Adsorption device based on DNA composite gel microspheres, preparation method and application thereof

Abstract

The present invention relates to an adsorption device based on DNA composite gel microspheres, a preparation method thereof and an application. The adsorption device comprises a PDMS chip and DNA composite gel microspheres having the function of adsorbing chemotherapeutic drugs. The preparation method of the adsorption device comprises the following steps: preparing porous gel microspheres through a microfluidic emulsification technique; adding the obtained porous gel microspheres into a DNA solution to obtain DNA composite gel microspheres having the function of adsorbing chemotherapeutic drugs; putting the obtained DNA composite gel microspheres into a PDMS chip to assemble an adsorption device based on DNA composite gel microspheres. The adsorption device is used for adsorbing chemotherapeutic drugs. Compared with the prior art, the adsorption device based on DNA composite gel microspheres provided by the present invention has the function of efficiently adsorbing various chemotherapeutic drugs, and has the advantages of low cost, convenient operation, etc.

Description

Technical Field

[0001] The present invention relates to the field of biomedical engineering, and particularly to an adsorption device based on DNA composite gel microspheres, a preparation method thereof, and an application thereof. Background Art

[0002] As one of the most effective treatment methods, chemotherapeutic drugs have been widely used in the field of tumor treatment. However, in order to achieve the desired therapeutic effect, the chemotherapeutic drugs currently used in clinical practice are usually in excess, which results in too high blood drug concentration of chemotherapeutic drugs. At the same time, normal cells will also take in chemotherapeutic drugs during the treatment process, causing serious systemic toxicity and reducing the survival rate of patients. In order to maximize the therapeutic effect and avoid serious side effects on healthy cells and tissues, some materials for adsorbing and removing excess chemotherapeutic drugs in the blood have been developed. However, these materials are usually in bulk, and their effective adsorption sites are relatively few, so it is difficult to achieve a satisfactory adsorption effect.

[0003] Microspheres have been widely used in the fields of biomedicine, cosmetics, and food safety detection. In particular, microspheres have an extremely high specific surface area and are more advantageous than traditional bulk materials in the adsorption of small molecules. Because of this, microspheres have attracted attention in the fields of water treatment, metabolite adsorption, etc. For example, the invention patent CN102532390B discloses triazine herbicides and their metabolite molecularly imprinted polymer microspheres, their preparation methods and applications, which relate to molecularly imprinted polymer microspheres, preparation methods and applications, and solve the problem that existing molecularly imprinted polymers can only specifically adsorb one substance. The product has strong specificity, high adsorption capacity, wide application range, simple method and low cost. It has high class-specific separation and enrichment characteristics for triazine herbicides, and the recovery rate is between 65% and 110%, which can meet the requirements of multi-residues. However, there is currently no research on drug adsorption based on microspheres, especially on the adsorption of chemotherapeutic drugs. Summary of the Invention

[0004] The purpose of the present invention is to overcome the above-mentioned defects existing in the prior art and provide an adsorption device based on DNA composite gel microspheres, a preparation method thereof, and an application thereof. By preparing a DNA composite gel microsphere with good biocompatibility and using it as the main body, an adsorption device for adsorbing chemotherapeutic drugs based on DNA composite gel microspheres is designed, which is used for adsorbing excess drugs during chemotherapy, can provide more adsorption sites, and achieve a satisfactory adsorption effect.

[0005] The purpose of the present invention can be achieved by the following technical solutions:

[0006] The first purpose of the present invention is to provide a preparation method of an adsorption device based on DNA composite gel microspheres. The preparation method of the adsorption device includes the following steps:

[0007] S1. Prepare porous gel microspheres with a positive charge on the surface through a microfluidic emulsification technique;

[0008] S2. After washing the porous gel microspheres obtained in step S1, add them to a DNA solution. After the microspheres completely adsorb the DNA, obtain DNA composite gel microspheres with the function of adsorbing chemotherapeutic drugs;

[0009] S3. Place the DNA composite gel microspheres obtained in step S2 into a PDMS chip to assemble an adsorption device based on the DNA composite gel microspheres.

