CN114259478A - Preparation method of pollen hydrogel hybrid drug-loaded microspheres and application of pollen hydrogel hybrid drug-loaded microspheres in drug responsive release under photo-thermal stimulation - Google Patents
Preparation method of pollen hydrogel hybrid drug-loaded microspheres and application of pollen hydrogel hybrid drug-loaded microspheres in drug responsive release under photo-thermal stimulation Download PDFInfo
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- CN114259478A CN114259478A CN202210138101.XA CN202210138101A CN114259478A CN 114259478 A CN114259478 A CN 114259478A CN 202210138101 A CN202210138101 A CN 202210138101A CN 114259478 A CN114259478 A CN 114259478A
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Abstract
The invention provides a pollen hydrogel hybrid drug-loaded microsphere, which comprises the following steps: step one, preparing pollen cavity microspheres with thorn-shaped structures; step two, drug loading: soaking the pollen cavity microspheres prepared in the step one in a hydrogel solution containing a drug and a near-infrared light conversion material, filling hydrogel into cavities of pollen in a vacuum decompression mode, curing the hydrogel through ultraviolet irradiation and locking cytokines to obtain pollen hydrogel hybrid drug-loaded microspheres; the drug is a cytokine; the near infrared light conversion material is one or more of BP, BPNSs and BPQDs. The pollen hydrogel hybrid drug-loaded microsphere provided by the invention can be loaded with small-molecule drugs and protein drugs, and can release the drugs controllably through photo-thermal stimulation, thereby realizing intelligent drug delivery.
Description
Technical Field
The invention relates to the field of biological materials, in particular to a preparation method of a pollen hydrogel hybrid drug-loaded microsphere and application of the pollen hydrogel hybrid drug-loaded microsphere in responsive drug release under photo-thermal stimulation.
Background
Smart responsive biomaterials have been used to deliver bioactive substances to treat cancer. As one of these innovative materials, stimulus-responsive hydrogels are capable of undergoing structural changes under external environmental stimuli, such as light, temperature, pH, and ions. The near-infrared light is a common external stimulation signal, photoresponse potential is given by means of the thermoacoustics of the hydrogel, namely the near-infrared light is converted into heat energy through a photo-thermal conversion material, so that the temperature-sensitive hydrogel material shrinks or expands in volume, and therefore the medicine and the active factors wrapped in the hydrogel are released, and near-infrared light intelligent response is achieved. Black Phosphorus Nanoplates (BPNSs) are a last-but-one of the two-dimensional materials other than graphene, and have application values in photocatalysis, biomedicine, energy storage and conversion, and electronic and optoelectronic devices. Based on the excellent biocompatibility, biodegradability and photothermal and photosensitive properties of BPNSs, BPNSs also have great potential in biological applications, and the non-toxic degradation products thereof can be further used for bone regeneration through biomineralization. However, the in vivo application of the stimuli-responsive hydrogel is gradually disintegrated under the influence of the in vivo physiological environment, and how to prolong the in vivo residence time of the hydrogel so as to realize long-acting release is still to be further studied.
Pollen grains are a micro-particulate material derived from natural biomass and have received increasing attention due to their ready availability, biodegradability and low cost. In particular, pollen grains exhibit a unique morphology, with surface stings and cavities inside, which give them a large contact area and encapsulation capacity. Based on this, pollen grains have been used as novel carrier materials and show good potential in the biomedical field, including bioadhesion, drug delivery, organic adsorption, multiplex bioassay, etc. However, pollen grains are rarely used as a vehicle for immunotherapy and their use in enhancing cellular immunotherapy has not yet been explored. Given that mature DCs also have complex irregular shapes and thin dendrites, the similar spiky structure of pollen grains may be advantageous for efficient identification and target adhesion. Therefore, the pollen grains can be constructed into a biological heuristic immunopharmaceutical delivery bracket for stimulating the immune system of tumor patients and enhancing the tumor inhibition capability of the tumor patients.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a preparation method of a pollen hydrogel hybrid drug-loaded microsphere, which comprises the following steps:
step one, preparing pollen cavity microspheres with thorn-shaped structures;
step two, drug loading: soaking the pollen cavity microspheres prepared in the step one in a hydrogel solution containing a drug and a near-infrared light conversion material, filling hydrogel into cavities of the pollen cavity microspheres in a vacuum decompression mode, curing the hydrogel through ultraviolet irradiation and locking cytokines to obtain pollen hydrogel hybrid drug-loaded microspheres;
the drug is a cytokine;
the near infrared light conversion material is one or more of BP, BPNSs and BPQDs.
