阅读说明：本技术 一种非离体胰岛素控释药物的包载设备及包载方法 (Entrapment equipment and entrapment method for non-isolated insulin controlled-release drug ) 是由 夏栋林 冯灵子 李佳 陈超 顾海鹰 于 2021-09-02 设计创作，主要内容包括：本发明公开了一种非离体胰岛素控释药物的包载设备,该包载设备用于血液透析器,包括：红细胞载药段,引入动脉全血,进行胰岛素包载,送至药物开关组装段；药物开关组装段,对包载胰岛素后的血液进行葡萄糖酶修饰,送至血液透析器。本发明以非离体方式进行胰岛素药物包载给药,利用患者自身的红细胞作为药物载体,充分利用了红细胞药物载体的优势,有效延长药物作用时间,同时避免了输血感染问题的出现。(The invention discloses an entrapment device of non-isolated insulin controlled release medicine, which is used for a hemodialyzer and comprises: the red blood cell loading section introduces whole arterial blood, carries out insulin entrapment and sends the whole arterial blood to the drug switch assembly section; and a drug switch assembly section for carrying out glucolase modification on the blood after the insulin is encapsulated and sending the blood to a hemodialyzer. The invention carries out insulin drug loading and administration in a non-isolated mode, utilizes the erythrocytes of patients as drug carriers, fully utilizes the advantages of the erythrocyte drug carriers, effectively prolongs the drug action time, and simultaneously avoids the occurrence of transfusion infection.)

1. An entrapment device for controlled release of non-ex vivo insulin for use in a hemodialyzer, comprising:

the red blood cell loading section introduces whole arterial blood, carries out insulin entrapment and sends the whole arterial blood to the drug switch assembly section;

and a drug switch assembly section for carrying out glucolase modification on the blood after the insulin is encapsulated and sending the blood to a hemodialyzer.

2. The entrapment device of claim wherein the red blood cell drug-loaded section comprises a red blood cell drug-loaded reactor, a peristaltic pump, a hypotonic reactor, a hypertonic reactor, an isotonic reactor; the drug switch assembly section comprises a GOx reactor; a semi-permeable bag is arranged in the erythrocyte drug-loaded reactor, and two ends of the semi-permeable bag extend out of the erythrocyte drug-loaded reactor through a guide tube and are respectively communicated with the arterial blood taking end and the GOx reactor; an insulin injection port is arranged on the semi-permeable bag; an insulin entrapment circulation outlet, an insulin entrapment circulation inlet and a GOx reaction outlet are arranged on the erythrocyte drug loading reactor; the insulin entrapment circulation outlet is divided into three paths after passing through a peristaltic pump, and is communicated with the insulin entrapment circulation inlet after passing through a hypotonic reactor, a hypertonic reactor and an isotonic reactor respectively through a conduit; two ends of the GOx reactor are respectively communicated with the GOx reaction outlet and an arterial blood taking end of a hemodialyzer through a conduit; the semi-permeable bag is prepared by adopting a semi-permeable membrane; and catheter clamps are arranged on catheters at two ends of the hypotonic reactor, the hypertonic reactor and the isotonic reactor, catheters at two ends of the GOx reactor and catheters connecting the semi-permeable bag and the arterial blood taking end.

3. The entrapment device of claim 2, wherein the hypotonic reactor, the hypertonic reactor, and the isotonic reactor are provided with reaction liquid injection ports; and a reaction liquid injection port is arranged on the GOx reactor.

4. The entrapment device of claim 3, wherein the red blood cell drug-loaded reactor is in the form of a bag; the hypotonic reactor, the hypertonic reactor and the isotonic reactor are packaged; the GOx reactor adopts a medical plastic tube.

5. The entrapment device of claim 4, wherein the entrapment device further comprises a hypotonic temperature control device, a hypertonic temperature control device, and a GOx reaction temperature control device for controlling the temperature of the hypotonic reactor, the hypertonic reactor, and the GOx reactor, respectively.

6. The entrapment equipment of claim 5 wherein the hypotonic temperature control equipment and the GOx reaction temperature control equipment are thermostated containers, and the hypotonic reactor and the GOx reactor are both placed in the thermostated containers; the high-permeability temperature control equipment adopts a water bath, and the high-permeability reactor is arranged in the water bath.

