, placing the platelet suspension in a silent mixer, polymerizing for 2 hours at room temperature to obtain platelet micro-nano motor suspension, centrifuging at 3000rpm for 10min to take down a lower layer precipitate, adding PBS buffer solution, mixing uniformly, centrifuging, washing, and dispersing in the PBS buffer solution for later use.
(2) Preparing a platelet drug-loaded micro-nano motor:
2mg of mitomycin was weighed and dissolved in 2mL of PBS buffer. Regulating the density of the platelets in the platelet micro-nano motor suspension containing the polydopamine coating in the step (1) to be 1 multiplied by 10 by a cell counting method8Mixing 1mL of the mitomycin solution with 1mL of the mitomycin solution, carrying out a light-shielding reaction for 2 hours at room temperature to obtain a mitomycin-loaded platelet drug-loaded motor suspension, centrifuging at 3000rpm for 10min to take a lower-layer precipitate, adding a PBS (phosphate buffer solution) to the lower-layer precipitate, uniformly mixing, centrifuging, washing, and dispersing in the PBS for storage for later use.
Example 5
Example 5 the same preparation method as example 1, but differentThe concentration of the adriamycin hydrochloride drug is 0.1mg/mL, the volume ratio of the adriamycin hydrochloride drug to platelet micro-nano motor suspension containing the polydopamine coating is 4:1, and the density of the platelets is 1 multiplied by 107pieces/mL, mixed at room temperature for 4 hours.
Example 6
Example 6 is the same as example 1 except that the concentration of the adriamycin hydrochloride drug is 2mg/mL, the volume ratio of the adriamycin hydrochloride drug to the platelet micro-nano motor suspension containing the polydopamine coating is 5:1, and the density of the platelets is 1 x 107pieces/mL, mixed at room temperature for 12 hours.
In the preparation of the drug for preventing cancer metastasis and recurrence, the platelet drug-loaded micro-nano motor prepared by the embodiment of the invention can automatically tend to the wound after surgical resection, is stimulated by inflammatory environment or tumor microenvironment, activates and releases platelet-derived microparticles with nanometer sizes, and promotes the release of chemotherapeutic drugs such as adriamycin. The invention utilizes the asymmetry of the platelet by the near infrared laser irradiation, and the poly dopamine coating with the photothermal effect can form uneven thermophoresis under the irradiation of the near infrared light, thereby promoting the movement of the platelet drug-carrying micro-nano motor, promoting the uptake of platelet derived particles by cancer cells, and better playing the drug effect of adriamycin, nimustine or mitomycin.
Claims (8)
1. The platelet drug-carrying micro-nano motor is characterized by comprising a polydopamine coating wrapped on the outer side of platelets and amine-containing compounds or aromatic compound drug molecules combined with the polydopamine, and the platelet drug-carrying micro-nano motor is formed.
2. The platelet-loaded micro-nano motor according to claim 1, wherein the thickness of the polydopamine coating is 0.1-1 μm.
3. A preparation method of a platelet drug-loaded micro-nano motor is characterized by comprising the following steps:
(1) dispersing the extracted Platelets (PLTs) in a PBS buffer solution to obtain a platelet suspension, and storing at room temperature for later use;
(2) dissolving dopamine hydrochloride in a buffer solution, mixing the dopamine hydrochloride with the platelet suspension, polymerizing the mixture under illumination to obtain platelet micro-nano motor suspension containing a polydopamine coating, centrifugally washing the platelet micro-nano motor suspension, and dispersing the platelet micro-nano motor suspension in a PBS buffer solution for storage and later use;
(3) dissolving a needed amine compound or aromatic compound-containing drug in a PBS buffer solution, and oscillating and mixing the PBS buffer solution with the platelet micro-nano motor suspension containing the polydopamine coating at room temperature to obtain drug-loaded platelet micro-nano motor suspension, namely the platelet micro-nano motor.
4. The preparation method according to claim 3, wherein the step (1) of extracting platelets is preferably to separate and extract platelets from fresh whole blood by using a gradient centrifugation method, wherein the gradient centrifugation method comprises centrifugation at 1500rpm for 15-20 minutes at 1000-.
5. The method according to claim 3, wherein the concentration of the Tris-HCl buffer solution of dopamine hydrochloride in the step (2) is 0.1-2mg/mL, the volume ratio of the buffer solution to the platelet suspension is 1:1-1:10, and the density of the platelets in the platelet suspension is 1 x 107-8Per mL; the polymerization reaction time is 2-6 h.
6. The method according to claim 3, wherein the drug in step (3) is an amine-containing compound or an aromatic compound, such as doxorubicin, nimustine or mitomycin.
7. The preparation method according to claim 3, wherein the drug concentration in the step (3) is 0.1-2mg/mL, the volume ratio of the drug concentration to the platelet micro-nano motor suspension containing the polydopamine coating is 1:1-5:1, and the drug concentration is mixed with the platelet micro-nano motor suspension containing the polydopamine coating at room temperature for 2-12 hours.
8. The use of the platelet-loaded micro-nano motor according to claim 1, wherein the drug is doxorubicin, nimustine or mitomycin, for the preparation of a drug for preventing metastasis and recurrence of cancer.
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