[0010] Further, the preparation steps of the porous gel microspheres in step S1 are specifically as follows: Adjust the flow rates of the internal phase prepolymer and the external phase castor oil in the microfluidic chip to obtain prepolymer droplets. Place the obtained prepolymer droplets in a -20°C pre-cooling, and then immerse them in liquid nitrogen. After ultraviolet curing, obtain porous gel microspheres with a positive charge on the surface.

[0011] Further, the prepolymer droplets include a positively charged polymer, graphene oxide (GO), N-isopropylacrylamide (NIPAM), N,N'-methylenebisacrylamide (Bis), methacrylated hydrogel (GelMA), and 2-hydroxy-2-methyl-1-phenyl-1-propanone (HMPP).

[0012] Further preferably, the positively charged polymer is selected from one or a combination of polyallylamine (PAH) and chitosan.

[0013] Further preferably, the concentration of the positively charged polymer is 0.1 - 10 mg / mL, and the adsorption capacity of the porous gel microspheres for DNA can be adjusted by changing the concentration of the positively charged polymer.

[0014] Further preferably, the concentration of the GO is 1 - 5 mg / mL; the concentration of the 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%.

[0015] Further, the diameter of the porous gel microspheres in step S1 is 100 - 300 μm, and the diameter of the porous gel microspheres can be adjusted by adjusting the flow rates of the internal and external phases in the microfluidic chip.

[0016] Further, the pore size on the surface of the porous gel microspheres can be adjusted by the pre-cooling time at -20°C.

[0017] Further, the DNA solution described in step S2 is a pre-prepared DNA solution, and its preparation steps are as follows: Dissolve the DNA powder in ultrapure water and vortex until completely mixed to obtain the DNA solution.

[0018] Further preferably, the DNA powder is salmon sperm DNA powder.

[0019] Further, the DNA composite gel microspheres are assembled by the electrostatic interaction between the positive charges on the surface of the porous gel microspheres and DNA molecules.

[0020] Further preferably, the PDMS chip described in step S3 is a chip formed by bonding a fishbone chip and a cylindrical chip.

[0021] Further preferably, the geometric parameters of the PDMS chip can be designed according to the size of the DNA composite gel microspheres to accommodate the DNA composite gel microspheres.

[0022] Further, the assembly method in step S3 is to lay the DNA composite gel microspheres flat inside the cylindrical chip, press moderately after fitting the cylindrical chip and the fishbone chip to complete chip bonding to obtain the PDMS chip. At the same time, the PDMS chip and the DNA composite gel microspheres are assembled to obtain an adsorption device based on DNA composite gel microspheres.

[0023] The second object of the present invention is to provide an adsorption device based on DNA composite gel microspheres, characterized in that the adsorption device is prepared by the preparation method of the above-mentioned adsorption device based on DNA composite gel microspheres.

[0024] Further, the adsorption device includes a PDMS chip and DNA composite gel microspheres with the function of adsorbing chemotherapy drugs.

[0025] The third object of the present invention is to provide an application of an adsorption device based on DNA composite gel microspheres, using the adsorption device based on DNA composite gel microspheres prepared by the above preparation method to adsorb chemotherapy drugs.

[0026] Further, the chemotherapy drugs are selected from one or a combination of doxorubicin hydrochloride, epirubicin hydrochloride and cisplatin.

[0027] Further, the method of adsorbing chemotherapy drugs includes in vitro adsorption and hemodialysis.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] 1) The present invention constructs DNA composite gel microspheres capable of adsorbing and filtering chemotherapeutic drugs with responsive gels as carriers. In addition, by loading them into a fishbone adsorption device, the contact time between the fluid and the microspheres can be extended, the adsorption effect of the DNA composite gel microspheres on chemotherapeutic drugs can be improved, the preparation method is simple, the operation is easy, and the control of the adsorption process of chemotherapeutic drugs can be achieved.

[0030] 2) The DNA composite gel microspheres of the present invention have GelMA as the main body, have the characteristics of good biocompatibility and wide application range, and achieve the adsorption of chemotherapeutic drugs through the specific interaction between DNA and chemotherapeutic drugs, and have a high clearance efficiency for chemotherapeutic drugs.