7. Method for the non-ex vivo entrapment of controlled release insulin drugs using the entrapment device of any of the claims 2 to 6, wherein the entrapment method comprises the steps of:

(1) insulin entrapment: leading blood from an artery into a semi-permeable bag of the erythrocyte drug-loaded reactor, injecting an insulin solution into the semi-permeable bag, circularly leading the hypotonic solution into the erythrocyte drug-loaded reactor through the hypotonic reactor, and circularly reacting for 2-3 hours; then the hypertonic solution is circularly introduced into the erythrocyte drug-loaded reactor through the hypertonic reactor, and the circular reaction lasts for 0.5 to 1 hour; finally, introducing the isotonic solution into the erythrocyte drug-loaded reactor through an isotonic reactor in a circulating way, and carrying out circulating reaction for 5-10 min;

(2) assembling a medicine switch: after the isotonic fluid circulation reaction is finished, injecting the blood in the semi-permeable bag into a GOx reactor for biotin-glucolase modification;

(3) and injecting the modified drug-loaded red blood cells into a hemodialyzer, and removing biotin-glucolase which is not modified on the surfaces of the red blood cells.

8. The entrapment method of claim 7, wherein the reaction temperature of the hypotonic cycling reaction is 4 ℃; the reaction temperature of the hypertonic cyclic reaction is 37 ℃; when the artery is used for introducing blood, 2-10mL of whole blood is introduced, 100-150U/mL of insulin solution is injected into the semi-permeable bag, and the volume ratio of the insulin solution to the whole blood is 1: 10.

9. The entrapment method of claim 8, wherein when the artery is bled, 10mL of whole blood is introduced, and 1mL of 100U/mL or 150U/mL of insulin solution is injected into the semi-permeable bag.

10. According to the claimsThe entrapment method of claim 9, wherein the hypotonic solution is: 250mM NaCl, 12.5mM glucose, 12.5mM sodium pyruvate, 12.5mM inosine, 12.5mM NaH₂PO₄·2H₂O, 0.63mM adenine, 550mOsm/Kg, pH 8; the hypertonic solution is as follows: 250mM NaCl, 12.5mM glucose, 12.5mM sodium pyruvate, 12.5mM inosine, 12.5mM NaH₂PO₄·2H₂O, 0.63mM adenine, 550mOsm/Kg, pH 8; the isotonic solution adopts normal saline; injecting GOx reaction liquid into the GOx reactor when biotin-glucolase modification is carried out; the GOx reaction liquid is prepared by the following method: dissolving 10mg of biotin in 1mL of dimethylformamide, adding 5mL of 2 mg/mL-concentration glucoamylase solution and 10mg of N, N' -carbonyldiimidazole, stirring at room temperature for 1 hour, putting the solution into a dialysis bag, and dialyzing at room temperature for 24 hours; the reaction time of biotin-glucosidase modification in the step (3) is 0.2-0.5 h.

Technical Field

The invention relates to a drug loading technology which can adopt a non-isolated erythrocyte insulin administration mode.

Background

Diabetic nephropathy is one of the most common microvascular complications of diabetes. Hemodialysis can delay renal failure, help kidney to excrete waste poison produced by human metabolism, and maintain life. Hemodialysis is one of the kidney replacement treatment modes of patients with acute and chronic renal failure. The blood and electrolyte solution (dialysate) with similar concentration of organism are arranged inside and outside one hollow fiber by draining the blood in vivo to the outside of the body through a dialyzer consisting of a plurality of hollow fibers, and the material exchange is carried out by dispersion/convection, thereby removing the metabolic waste in the body and maintaining the balance of electrolyte and acid-base; and simultaneously, excessive water in the body is removed. Hemodialysis, however, only temporarily controls blood glucose. Therefore, the diabetic nephropathy patients need to be treated by dialysis regularly, and the blood sugar control also needs to be controlled by regular insulin injection.

The erythrocyte drug-loading system is a novel drug-loading mode, and has the advantages of prolonging the whole-body action time of the drug, reducing the adverse reaction of the drug and the like. However, the existing erythrocyte drug-carrying system adopts an in vitro mode, so that the erythrocyte source is tense, and the problems of transfusion infection and the like can occur after the drug is taken.

