CN116139096A - Adsorption device based on DNA composite gel microsphere and preparation method and application thereof - Google Patents
Adsorption device based on DNA composite gel microsphere and preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to an adsorption device based on DNA composite gel microspheres, a preparation method and application thereof. The preparation method of the adsorption device comprises the following steps: preparing porous gel microspheres by a microfluidic emulsification technology; adding the obtained porous gel microsphere into a DNA solution to obtain a DNA composite gel microsphere with the function of adsorbing the chemotherapeutic drugs; and (3) placing the obtained DNA composite gel microsphere into a PDMS chip, and assembling to obtain the adsorption device based on the DNA composite gel microsphere. The adsorption device is used for adsorbing chemotherapeutic drugs. Compared with the prior art, the adsorption device based on the DNA composite gel microsphere has the function of efficiently adsorbing various chemotherapeutic drugs, and has the advantages of low cost, convenience in operation and the like.
Description
Technical Field
The invention relates to the field of biomedical engineering, in particular to an adsorption device based on DNA composite gel microspheres, and a preparation method and application thereof.
Background
Chemotherapeutic agents have been widely used in the field of tumor therapy as one of the most effective treatments. However, in order to achieve the desired therapeutic effect, the chemotherapy drugs currently used in clinic are often excessive, which results in excessive blood concentrations of the chemotherapy drugs. At the same time, normal cells also ingest chemotherapeutic drugs during treatment, causing serious systemic toxicity and reducing patient survival. In order to maximize therapeutic effects while avoiding serious side effects on healthy cells and tissues, materials for adsorbing and removing excessive amounts of chemotherapeutic drugs in the blood have been developed. However, these materials are generally bulk, with relatively few effective adsorption sites, and thus, satisfactory adsorption results are difficult to achieve.
The microsphere has wide application in the fields of biomedicine, cosmetics and food safety detection. In particular, the microsphere has extremely high specific surface area, and has more advantages in the aspect of adsorption of small molecules compared with the traditional bulk material. As such, microspheres have gained attention in the fields of water treatment, metabolite adsorption, and the like. The invention patent CN102532390B discloses triazine herbicide and metabolite molecular engram polymer microsphere thereof, a preparation method and application thereof, relates to molecular engram polymer microsphere, and solves the problem that the existing molecular engram polymer can only specifically adsorb one substance, and has the advantages of strong product specificity, high adsorption quantity, wide application range, simple method and low cost. However, there is currently no research on microsphere-based drug adsorption, particularly chemotherapy drug adsorption.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide an adsorption device based on DNA composite gel microspheres, a preparation method and application thereof, wherein the adsorption device based on DNA composite gel microspheres with good biocompatibility is designed by taking the DNA composite gel microspheres as a main body and used for adsorbing excessive drugs in the chemotherapy process, so that more adsorption sites can be provided, and a satisfactory adsorption effect is achieved.
The aim of the invention can be achieved by the following technical scheme:
the first object of the present invention is to provide a method for preparing an adsorption device based on DNA composite gel microspheres, comprising the steps of:
s1, preparing porous gel microspheres with positive charges on the surfaces through a microfluidic emulsification technology;
s2, cleaning the porous gel microspheres obtained in the step S1, adding the porous gel microspheres into a DNA solution, and obtaining the DNA composite gel microspheres with the function of adsorbing the chemotherapeutic drugs after the microspheres completely adsorb the DNA;
s3, placing the DNA composite gel microsphere obtained in the step S2 into a PDMS chip, and assembling to obtain the adsorption device based on the DNA composite gel microsphere.
Further, the preparation steps of the porous gel microsphere in the step S1 are specifically as follows: and (3) regulating the flow rates of internal phase prepolymer and external phase castor oil in the microfluidic chip to obtain prepolymer droplets, pre-cooling the obtained prepolymer droplets at-20 ℃, immersing the prepolymer droplets in liquid nitrogen, and carrying out ultraviolet curing to obtain the porous gel microspheres with positive charges on the surfaces.
