Minimally swellable biocompatible membrane and preparation method thereof

Abstract

The present invention relates to a biocompatible membrane, specifically to a minimally swellable biocompatible membrane and the preparation method thereof. The preparation method of the minimally swellable biocompatible membrane comprises the following steps: synthesis of a copolymer containing a skeleton and a hydrophilic group, the introduction of a biocompatible property, the preparation of a biocompatible membrane solution, and the coating of the biocompatible membrane. The present invention can effectively regulate glucose, and has high biocompatibility (long service life) as well, thereby improving the sensitivity, accuracy, reproducibility, stability, specificity and anti-interference ability in a continuous glucose monitoring (CGM) system, prolonging the life time of the CGM, and greatly reducing the cost of the CGM.

Claims

1. A preparation method of a swellable biocompatible membrane, characterized in that the method comprises the following steps: A. synthesis of a copolymer containing a hydrophobic skeleton and hydrophilic moieties; A1. dissolving a hydrophobic monomer and a monomer containing a hydrophilic group in ethanol, and removing oxygen with nitrogen; A2. adding azodiisobutyronitrile, and placing the obtained solution in an airtight container for reaction; and A3. separating and purifying to obtain a precipitate, which is the copolymer containing the hydrophobic skeleton and hydrophilic moieties; B. introduction of biocompatibility and preparation of a biocompatible membrane solution; B1. dissolving the copolymer containing the hydrophobic skeleton and hydrophilic moieties in ethanol; and B2. adding a biocompatible substance and a crosslinking agent, mixing thoroughly, and then reacting in a water bath to obtain the biocompatible membrane solution; and C. coating of the biocompatible membrane; coating the biocompatible membrane solution on a biosensor directly or after dilution of the biocompatible membrane solution, and drying at room temperature to form a membrane, thus obtaining the swellable biocompatible membrane, wherein the separating and purifying operation in A3 is specifically as follows: adding water to precipitate the copolymer containing the hydrophobic membrane skeleton and hydrophilic moieties, centrifuging, removing a supernatant, adding ethanol to dissolve the precipitate, and repeating for 2-6 times.

2. The preparation method of a swellable biocompatible membrane according to claim 1, characterized in that: the hydrophobic monomer is styrene, vinylpyridine, acrylate, or acrylamide and the derivatives thereof.

3. The preparation method of a swellable biocompatible membrane according to claim 1, characterized in that: the monomer containing a hydrophilic group is vinylpyrrolidone, vinylized polyethylene glycol, an acrylate with an ethylene glycol group, or an olefin with an ethylene glycol group.

4. The preparation method of a swellable biocompatible membrane according to claim 1, characterized in that: the biocompatible substance is a polymer with hydrophilicity and biocompatibility or an aminated monomer with high biocompatibility.

5. The preparation method of a swellable biocompatible membrane according to claim 4, characterized in that: the high-molecular-weight polymer with high hydrophilicity and biocompatibility is polyethylene oxide, a copolymer containing polyethylene oxide, polypropylene oxide, a copolymer containing polypropylene oxide, polyvinyl alcohol, polylactic acid, hyaluronic acid and the derivatives thereof, chitosan and the derivatives thereof, cellulose and the derivatives thereof, alginic acid and the derivatives thereof, or aminated polyethylene glycol; and the monomer with high biocompatibility is choline, betaine, amino acid, ethylene oxide, or propylene oxide.

6. The preparation method of a swellable biocompatible membrane according to claim 1, characterized in that: the crosslinking agent is triglycidyl p-aminophenol, glycidyl ether and the derivatives thereof, polypropylene glycol glycidyl ether and the derivatives thereof, polyethylene glycol diglycidyl ether and the derivatives thereof, or glutaraldehyde.

7. The preparation method of a swellable biocompatible membrane according to claim 1, characterized in that: the biocompatible membrane solution is dissolved in methanol, ethanol, propanol, isopropanol, water, N,N-dimethylacrylamide, dimethyl sulfoxide, sulfolane, tetrahydrofuran, or dioxane for storage.

8. A swellable biocompatible membrane prepared by the method according to claim 1.

9. A glucose biosensor, characterized in that: it includes the swellable biocompatible membrane according to claim 8.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows the change in volume of the biocompatible membrane before and after being soaked.

(2) FIG. 2 schematically shows the structure of the glucose biosensor in Example 1.

(3) FIG. 3 shows the relationship between the current and the number of coatings of the glucose biosensor in Example 1 in a PBS buffer solution containing 10 mmol/L glucose.

(4) FIG. 4 shows the change in stability of the glucose biosensor coated with a three-layer biocompatible membrane in Example 1 in a PBS buffer solution containing 20 mmol/L glucose.