Disclosure of Invention

The invention aims to solve the defects in the prior art and provide a novel medicine loading mode which can effectively control the release of medicines and avoid the problems of blood transfusion infection and the like.

In order to achieve the above object, the present invention provides an entrapment device for a non-ex vivo insulin controlled release drug, the entrapment device being used in a hemodialyzer, comprising:

the red blood cell loading section introduces whole arterial blood, carries out insulin entrapment and sends the whole arterial blood to the drug switch assembly section;

and a drug switch assembly section for carrying out glucolase modification on the blood after the insulin is encapsulated and sending the blood to a hemodialyzer.

The non-in-vitro medicine carrying device is used for carrying out insulin administration operation while the blood of a patient is permeated, the advantages of a red blood cell medicine carrying system are fully utilized, meanwhile, the red blood cells of the patient are used for carrying medicine, the condition of transfusion infection is avoided, and the controlled release effect of blood sugar is achieved by carrying out glucolase modification on the surfaces of the red blood cells.

Furthermore, the red blood cell drug-loading section comprises a red blood cell drug-loading reactor, a peristaltic pump, a hypotonic reactor, a hypertonic reactor and an isotonic reactor; the drug switch assembly section comprises a GOx reactor; a semi-permeable bag is arranged in the erythrocyte drug-loaded reactor, and two ends of the semi-permeable bag extend out of the erythrocyte drug-loaded reactor through a guide tube and are respectively communicated with the arterial blood taking end and the GOx reactor; the semipermeable bag is provided with an insulin injection port; an insulin entrapment circulation outlet, an insulin entrapment circulation inlet and a GOX reaction outlet are arranged on the erythrocyte drug-loaded reactor; the insulin entrapment circulation outlet is divided into three paths after passing through a peristaltic pump, and is communicated with the insulin entrapment circulation inlet after passing through a hypotonic reactor, a hypertonic reactor and an isotonic reactor respectively through a conduit; two ends of the GOx reactor are respectively communicated with the GOx reaction outlet and an arterial blood taking end of a hemodialyzer through a conduit; the semi-permeable bag is prepared by adopting a semi-permeable membrane; catheter clamps are arranged on catheters at two ends of the hypotonic reactor, the hypertonic reactor and the isotonic reactor, catheters at two ends of the GOx reactor and catheters connecting the semi-permeable bag and the arterial blood taking end.

According to the invention, the insulin is encapsulated by the erythrocytes through hypotonic-hypertonic-isotonic treatment, and the steps of hypotonic-hypertonic-isotonic treatment can be sequentially subjected to circulating flow type reaction by utilizing the built-in semi-permeable bag of the erythrocyte drug-loaded reactor and combining the arrangement of the catheter clamp, the hypotonic reactor, the hypertonic reactor and the isotonic reactor, so that the reaction time is shortened, and the blood permeation time of a patient is effectively synchronized.

In some embodiments, preferably, the hypotonic reactor, the hypertonic reactor and the isotonic reactor are all provided with reaction liquid injection ports; and a reaction liquid injection port is arranged on the GOx reactor.

The corresponding reaction liquid can be effectively injected by the arrangement of each reaction liquid injection port. The reaction liquid injection port can adopt a rubber plug type injection port for an injection bag, so that liquid can be conveniently injected, and meanwhile, the sealing performance can be kept.

In some embodiments, it is preferred that the red blood cell drug-loaded reactor is in the form of a pouch; the hypotonic reactor, the hypertonic reactor and the isotonic reactor are packaged; the GOx reactor uses plastic tubes.

The non-isolated insulin controlled release drug entrapment device is disposable, and cross infection of patients is avoided. The erythrocyte medicine-carrying reactor, the hypotonic reactor, the hypertonic reactor and the isotonic reactor are designed in a bag type mode, the GOx reactor is designed in a medical plastic tube type mode, and cost is low.

In some embodiments, the encapsulation device further comprises a hypotonic temperature control device and a hypertonic temperature control device, which are used for controlling the temperature of the hypotonic reactor and the hypertonic reactor respectively.

In some embodiments, it is preferable that the hypotonic temperature control device is an incubator, and the hypotonic reactor is placed in the incubator; the high-permeability temperature control equipment adopts a water bath kettle, and the high-permeability reactor is arranged in the water bath kettle.