Further, the prepolymer droplets include positively charged polymers, graphene Oxide (GO), N-isopropylacrylamide (NIPAM), N' -methylenebisacrylamide (Bis), methacrylated hydrogels (GelMA), and 2-hydroxy-2-methyl-1-phenyl-1-propanone (HMPP).
Further preferably, the positively charged polymer is selected from one or more of Polyallylamine (PAH), chitosan.
Further preferably, the concentration of the positively charged polymer is 0.1-10mg/mL, and the adsorption capacity of the porous gel microspheres to DNA can be adjusted by changing the concentration of the positively charged polymer.
Further preferably, the concentration of GO is 1-5 mg/mL; the concentration of NIPAM is 5-30 mg/mL; the concentration of the Bis is 0.2-0.5 mg/mL; the concentration of the GelMA is 2-3 mg/mL; the concentration of the HMPP is 0.5-1.5 v%.
Further, the diameter of the porous gel microsphere in the step S1 is 100-300 μm, and the diameter of the porous gel microsphere can be adjusted by adjusting the flow rate of the inner phase and the outer phase in the microfluidic chip.
Further, the pore size of the porous gel microsphere surface can be adjusted by pre-cooling for a period of time at-20 ℃.
Further, the DNA solution in step S2 is a pre-configured DNA solution, and the configuration steps are as follows: dissolving DNA powder in ultrapure water, and vortex oscillating until the DNA powder is completely mixed to obtain DNA solution.
Further preferably, the DNA powder is salmon sperm DNA powder.
Further, the DNA composite gel microsphere is assembled by the positive charges on the surface of the porous gel microsphere and the electrostatic action between DNA molecules.
Further preferably, in step S3, the PDMS chip is a chip formed by bonding a fish bone chip and a cylindrical chip.
Further preferably, the geometric parameters of the PDMS chip may be sized with the DNA composite gel microspheres to accommodate the DNA composite gel microspheres.
Further, in the step S3, the assembling method is that the DNA composite gel microsphere is tiled inside the cylindrical chip, the cylindrical chip and the fishbone chip are properly pressed after being attached, and chip bonding is completed, so as to obtain a PDMS chip, and meanwhile, the PDMS chip and the DNA composite gel microsphere are assembled to obtain the adsorption device based on the DNA composite gel microsphere.
The second object of the invention is to provide an adsorption device based on DNA composite gel microspheres, which is characterized in that the adsorption device is prepared by the preparation method of the adsorption device based on DNA composite gel microspheres.
Further, the adsorption device comprises a PDMS chip and DNA composite gel microspheres with the function of adsorbing chemotherapeutic drugs
The third object of the invention is to provide an application of an adsorption device based on DNA composite gel microspheres, wherein the adsorption device based on DNA composite gel microspheres prepared by the preparation method is used for adsorbing chemotherapeutic drugs.
Further, the chemotherapeutic drug is selected from the group consisting of doxorubicin hydrochloride, epirubicin hydrochloride, and cisplatin.
Further, the modes of adsorbing the chemotherapeutic drug include in vitro adsorption and hemodialysis.
Compared with the prior art, the invention has the following beneficial effects:
1) The invention constructs DNA composite gel microspheres capable of adsorbing and filtering chemotherapeutic drugs by taking the responsive gel as a carrier; in addition, the contact time of the fluid and the microspheres can be prolonged by loading the DNA composite gel microspheres into the fishbone adsorption device, the adsorption effect of the DNA composite gel microspheres on the chemotherapeutic drugs is improved, the preparation method is simple, the operation is easy, and the control of the chemotherapeutic drug adsorption process can be realized.
2) The DNA composite gel microsphere takes GelMA as a main body, has the characteristics of good biocompatibility and wide application range, realizes the adsorption of chemotherapeutic drugs through the specific action between DNA and the chemotherapeutic drugs, and has high removal efficiency on the chemotherapeutic drugs.