[0031] 3) The present invention realizes the porous treatment of the structure of DNA composite gel microspheres by crosslinking in liquid nitrogen. This porous structure increases the surface area of the DNA composite gel microspheres and enhances their adsorption effect on various chemotherapeutic drugs.

[0032] 4) The DNA composite gel microspheres prepared by the present invention can achieve periodic relaxation and contraction under the stimulation of external near-infrared laser (NIR), which can promote the adsorption of chemotherapeutic drugs.

[0033] 5) The present invention manufactures an adsorption device based on DNA composite gel microspheres by combining microfluidic technology and 3D printing manufacturing technology, which can adsorb chemotherapeutic drugs and can achieve high-throughput production, and has strong practicability. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 Schematic diagrams of the manufacturing process and adsorption process of the DNA composite gel microspheres provided by this technical solution.

[0035] Figure 2 Light microscope image of the DNA composite gel microspheres provided in Example 1.

[0036] Figure 3 Engineering drawing of the fishbone chip provided by this technical solution

[0037] Figure 4 Engineering drawing of the cylindrical chip provided by this technical solution.

[0038] Figure 5 Microscopic structure diagram of the fishbone chip provided by this technical solution.

[0039] Figure 6 Microscopic structure diagram of the cylindrical chip provided by this technical solution. DETAILED DESCRIPTION OF THE INVENTION

[0040] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but do not limit the present invention in any form. It should be noted that those of ordinary skill in the art can make several modifications and improvements without departing from the concept of the present invention. These all belong to the protection scope of the present invention.

[0041] In this technical solution, features such as preparation means, materials, structures or composition ratios that are not clearly stated are regarded as common technical features disclosed in the prior art.

[0042] The DNA solution used in Examples 1 to 3 is a salmon sperm DNA solution, which is pre-prepared, and the preparation steps are as follows: Dissolve 10 mg of salmon sperm DNA powder (RHAWN, CAS: 438545-06-3) in 10 mL of ultrapure water, and vortex until completely mixed to obtain a salmon sperm DNA solution with a concentration of 1 mg / mL.

[0043] As Figure 1 shown, this technical solution provides a preparation method of an adsorption device based on DNA composite gel microspheres. The preparation method of the adsorption device includes the following steps:

[0044] S1. Prepare porous gel microspheres with a positive charge on the surface through microfluidic emulsification technology;

[0045] S2. After washing the porous gel microspheres obtained in step S1, add them to the DNA solution. After the microspheres are completely adsorbed with DNA, DNA composite gel microspheres with the function of adsorbing chemotherapeutic drugs are obtained;

[0046] S3. Put the DNA composite gel microspheres obtained in step S2 into a PDMS chip and assemble an adsorption device based on DNA composite gel microspheres.

[0047] Example 1

[0048] This example provides an adsorption device based on DNA composite gel microspheres. The preparation method of the adsorption device includes the following steps:

[0049] S1. Prepare a mixed solution containing 1 mg / mL PAH, 2 mg / mL GO, 10 mg / mL NIPAM, 0.34 mg / mL Bis, 2.5 mg / mL GelMA, and 1 v% HMPP. Introduce the obtained mixed solution into the inner tube of a capillary microfluidic chip at a flow rate of 50 μL / h, and introduce castor oil into the outer tube of the capillary microfluidic chip at a flow rate of 1.5 mL / h to prepare uniform prepolymer droplets. After collecting the obtained prepolymer droplets in a plastic dish, place them in a -20 °C refrigerator for precooling for 30 min. After the precooling is completed, continuously add liquid nitrogen to the culture dish until the droplets are completely solidified. Finally, place them under an ultraviolet lamp for curing, and continuously add liquid nitrogen during the process to ensure that the microspheres remain solid during the curing process, obtaining porous gel microspheres with a diameter of 200 μm.