(5) FIG. 5 shows the relationship between the current and glucose concentration of the glucose biosensor coated with a four-layer biocompatible membrane in Example 1 (the change gradient of the glucose concentration: 5 mmol/L).

(6) FIG. 6 shows an in vivo test of the glucose biosensor coated with the three-layer biocompatible membrane in Example 1 in a CGM.

(7) Description of reference numerals: 1. Biocompatible membrane; 2. carbon counter electrode; 3. polyethylene terephthalate substrate; 4. carbon working electrode; 5. glucose sensing membrane; and 6. silver/silver chloride reference electrode.

DETAILED DESCRIPTION OF THE EMBODIMENTS

(8) The present invention will be further described below in conjunction with the accompanying drawings and specific examples, so that those skilled in the art can better understand and implement the present invention, while the examples are not intended to limit the present invention.

Example 1

(9) The preparation method of the minimally swellable biocompatible membrane comprised the following steps: A. Synthesis of a copolymer containing the hydrophobic skeleton and hydrophilic moieties. A1. mixing 10-100 mL styrene, 5-50 mL vinylpyrrolidone and 20-200 mL absolute ethanol, and removing oxygen with nitrogen for 20-60 min; A2. adding 10-200 mg azodiisobutyronitrile, placing the obtained solution in an airtight container, and reacting at 40-70 C. for 12-24 h; A3. adding 500-5000 mL water to precipitate the copolymer of styrene and vinylpyrrolidone, and centrifuging for separation; A4. adding ethanol to the precipitate obtained in step A3 to dissolve the precipitate, then adding 500-5000 mL water for precipitation, and centrifuging; and A5. repeating step A4 for 2-6 times, drying the obtained precipitate under vacuum at 60-120 C. for 12-40 h to obtain the polymer containing the hydrophobic skeleton and hydrophilic moieties; B. introduction of the biocompatible property and preparation of a biocompatible membrane solution B1. dissolving the copolymer containing the hydrophobic skeleton and hydrophilic moieties in ethanol to obtain a 100-300 mg/mL solution; and B2. adding 10-30 mg/mL aminated polyethylene glycol and 0.2-5 mg/mL triglycidyl p-aminophenol crosslinking agent, mixing thoroughly, then reacting in a water bath at 60 C. for 20-60 min to obtain the biocompatible membrane solution. After the crosslinking reaction, adding 5-20 mg/mL amino acid to stop the crossing linking reaction, so as to obtain a highly stable solution, and dissolving the biocompatible material in methanol, ethanol, propanol, isopropanol, water, N,N-dimethylacrylamide, dimethyl sulfoxide, sulfolane, tetrahydrofuran or dioxane for storage and indefinite use; and C. coating of a biocompatible membrane

(10) coating the biocompatible membrane solution evenly on the biosensor by a dip coating method or a spin coating method directly, or evenly on the biosensor by a spraying method after dilution of the biocompatible membrane solution, drying at room temperature to form a membrane, then repeating the coating, and drying for 1-10 times.

Examples 2-3

(11) On the basis of Example 1, the vinylpyrrolidone in step A1 was replaced by vinylized polyethylene glycol, an acrylate with an ethylene glycol group, and an olefin with an ethylene glycol group, respectively.

Examples 4-12

(12) On the basis of Example 1, the styrene in step A1 was replaced by vinylpyridine, acrylate, acrylamide, methacrylamide, diacetone acrylamide, cinnamamide, N-isopropylacrylamide, N-phenylacrylamide, and N,N-dimethylacrylamide, respectively.

Examples 13-32

(13) On the basis of Example 1, the aminated polyethylene glycol in step B2 was replaced by polyethylene oxide, a copolymer containing polyethylene oxide, polypropylene oxide, a copolymer containing polypropylene oxide, polyvinyl alcohol, polylactic acid, hyaluronic acid and the derivatives thereof, chitosan and the derivatives thereof, cellulose and the derivatives thereof, alginic acid and the derivatives thereof, aminated choline, aminated betaine, aminated amino acid, aminated ethylene oxide, aminated propylene oxide, and aminated vinylpyrrolidone, respectively.

Examples 33-38

(14) On the basis of Example 1, the triglycidyl p-aminophenol in step B2 was replaced by glycidyl ether, polypropylene glycol glycidyl ether and the derivatives thereof, polyethylene glycol diglycidyl ether and the derivatives thereof, and glutaraldehyde, respectively.