3) The invention realizes the porous treatment of the DNA composite gel microsphere structure by crosslinking in liquid nitrogen, and the porous structure increases the surface area of the DNA composite gel microsphere and enhances the adsorption effect of the DNA composite gel microsphere on various chemotherapeutics.
4) The DNA composite gel microsphere prepared by the invention can realize periodic diastole and systole under the stimulation of external near infrared laser (NIR), and can promote the adsorption of chemotherapeutic drugs.
5) The adsorption device based on the DNA composite gel microsphere is manufactured by combining a microfluidic technology and a 3D printing manufacturing technology, can adsorb chemotherapeutic drugs, can realize high-flux production, and has strong practicability.
Drawings
Fig. 1 is a schematic diagram of a DNA composite gel microsphere manufacturing process and an adsorption process according to the present technical scheme.
FIG. 2 is a photomicrograph of the DNA composite gel microsphere provided in example 1.
FIG. 3 is an engineering drawing of a fish bone chip according to the present embodiment
Fig. 4 is an engineering drawing of a cylindrical chip provided by the technical scheme.
Fig. 5 is a microstructure of the fish bone chip according to the present embodiment.
Fig. 6 is a microstructure of the cylindrical chip according to the present embodiment.
Detailed Description
The invention will now be described in detail with reference to the drawings and specific examples. The following examples will assist those skilled in the art in further understanding the present invention, but are not intended to limit the invention in any way. It should be noted that variations and modifications could be made by those skilled in the art without departing from the inventive concept. These are all within the scope of the present invention.
In the technical scheme, the characteristics of preparation means, materials, structures or composition ratios and the like which are not explicitly described are regarded as common technical characteristics disclosed in the prior art.
The DNA solution used in examples 1 to 3 was a salmon sperm DNA solution prepared in advance, and the preparation steps were as follows: 10mg of salmon sperm DNA powder (RHIWN, CAS: 438545-06-3) was dissolved in 10mL of ultrapure water, and vortexed until completely mixed to obtain a salmon sperm DNA solution having a concentration of 1 mg/mL.
As shown in fig. 1, the present technical solution provides a method for preparing an adsorption device based on DNA composite gel microspheres, the method for preparing the adsorption device comprising the following steps:
s1, preparing porous gel microspheres with positive charges on the surfaces through a microfluidic emulsification technology;
s2, cleaning the porous gel microspheres obtained in the step S1, adding the porous gel microspheres into a DNA solution, and obtaining the DNA composite gel microspheres with the function of adsorbing the chemotherapeutic drugs after the microspheres completely adsorb the DNA;
s3, placing the DNA composite gel microsphere obtained in the step S2 into a PDMS chip, and assembling to obtain the adsorption device based on the DNA composite gel microsphere.
Example 1
The embodiment provides an adsorption device based on DNA composite gel microspheres, and the preparation method of the adsorption device comprises the following steps:
s1, preparing a mixed solution of 1mg/mL PAH, 2mg/mL GO, 10mg/mL NIPAM, 0.34mg/mL Bis and 2.5mg/mL GelMA and 1v% HMPP, introducing the obtained mixed solution into an inner tube of a capillary microfluidic chip at a flow rate of 50 mu L/h, introducing castor oil into an outer tube of the capillary microfluidic chip at a flow rate of 1.5mL/h, preparing uniform prepolymer liquid drops, collecting the obtained prepolymer liquid drops in a plastic dish, placing the plastic dish in a refrigerator for precooling for 30min, continuously adding liquid nitrogen into the dish after precooling until the liquid drops are completely solidified, finally placing the plastic dish under an ultraviolet lamp for solidification, and continuously adding the liquid nitrogen during the solidification to ensure that the microspheres keep solidifying in the solidification process, thereby obtaining porous gel microspheres with the diameters of 200 mu m;
s2, adding the porous gel microspheres obtained in the step S1 into a centrifuge tube, washing with absolute ethyl alcohol, repeating for three times, adding 1mg/mL salmon sperm DNA solution which is uniformly mixed by vibration into the centrifuge tube, standing until the porous gel microspheres adsorb DNA molecules, and removing the redundant DNA solution to obtain the DNA composite gel microspheres with the function of adsorbing chemotherapeutic drugs, wherein the figure 2 shows;