[0050] S2. Add the porous gel microspheres obtained in step S1 into a centrifuge tube and wash them with absolute ethanol three times. Subsequently, add a salmon sperm DNA solution of 1 mg / mL that has been shaken and mixed evenly into the centrifuge tube. After standing until the porous gel microspheres have adsorbed DNA molecules completely, remove the excess DNA solution to obtain DNA composite gel microspheres with the function of adsorbing chemotherapeutic drugs, as Figure 2 shown.

[0051] S3. Mix PDMS prepolymer A (also known as PDMS precursor) and curing agent B (tetraethyl orthosilicate) at a mass ratio of 10:1 and stir well. After centrifugation and evacuation to remove excess bubbles, add them into a fishbone chip and a cylindrical chip mold fabricated by 3D printing, evacuate to remove bubbles, and cure at 70 °C for 60 min. After curing is completed, demold to obtain a fishbone chip and a cylindrical chip (the geometric parameters of the fishbone chip and the cylindrical chip are respectively as Figure 3 , 4 shown, and the microstructures are respectively as Figure 5 , 6 shown). Place the bonding surfaces of the two chips facing up, put them into a plasma surface treatment instrument for treatment for 3 min. Subsequently, spread the DNA composite gel microspheres obtained in step S2 inside the cylindrical chip, press the two chips together moderately to complete chip bonding, obtaining a PDMS chip. At the same time, assemble the PDMS chip and the DNA composite gel microspheres to obtain an adsorption device based on DNA composite gel microspheres.

[0052] Insert the hose into the inlet and outlet of the above-mentioned adsorption device based on DNA composite gel microspheres. Subsequently, inject a 50 μg / mL doxorubicin hydrochloride solution into the adsorption device at a flow rate of 167 μL / min, and collect it with a centrifuge tube at the outlet. At the beginning of the injection, irradiate the microspheres inside the chip with near-infrared laser (NIR) in a cycle (NIR irradiation for 1 min, followed by 5 min without NIR irradiation), and repeat 5 times. Then, use an ultraviolet spectrophotometer to measure the content of doxorubicin hydrochloride in the solution after adsorption, and it is found that the content of doxorubicin hydrochloride has decreased by 26% compared with the initial concentration, indicating that the adsorption device based on DNA composite gel microspheres has successfully adsorbed the chemotherapeutic drug doxorubicin hydrochloride and achieved the filtering function.

[0053] Example 2

[0054] This example provides an adsorption device based on DNA composite gel microspheres. The preparation method of the adsorption device includes the following steps:

[0055] S1. Prepare a mixed solution of 1.5 mg / mL PAH, 2.5 mg / mL GO, 15 mg / mL NIPAM, 0.3 mg / mL Bis, 3 mg / mL GelMA, and 1 v% HMPP. Pass the obtained mixed solution into the inner tube of the capillary microfluidic chip at a flow rate of 50 μL / h, and pass castor oil into the outer tube of the capillary microfluidic chip at a flow rate of 1.5 mL / h to prepare uniform prepolymer droplets. After collecting the obtained prepolymer droplets in a plastic dish, place them in a -20 °C refrigerator for precooling for 30 min. After the precooling is completed, continuously add liquid nitrogen to the culture dish until the droplets are completely solidified. Finally, place them under an ultraviolet lamp for curing, and continuously add liquid nitrogen during the process to ensure that the microspheres remain solid during the curing process, obtaining porous gel microspheres with a diameter of 200 μm.

[0056] S2. Add the porous gel microspheres obtained in step S1 to a centrifuge tube and wash them with absolute ethanol three times. Subsequently, add a salmon sperm DNA solution of 1 mg / mL that has been shaken and mixed evenly to the centrifuge tube. After standing until the porous gel microspheres have adsorbed DNA molecules completely, remove the excess DNA solution to obtain DNA composite gel microspheres with the function of adsorbing chemotherapeutic drugs.