In order to optimize the technical scheme, the specific measures adopted further comprise:
further, in the first step, the pollen cavity microsphere is a sunflower pollen cavity microsphere.
Further, in the step one, the preparation method of the pollen cavity microsphere comprises the following steps: degreasing pollen grains by using acetone, and then cracking the pollen grains by using KOH to prepare the pollen cavity microsphere with the thorn-shaped structure.
Further, in the second step, the hydrogel solution is an aqueous solution mixed by one or more than two of enamide, PEG8000 and acrylic acid.
Further, the concentration of acrylamide and acrylic acid is 5-10 v/v%.
Further, in the second step, a photoinitiator is mixed in the hydrogel solution, and the addition amount of the photoinitiator is 1% of the volume of the hydrogel solution.
Further, in step two, the cytokine is one or more of IL-2, IL-15 and IL-21, and the concentration of the cytokine is 100-1000 ng/ml.
Further, in the second step, the concentration of the near infrared light conversion material is 100-500 ug/ml.
The pollen hydrogel hybrid drug-loaded microsphere is used for responding to release drugs under photo-thermal stimulation.
The invention has the beneficial effects that:
the invention provides pollen hydrogel hybrid drug-loaded microspheres based on pollen grains. The pollen hydrogel hybrid drug-loaded microsphere is formed by mixing and assembling pollen grains, temperature-sensitive hydrogel and near-infrared light conversion materials. The pollen grains are resistant to the digestion of human digestive juice, have stable structure and good biocompatibility, can maintain the stability of the loaded drug, delay the degradation of hydrogel in vivo, and are carriers with drug delivery potential; the temperature-sensitive hydrogel is sensitive to temperature, the volume of the temperature-sensitive hydrogel gradually expands along with the increase of the temperature, and the BPNSs doped into the temperature-sensitive hydrogel endows the temperature-sensitive hydrogel with intelligent responsiveness. The irradiation of near infrared light can increase the temperature of BPNSs, further increase the temperature of the temperature-sensitive hydrogel doped with BPNSs and enable the temperature-sensitive hydrogel to expand in volume and change in form so as to release the internally wrapped medicine, thereby realizing the controlled release process of the medicine in time, space and dosage, reducing the side effect caused by excessive administration, reducing the administration frequency and reducing the pain of patients.
Drawings
FIG. 1 is a brightfield image of pollen cavity microspheres;
fig. 2 is a preparation diagram of a near-infrared light conversion material, wherein (a) is a real object diagram of a black phosphorus nanosheet, (b) is a transmission electron microscope diagram of the black phosphorus nanosheet, (c) is a particle size distribution diagram of the black phosphorus nanosheet, and (d) shows a temperature change of the black phosphorus nanosheet under irradiation of near-infrared light;
FIG. 3 is a preparation diagram of a temperature-sensitive hydrogel, wherein (a) shows temperature-sensitive hydrogels at different temperatures, (b) shows light transmittances of the temperature-sensitive hydrogels at different temperatures, (c) shows a trend of volume of the temperature-sensitive hydrogel changing with temperature, and (d) shows temperature changes and volume changes of the temperature-sensitive hydrogel under near infrared light irradiation;
FIG. 4 is a scanning electron microscope image of pollen hydrogel hybrid microspheres, wherein (a) are pollen cavity microspheres without hydrogel coating, and (b) are pollen cavity microspheres with hydrogel coating;
FIG. 5 is a drug release diagram of the pollen hydrogel hybrid drug-loaded microspheres driven by near infrared light.
Detailed Description
The present invention will now be described in further detail with reference to the accompanying drawings.
The invention provides a preparation method of a pollen hydrogel hybrid drug-loaded microsphere, which comprises the following steps:
step one, preparing the pollen cavity microsphere with the thorn-shaped structure.