s3, mixing PDMS prepolymer A (also called PDMS precursor) and curing agent B (ethyl orthosilicate) according to the mass ratio of 10:1, fully stirring, centrifuging, vacuumizing to remove redundant bubbles, adding into a fishbone chip and a cylindrical chip die manufactured by 3D printing, vacuumizing to remove bubbles, heating and curing at 70 ℃ for 60min, demolding after curing to respectively obtain the fishbone chip and the cylindrical chip (the geometric parameters of the fishbone chip and the cylindrical chip are respectively shown in figures 3 and 4, and the microstructure is respectively shown in figures 5 and 6), placing the bonding surfaces of the two chips upwards, placing the bonding surfaces into a plasma surface treatment instrument for treatment for 3min, then laying the DNA composite gel microsphere obtained in the step S2 inside the cylindrical chip, moderately pressing after the two chips are bonded, and finishing chip bonding to obtain the PDMS chip, and assembling the PDMS chip and the DNA composite gel microsphere to obtain the adsorption device based on the DNA composite gel microsphere.
Inserting a hose into an inlet and an outlet of the adsorption device based on the DNA composite gel microsphere, injecting 50 mug/mL of doxorubicin hydrochloride solution into the adsorption device at a flow rate of 167 mug/min, collecting at an outlet by using a centrifuge tube, circularly irradiating the microsphere inside a chip by using near infrared laser (NIR irradiation for 1min and then for 5min without NIR irradiation at the beginning of injection, and measuring the content of doxorubicin hydrochloride in the solution after adsorption by using an ultraviolet spectrophotometer after repeating for 5 times, wherein the content of doxorubicin hydrochloride is reduced by 26% compared with the initial concentration, which indicates that the adsorption device based on the DNA composite gel microsphere successfully adsorbs the chemotherapeutic drug doxorubicin hydrochloride, thereby realizing the filtering function.
Example 2
The embodiment provides an adsorption device based on DNA composite gel microspheres, and the preparation method of the adsorption device comprises the following steps:
s1, preparing a mixed solution of 1.5mg/mL PAH, 2.5mg/mL GO, 15mg/mL NIPAM, 0.3mg/mL Bis, 3mg/mL GelMA and 1v% HMPP, introducing the obtained mixed solution into an inner tube of a capillary microfluidic chip at a flow rate of 50 mu L/h, introducing castor oil into an outer tube of the capillary microfluidic chip at a flow rate of 1.5mL/h, preparing uniform prepolymer droplets, collecting the obtained prepolymer droplets in a plastic dish, pre-cooling in a refrigerator at-20 ℃ for 30min, continuously adding liquid nitrogen into the culture dish after the pre-cooling is finished until the droplets are completely solidified, finally solidifying under an ultraviolet lamp, continuously adding the liquid nitrogen during the solidification process to ensure that the microspheres are kept solidified in the solidification process, and obtaining porous gel microspheres with the diameter of 200 mu m;
s2, adding the porous gel microspheres obtained in the step S1 into a centrifuge tube, then washing with absolute ethyl alcohol, repeating for three times, then adding 1mg/mL salmon sperm DNA solution which is uniformly mixed by vibration into the centrifuge tube, standing until the porous gel microspheres adsorb DNA molecules, and removing redundant DNA solution to obtain the DNA composite gel microspheres with the function of adsorbing chemotherapeutic drugs;
s3, mixing PDMS prepolymer A (also called PDMS precursor) and curing agent B (ethyl orthosilicate) according to the mass ratio of 10:1, fully stirring, centrifuging, vacuumizing to remove redundant bubbles, adding into a fishbone chip and a cylindrical chip die manufactured by 3D printing, vacuumizing to remove bubbles, heating and curing at 70 ℃ for 60min, demolding after curing to respectively obtain the fishbone chip and the cylindrical chip (the geometric parameters of the fishbone chip and the cylindrical chip are respectively shown in figures 3 and 4, and the microstructure is respectively shown in figures 5 and 6), placing the bonding surfaces of the two chips upwards, placing the bonding surfaces into a plasma surface treatment instrument for treatment for 3min, then laying the DNA composite gel microsphere obtained in the step S2 inside the cylindrical chip, moderately pressing after the two chips are bonded, and finishing chip bonding to obtain the PDMS chip, and assembling the PDMS chip and the DNA composite gel microsphere to obtain the adsorption device based on the DNA composite gel microsphere.