[0057] S3. Mix PDMS prepolymer A (also known as PDMS precursor) and curing agent B (tetraethyl orthosilicate) in a mass ratio of 10:1 and stir well. After centrifugation and evacuation to remove excess bubbles, add them to the fishbone chip and cylindrical chip molds fabricated by 3D printing, evacuate to remove bubbles, and cure at 70 °C for 60 min. After curing is completed, demold to obtain a fishbone chip and a cylindrical chip respectively (the geometric parameters of the fishbone chip and the cylindrical chip are as shown in Figure 3 、 4 shown, and the microstructures are as shown in Figure 5 、6 As shown in the figure, place the bonding surfaces of the two chips facing upwards into a plasma surface treatment instrument for 3 minutes of treatment. Subsequently, spread the DNA composite gel microspheres obtained in step S2 evenly inside the cylindrical chip, press the two chips together moderately to complete chip bonding, obtaining a PDMS chip. At the same time, assemble the PDMS chip with the DNA composite gel microspheres to obtain an adsorption device based on DNA composite gel microspheres.

[0058] Insert a hose into the inlet and outlet of the above-mentioned adsorption device based on DNA composite gel microspheres. Subsequently, inject a 50 μg / mL epirubicin hydrochloride solution into the adsorption device at a flow rate of 167 μL / min, and collect it with a centrifuge tube at the outlet. At the beginning of the injection, irradiate the microspheres inside the chip with near-infrared laser (NIR) in a cycle (NIR irradiation for 1 minute, followed by 5 minutes without NIR irradiation). After repeating 5 times, use an ultraviolet spectrophotometer to measure the content of epirubicin hydrochloride in the solution after adsorption, and it is found that the content of epirubicin hydrochloride has decreased by 32% compared to the initial concentration, indicating that the adsorption device based on DNA composite gel microspheres has successfully adsorbed the chemotherapy drug epirubicin hydrochloride and achieved the filtration function.

[0059] Example 3

[0060] This example provides an adsorption device based on DNA composite gel microspheres. The preparation method of this adsorption device includes the following steps:

[0061] S1. Prepare a mixed solution of 0.75 mg / mL PAH, 2 mg / mL GO, 15 mg / mL NIPAM, 0.3 mg / mL Bis, 2.5 mg / mL GelMA, and 1 v% HMPP. Pass the obtained mixed solution into the inner tube of a capillary microfluidic chip at a flow rate of 50 μL / h, and pass castor oil into the outer tube of the capillary microfluidic chip at a flow rate of 1.5 mL / h to prepare uniform prepolymer droplets. After collecting the obtained prepolymer droplets in a plastic dish, place them in a -20°C refrigerator for 30 minutes of pre-cooling. After the pre-cooling ends, continuously add liquid nitrogen to the culture dish until the droplets are completely solidified. Finally, place them under an ultraviolet lamp for curing, and continuously add liquid nitrogen during the process to ensure that the microspheres remain solid during the curing process, obtaining porous gel microspheres. The diameter of the obtained porous gel microspheres is 200 μm.

[0062] S2. Add the porous gel microspheres obtained in step S1 into a centrifuge tube and wash them with absolute ethanol three times. Subsequently, add a 1 mg / mL salmon sperm DNA solution that has been shaken and mixed evenly to the centrifuge tube. After standing until the porous gel microspheres have adsorbed DNA molecules completely, remove the excess DNA solution to obtain DNA composite gel microspheres with the function of adsorbing chemotherapy drugs.

[0063] S3. Mix the PDMS prepolymer A (also known as the PDMS precursor) and the curing agent B (tetraethyl orthosilicate) in a mass ratio of 10:1 and stir well. After centrifugation and evacuation to remove excess bubbles, add them into the fishbone chip and cylindrical chip molds fabricated by 3D printing. Evacuate to remove bubbles again, heat and cure at 70 °C for 60 min. After curing is completed, demold to obtain the fishbone chip and the cylindrical chip respectively (the geometric parameters of the fishbone chip and the cylindrical chip are as shown in Figure 3 , 4 respectively, and the microstructures are as shown in Figure 5 , 6 respectively). Place the bonding surfaces of the two chips upward, put them into a plasma surface treatment instrument for 3 min. Subsequently, spread the DNA composite gel microspheres obtained in step S2 inside the cylindrical chip. Press moderately after fitting the two chips together to complete chip bonding, obtaining the PDMS chip. At the same time, assemble the PDMS chip and the DNA composite gel microspheres to obtain an adsorption device based on DNA composite gel microspheres.