Test Example 1

(15) Biocompatible membrane beads above 10 mm were prepared by the dip-pull method from the biocompatible membrane solution obtained in Example 1, and then they were soaked in a PBS buffer solution containing 10 mmol/L glucose for 7 days. The volumes of the biocompatible membrane beads before and after soaking are compared, with the results shown in FIG. 1. It can be seen that the biocompatible membrane beads, after being soaked in the PBS buffer solution containing 10 mmol/L glucose for 7 days, only have less than 10% of swelling, which fundamentally ensures that the biocompatible membrane will not be significantly affected in the mechanical properties by swelling after being implanted. Besides, because the crosslinking reaction is completely terminated by amino acids, their stability and pot life are greatly improved, making it possible to prepare CGMs with high consistency.

Application Example 1

(16) The biocompatible membrane solution obtained in Example 1 was evenly coated on the glucose biosensors as shown in FIG. 2 or 3 by the dip coating method, the glucose biosensors comprising a polyethylene terephthalate substrate 3, a carbon counter electrode 2 and a carbon working electrode 4 respectively covering the two sides of the polyethylene terephthalate substrate 3, a glucose sensing membrane 5 coated on the carbon working electrode 4, a silver/silver chloride reference electrode 6 disposed outside the carbon working electrode 4, and a biocompatible membrane 1 covering the carbon counter electrode 2, the silver/silver chloride reference electrode 6 and the carbon working electrode 4. These glucose biosensors were then dried to form a membrane in a strictly controlled environment. After the solvent evaporated completely, the surface of these glucose biosensors were completely coated with biocompatible membranes. In order to increase the thickness of the biocompatible membrane, the above process could be repeated at will, usually 3-4 times, to achieve the required thickness. Since this biocompatible membrane was formed through multiple membrane-forming processes, its final performance of regulating oxygen and glucose could reach the desired level very conveniently and effectively through optimization of the thickness of the membrane (by controlling the number of dip coatings) and the formulation of the biocompatible membrane solution.

(17) As shown in FIG. 3, when the glucose biosensor was completely coated with the biocompatible membrane, its catalytic oxidation current for glucose decreased exponentially with the increase of the thickness of the membrane (the increase of the number of dip coatings); after four cycles of dip coating and drying, the current of the glucose biosensor decreased to less than 1% of the original. This result shows that the biocompatible membrane of the present invention can regulate glucose very efficiently. Further, the glucose biosensor coated with the biocompatible membrane exhibited excellent stability during continuous testing for up to three weeks (as shown in FIG. 4).

(18) Precise regulation of oxygen and glucose with high stability has been successfully achieved by covering the glucose biosensors with a biocompatible membrane; however, in order to obtain a glucose biosensor with high accuracy, reproducibility and stability, it is necessary to ensure that these sensors have a wide enough linear response range, which can be achieved by optimizing the biocompatible membrane on the glucose biosensors. For example, compared with the glucose biosensor without being coated with any biocompatible membrane, the current of the glucose biosensor that had been subjected to four cycles of coating was well regulated by the biocompatible membrane; in addition, the monitorable range of glucose was successfully extended from 10 mmol/L to 50 mmol/L (as shown in FIG. 5), the glucose biosensor coated with the biocompatible membrane has the widest linear response range so far for in vivo use, fully satisfying the needs of diabetics for glucose monitoring.

(19) Although the biocompatible membrane of the present invention exhibits superior performance in in vitro tests as demonstrated by the above experimental results, its performance in in vivo monitoring is the most powerful proof of its biocompatibility. Therefore, on the basis of the in vitro work, the glucose biosensor coated with the biocompatible membrane was applied to an CGM, which showed no significant decrease in sensitivity (baseline) in a 21-day in vivo trial (as shown in FIG. 6), the longest life time of CGM ever manufactured; moreover, the glucose concentration results obtained by the CGM were highly consistent with those obtained by the finger blood glucose testing.

(20) To sum up, by being coated with the minimally swellable biocompatible membrane of the present invention, the glucose biosensor developed based on the third-generation biosensing technology can regulate glucose very effectively and accurately; more importantly, the existence of this biocompatible membrane significantly extends the monitorable range of glucose, greatly improves the stability and biocompatibility of the glucose biosensor in vivo, which fully meets the requirements of calibration-free (factory-calibrated) CGMs, laying a solid foundation for the mass production of the calibration-free CGMs. In addition, this biocompatible membrane can also be applied to other implantable continuous monitoring systems, for example, continuous monitoring of lactic acid and blood ketone.

(21) Obviously, the above examples are only for clear description, rather than limiting the embodiments. For those of ordinary skill in the art, other various alterations or modifications can also be made on the basis of the above description. It is unnecessary and impossible to enumerate all the embodiments here. However, the obvious alterations or modifications thus derived are still within the protection scope of the present invention.