Inserting a hose into an inlet and an outlet of the adsorption device based on the DNA composite gel microspheres, injecting 50 mu g/mL epirubicin hydrochloride solution into the adsorption device at a flow rate of 167 mu L/min, collecting at an outlet by using a centrifuge tube, circularly irradiating the microspheres in a chip by using near infrared laser (NIR) at the beginning of injection (1 min of irradiation by NIR and no NIR irradiation for 5min later), measuring the content of epirubicin hydrochloride in the solution after adsorption by using an ultraviolet spectrophotometer after repeating for 5 times, and finding that the content of epirubicin hydrochloride is reduced by 32% compared with the initial concentration, thereby indicating that the adsorption device based on the DNA composite gel microspheres successfully adsorbs chemotherapeutic medicine epirubicin hydrochloride and realizing the filtering function.
Example 3
The embodiment provides an adsorption device based on DNA composite gel microspheres, and the preparation method of the adsorption device comprises the following steps:
s1, preparing a mixed solution of 0.75mg/mL PAH, 2mg/mL GO, 15mg/mL NIPAM, 0.3mg/mL Bis and 2.5mg/mL GelMA and 1v% HMPP, introducing the obtained mixed solution into an inner tube of a capillary microfluidic chip at a flow rate of 50 mu L/h, introducing castor oil into an outer tube of the capillary microfluidic chip at a flow rate of 1.5mL/h, preparing uniform prepolymer droplets, collecting the obtained prepolymer droplets in a plastic dish, pre-cooling in a refrigerator at-20 ℃ for 30min, continuously adding liquid nitrogen into the culture dish after the pre-cooling is finished until the droplets are completely solidified, finally solidifying under an ultraviolet lamp, continuously adding the liquid nitrogen during the solidification process to ensure that the microspheres are kept solidified in the solidification process, and obtaining porous gel microspheres with the diameter of 200 mu m;
s2, adding the porous gel microspheres obtained in the step S1 into a centrifuge tube, then washing with absolute ethyl alcohol, repeating for three times, then adding 1mg/mL salmon sperm DNA solution which is uniformly mixed by vibration into the centrifuge tube, standing until the porous gel microspheres adsorb DNA molecules, and removing redundant DNA solution to obtain the DNA composite gel microspheres with the function of adsorbing chemotherapeutic drugs;
s3, mixing PDMS prepolymer A (also called PDMS precursor) and curing agent B (ethyl orthosilicate) according to the mass ratio of 10:1, fully stirring, centrifuging, vacuumizing to remove redundant bubbles, adding into a fishbone chip and a cylindrical chip die manufactured by 3D printing, vacuumizing to remove bubbles, heating and curing at 70 ℃ for 60min, demolding after curing to respectively obtain the fishbone chip and the cylindrical chip (the geometric parameters of the fishbone chip and the cylindrical chip are respectively shown in figures 3 and 4, and the microstructure is respectively shown in figures 5 and 6), placing the bonding surfaces of the two chips upwards, placing the bonding surfaces into a plasma surface treatment instrument for treatment for 3min, then laying the DNA composite gel microsphere obtained in the step S2 inside the cylindrical chip, moderately pressing after the two chips are bonded, and finishing chip bonding to obtain the PDMS chip, and assembling the PDMS chip and the DNA composite gel microsphere to obtain the adsorption device based on the DNA composite gel microsphere.