[0064] Insert a hose into the inlet and outlet of the above-mentioned adsorption device based on DNA composite gel microspheres. Then inject a 50 μg / mL cisplatin solution into the adsorption device at a flow rate of 167 μL / min, and collect it with a centrifuge tube at the outlet. At the beginning of injection, irradiate the microspheres inside the chip with near-infrared laser (NIR) in a cycle (NIR irradiation for 1 min, and no NIR irradiation for the subsequent 5 min), and repeat 5 times. Then use ICP-MS to measure the content of cisplatin in the solution after adsorption, and it is found that the content of cisplatin has decreased by 33% compared with the initial concentration, indicating that the adsorption device based on DNA composite gel microspheres has successfully adsorbed the chemotherapeutic drug cisplatin and achieved the filtration function.

[0065] The above description of the embodiments is for the convenience of those of ordinary skill in the art to understand and use the invention. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative labor. Therefore, the present invention is not limited to the above embodiments, and all improvements and modifications made by those skilled in the art without departing from the scope of the present invention according to the disclosure of the present invention should be within the protection scope of the present invention.

Claims

1. A preparation method of an adsorption device based on DNA composite gel microspheres, characterized in that, The preparation method of the adsorption device comprises the following steps: S1. Prepare porous gel microspheres with a positive charge on the surface through microfluidic emulsification technology; S2. After washing the porous gel microspheres obtained in step S1, add them to a DNA solution. After the microspheres are completely adsorbed with DNA, DNA composite gel microspheres with the function of adsorbing chemotherapeutic drugs are obtained; S3. Place the DNA composite gel microspheres obtained in step S2 into a PDMS chip and assemble an adsorption device based on the DNA composite gel microspheres; The specific preparation steps of the porous gel microspheres in step S1 are as follows: Adjust the flow rates of the internal phase prepolymer and the external phase castor oil in the microfluidic chip to obtain prepolymer droplets. Place the obtained prepolymer droplets in a -20 °C pre-cooling, and then immerse them in liquid nitrogen. After ultraviolet curing, porous gel microspheres with a positive charge on the surface are obtained; The prepolymer droplets include a positively charged polymer, graphene oxide, N-isopropylacrylamide, N,N'-methylenebisacrylamide, methacrylated hydrogel, and 2-hydroxy-2-methyl-1-phenyl-1-propanone; The positively charged polymer is selected from one or a combination of polyallylamine, chitosan, etc.; The concentration of the positively charged polymer is 0.1 - 10 mg / mL, and the adsorption ability of the porous gel microspheres to DNA is adjusted by changing the concentration of the positively charged polymer; The prepared DNA composite gel microspheres achieve periodic relaxation and contraction under the stimulation of an external near-infrared laser to promote the adsorption of chemotherapeutic drugs.

2. The preparation method of an adsorption device based on DNA composite gel microspheres according to claim 1, wherein, The diameter of the porous gel microspheres in step S1 is 100 - 300 µm.

3. The preparation method of an adsorption device based on DNA composite gel microspheres according to claim 1, wherein, The PDMS chip in step S3 is a chip formed by bonding a fishbone chip and a cylindrical chip.

4. An adsorption device based on DNA composite gel microspheres prepared by the preparation method according to any one of claims 1-3, characterized in that, The adsorption device includes a PDMS chip and DNA composite gel microspheres with the function of adsorbing chemotherapeutic drugs.

5. Application of an adsorption device based on DNA composite gel microspheres prepared by the preparation method according to any one of claims 1 - 3 in the preparation of a device for adsorbing chemotherapeutic drugs.

6. The application of an adsorption device based on DNA composite gel microspheres according to claim 5, wherein, The chemotherapeutic drugs are selected from one or a combination of doxorubicin hydrochloride, epirubicin hydrochloride, and cisplatin.

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