The preparation of the pollen cavity microsphere with the thorn-shaped structure mainly comprises two steps: (1) degreasing pollen grains by using acetone and diethyl ether; (2) the pollen grains were subjected to a cracking treatment using KOH. Specifically, sunflower pollen grains are added into a vessel containing acetone, and stirred for 3 hours at the stirring temperature of 50 ℃ and the stirring speed of 200 rpm; centrifuging the obtained sunflower pollen granules, adding the precipitate into fresh acetone again, and magnetically stirring for 3 hr; adding the sunflower pollen granules treated by acetone into a vessel containing diethyl ether, and stirring overnight (about 12 hours) at a stirring temperature of 25 deg.C and a stirring speed of 200 rpm; the above degreasing process is repeated two to three times. Further, the defatted sunflower pollen grains were mixed with 10% (w/v) KOH solution, and stirred at 80 ℃ and 400rpm for 2 hours; centrifuging the obtained sunflower pollen granules, adding the precipitate into fresh 10% KOH again, magnetically stirring for 6 hours, and further centrifuging and collecting; and finally, washing the centrifugally collected sunflower pollen grains twice by using 10% KOH to prepare pollen cavity microspheres, adding the pollen cavity microspheres into deionized water, and storing in a refrigerator at 4 ℃ for subsequent experiments.
The scanning electron microscope picture of the pollen cavity microsphere is shown in figure 1, and it can be seen from the figure that the prepared pollen cavity microsphere is complete in shape, about 50 microns in size, and has a thorn-shaped bulge and a hole structure on the surface.
And step two, loading the medicine.
1) Preparation of near infrared light conversion material BPNSs
And preparing the black phosphorus nanosheet from the black phosphorus block by adopting an ultrasonic stripping method. Specifically, the black phosphorus blocks are dispersed in a solution of 1-methyl-2-pyrrolidone (NMP) and soaked in an ultrasonic water bath with the power of 720W for 6 hours; after the sonication was completed, the supernatant was carefully collected and centrifuged at 7000rpm for 20 minutes; the resulting precipitate was washed twice with deionized water and stored in a refrigerator at 4 ℃ for subsequent experiments.
The prepared BPNSs are shown in FIG. 2(a), and the BPNSs are seen to be light yellow and are well dispersed in an aqueous solution; FIGS. 2(b) and (c) show that BPNSs have a particle size around 170nm under transmission electron microscopy and dynamic light scattering observation; FIG. 2(d) shows that BPNSs can significantly increase the temperature under near infrared light irradiation, and the temperature increasing effect is positively correlated with the concentration of BPNSs used.
2) Preparation of temperature-sensitive hydrogel
Dissolving 250mg of acrylamide, 500mg of PEG8000, 250mg of acrylic acid, 10mg of Bis and 40ul of HMPP in 4ml of deionized water to form a pre-gel solution; the concentration of acrylamide and acrylic acid is 5-10 v/v%; the pre-gel solution contains a photoinitiator, and the volume of the photoinitiator is 1 percent of the volume of the pre-gel solution; and (3) crosslinking and curing the pre-gel solution for 10 minutes under the ultraviolet ray with the wavelength of 254nm to form the temperature-sensitive hydrogel. FIGS. 3(a) and 3(b) show that the transmittance of temperature-sensitive hydrogels is continuously increased with the increase of temperature.
Testing the photo-thermal response characteristics of the temperature-sensitive hydrogel: and (3) immersing the prepared temperature-sensitive hydrogel into water at a preset temperature, and observing the volume deformation of the temperature-sensitive hydrogel under a microscope. As shown in fig. 3(c), the hydrogel rapidly swells at a water temperature of 45 ℃ and changes in volume in response to temperature. In order to test the photo-thermal reaction characteristics of the hydrogel, in the process of preparing the temperature-sensitive hydrogel, a near infrared light conversion material BPNSs is dispersed in a pre-gel solution and irradiated by near infrared light of 808 nm. The temperature and area changes of the temperature-sensitive hydrogel containing BPNSs were measured with a thermal imager and a stereomicroscope, respectively. As shown in fig. 3(d), the temperature-sensitive hydrogel doped with the bpsss can significantly increase the temperature under the irradiation of the near infrared light, and the volume thereof increases correspondingly with the increase of the temperature, which indicates that the temperature-sensitive hydrogel doped with the bpsss can achieve the responsiveness of the near infrared light.
The near infrared light conversion material can also be one or more of BP, BPNSs and BPQDs.