Inserting a hose into an inlet and an outlet of the adsorption device based on the DNA composite gel microsphere, injecting 50 mug/mL of cisplatin solution into the adsorption device at a flow rate of 167 mug/min, collecting at an outlet by using a centrifuge tube, circularly irradiating the microsphere inside a chip by using near infrared laser (NIR) at the beginning of injection (1 min of NIR irradiation and 5min of NIR irradiation are not performed), repeating for 5 times, measuring the content of cisplatin in the adsorbed solution by using ICP-MS, and finding that the content of cisplatin is reduced by 33% compared with the initial concentration, thereby indicating that the adsorption device based on the DNA composite gel microsphere successfully adsorbs chemotherapeutic cisplatin and realizing the filtering function.
The previous description of the embodiments is provided to facilitate a person of ordinary skill in the art in order to make and use the present invention. It will be apparent to those skilled in the art that various modifications can be readily made to these embodiments and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above-described embodiments, and those skilled in the art, based on the present disclosure, should make improvements and modifications without departing from the scope of the present invention.
Claims (10)
1. The preparation method of the adsorption device based on the DNA composite gel microsphere is characterized by comprising the following steps of:
s1, preparing porous gel microspheres with positive charges on the surfaces through a microfluidic emulsification technology;
s2, cleaning the porous gel microspheres obtained in the step S1, adding the porous gel microspheres into a DNA solution, and obtaining the DNA composite gel microspheres with the function of adsorbing the chemotherapeutic drugs after the microspheres completely adsorb the DNA;
s3, placing the DNA composite gel microsphere obtained in the step S2 into a PDMS chip, and assembling to obtain the adsorption device based on the DNA composite gel microsphere.
2. The method for preparing an adsorption device based on a DNA composite gel microsphere according to claim 1, wherein the steps for preparing the porous gel microsphere in step S1 are specifically as follows: and (3) regulating the flow rates of internal phase prepolymer and external phase castor oil in the microfluidic chip to obtain prepolymer droplets, pre-cooling the obtained prepolymer droplets at-20 ℃, immersing the prepolymer droplets in liquid nitrogen, and carrying out ultraviolet curing to obtain the porous gel microspheres with positive charges on the surfaces.
3. The method for preparing an adsorption device based on a DNA composite gel microsphere according to claim 2, wherein the prepolymer droplets comprise positively charged polymers, graphene oxide, N-isopropylacrylamide, N' -methylenebisacrylamide, methacrylated hydrogels and 2-hydroxy-2-methyl-1-phenyl-1-propanone.
4. A method for preparing an adsorption device based on DNA composite gel microspheres according to claim 3, wherein the positively charged polymer is selected from one or more of polyallylamine and chitosan.
5. The method for preparing an adsorption device based on DNA composite gel microspheres according to claim 3, wherein the concentration of the positively charged polymer is 0.1-10mg/mL.
6. The method for preparing an adsorption device based on DNA composite gel microspheres according to claim 1, wherein the porous gel microspheres in step S1 have a diameter of 100-300 μm.
7. The method for preparing an adsorption device based on DNA composite gel microspheres according to claim 1, wherein in the step S3, the PDMS chip is a chip formed by bonding a fishbone chip and a cylindrical chip.
8. An adsorption device based on DNA composite gel microspheres prepared by the preparation method according to any one of claims 1 to 7, wherein the adsorption device comprises a PDMS chip and DNA composite gel microspheres having a function of adsorbing chemotherapeutic drugs.
9. Use of an adsorption device based on DNA composite gel microspheres prepared by the preparation method according to any one of claims 1-7 for adsorbing chemotherapeutic drugs.
10. The use of an adsorption device based on DNA composite gel microspheres according to claim 9, wherein the chemotherapeutic agent is selected from the group consisting of doxorubicin hydrochloride, epirubicin hydrochloride and cisplatin.
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