3) Preparation of pollen hydrogel hybrid drug-loaded microspheres
Adding the pollen cavity microspheres into a pre-gel solution containing IL-2 cell factors and BPNSs to form a hydrogel solution, wherein the concentration of IL-2 is 1000ng/ml, and the concentration of BPNSs is 500 ug/ml; mixing hydrogel solution containing pollen cavity microspheres by vortex, degassing by using a freeze dryer, and filling the hydrogel into cavities of the pollen cavity microspheres; collecting pollen microspheres coated by hydrogel by a centrifugal method, diluting the pollen microspheres with water, irradiating the crosslinked hydrogel by using ultraviolet light with the wavelength of 254nm, solidifying the hydrogel and locking a medicament (a cytokine IL-2) to obtain the pollen hydrogel hybrid medicament-carrying microspheres. As shown in fig. 4, the surface of the pollen cavity microsphere without hydrogel coating has a plurality of hole structures, and the hole structures of the surface of the pollen cavity microsphere coated with hydrogel are covered by hydrogel, which indicates that hydrogel can effectively enter the pollen cavity microsphere.
In the present embodiment, the near infrared light conversion material is one or more of BP, bpsss, and BPQDs; the pre-gel solution can be replaced by one or more than two mixed aqueous solutions of enamide, PEG8000 and acrylic acid; the cytokine is one or more of IL-2, IL-15 and IL-21.
Testing the slow release performance of the medicine:
to evaluate the drug release process, BSA-FITC as a model protein was loaded into pollen-hydrogel hybrid drug-loaded microspheres, irradiated with near infrared light, and observed along the Z-axis by confocal laser scanning microscopy (nikon a 1). The fluorescence intensity of the pollen hydrogel hybrid drug-loaded microspheres was further evaluated using image J software to calculate the gray value. As shown in figure 5, FITC is green fluorescence, after near-infrared light irradiation, hydrogel on the surface of the pollen hydrogel hybrid drug-loaded microsphere undergoes obvious volume change, and the fluorescent protein drug in the hydrogel hybrid drug-loaded microsphere can be gradually released along with the expansion of the hydrogel volume.
The above is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above-mentioned embodiments, and all technical solutions belonging to the idea of the present invention belong to the protection scope of the present invention. It should be noted that modifications and embellishments within the scope of the invention may be made by those skilled in the art without departing from the principle of the invention.
Claims (9)
1. The preparation method of the pollen hydrogel hybrid drug-loaded microsphere is characterized by comprising the following steps:
step one, preparing pollen cavity microspheres with thorn-shaped structures;
step two, drug loading: soaking the pollen cavity microspheres prepared in the step one in a hydrogel solution containing a drug and a near-infrared light conversion material, filling hydrogel into cavities of the pollen cavity microspheres in a vacuum decompression mode, curing the hydrogel through ultraviolet irradiation and locking cytokines to obtain pollen hydrogel hybrid drug-loaded microspheres;
the drug is a cytokine;
the near infrared light conversion material is one or more of BP, BPNSs and BPQDs.
2. The pollen hydrogel hybrid drug-loaded microsphere of claim 1, which is characterized in that:
in the first step, the pollen cavity microspheres are sunflower pollen cavity microspheres.
3. The pollen hydrogel hybrid drug-loaded microsphere of claim 1,
in the first step, the preparation method of the pollen cavity microsphere comprises the following steps: degreasing pollen grains by using acetone, and then cracking the pollen grains by using KOH to prepare the pollen cavity microsphere with the thorn-shaped structure.
4. The pollen hydrogel hybrid drug-loaded microsphere of claim 1,
in the second step, the hydrogel solution is a water solution mixed by one or more than two materials of acrylamide, PEG8000 and acrylic acid.
5. The pollen hydrogel hybrid drug-loaded microsphere of claim 4, which is characterized in that,
the concentration of the acrylamide and the concentration of the acrylic acid are both 5-10 v/v%.
6. The pollen hydrogel hybrid drug-loaded microsphere of claim 1,
in the second step, a photoinitiator is mixed in the hydrogel solution, and the addition amount of the photoinitiator is 1% of the volume of the hydrogel solution.
7. The pollen hydrogel hybrid drug-loaded microsphere of claim 1,
in the second step, the cytokine is one or more of IL-2, IL-15 and IL-21, and the concentration of the cytokine is 100-1000 ng/ml.
8. The pollen hydrogel hybrid drug-loaded microsphere of claim 1,
in the second step, the concentration of the near infrared light conversion material is 100-500 ug/ml.
9. The use of the pollen hydrogel hybrid drug-loaded microspheres of any one of claims 1-8 for the responsive release of drugs under photothermal stimulation.
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