The invention also provides a method for entrapping the non-isolated insulin controlled release medicament, which comprises the following steps:

(1) insulin entrapment: leading blood from an artery into a semi-permeable bag of the erythrocyte drug-loaded reactor, injecting an insulin solution into the semi-permeable bag, circularly leading the hypotonic solution into the erythrocyte drug-loaded reactor through the hypotonic reactor, and circularly reacting for 2-3 hours; then the hypertonic solution is circularly introduced into the erythrocyte drug-loaded reactor through the hypertonic reactor, and the circular reaction lasts for 0.5 to 1 hour; finally, introducing the isotonic solution into the erythrocyte drug-loaded reactor through an isotonic reactor in a circulating way, and carrying out circulating reaction for 5-10 min;

(2) assembling a medicine switch: after the isotonic fluid circulation reaction is finished, injecting the blood in the semi-permeable bag into a GOx reactor for biotin-glucolase modification;

(3) and injecting the modified drug-loaded red blood cells into a hemodialyzer, and removing biotin-glucolase which is not modified on the surfaces of the red blood cells.

The insulin entrapment process adopts a circulating flow type reaction, so that the entrapment process is accelerated.

In some embodiments, the reaction temperature of the hypotonic cycling reaction is preferably 4 ℃; the reaction temperature of the hypertonic cyclic reaction is 37 ℃; when the artery is used for introducing blood, 2-10mL of whole blood is introduced, and 100-150U/mL of insulin solution is injected into the semipermeable bag, wherein the volume ratio of the insulin solution to the whole blood is 1: 10.

In some embodiments, it is preferred that when introducing blood into an artery, 10mL of whole blood is introduced and 1mL of 100U/mL or 150U/mL of insulin solution is injected into the semipermeable bag.

The invention optimizes the insulin entrapment process to obtain higher insulin entrapment amount.

In some embodiments, the hypotonic solution is preferably: 250mM NaCl, 12.5mM glucose, 12.5mM sodium pyruvate, 12.5mM inosine, 12.5mM NaH₂PO₄·2H₂O, 0.63mM adenine, 550mOsm/Kg, pH 8; the hypertonic solution is as follows: 250mM NaCl, 12.5mM glucose, 12.5mM sodium pyruvate, 12.5mM inosine, 12.5mM NaH₂PO₄·2H₂O, 0.63mM adenine, 550mOsm/Kg, pH 8; the isotonic solution is: physiological saline (0.9% aqueous sodium chloride); injecting GOx reaction liquid into the GOx reactor when biotin-glucolase modification is carried out; the GOx reaction liquid is prepared by the following method: dissolving 10mg of biotin in 1mL of dimethylformamide, adding 5mL of a glucosidase solution (2mg/mL) and 10mg of N, N' -carbonyldiimidazole, stirring at room temperature for 1 hour, putting the solution into a dialysis bag, and dialyzing at room temperature for 24 hours; the reaction time of biotin-glucosidase modification in the step (3) is 0.2-0.5 h.

Compared with the prior art, the invention has the following advantages:

1. the invention carries out insulin drug loading and administration in a non-isolated mode, utilizes the erythrocytes of patients as drug carriers, fully utilizes the advantages of the erythrocyte drug carriers, effectively prolongs the drug action time, and simultaneously avoids the occurrence of transfusion infection.

2. The encapsulation equipment is used in combination with the hemodialyzer, and the insulin erythrocyte encapsulation process is optimized, so that the better encapsulation efficiency is achieved, the time of hemodialysis is adapted, the insulin drug action and the hemodialysis are carried out simultaneously, and convenience is brought to patients.

Drawings

FIG. 1 is a schematic structural diagram of a non-isolated insulin controlled release drug entrapping device in combination with a hemodialyzer;

FIG. 2 is a schematic view of the encapsulation and controlled release process of the non-isolated insulin controlled release drug of the present invention;

FIG. 3 is a SEM representation of a non-ex vivo controlled release insulin drug of the present invention;

FIG. 4 is a laser confocal image of a non-isolated insulin controlled release drug of the present invention;

in the figure, A is the fluorescence characterization of FITC marked insulin on erythrocytes, B is the fluorescence characterization of rhodamine B marked GOx on erythrocytes, and C is the fluorescence characterization of GOx-INS-ER;

FIG. 5 is a graph of insulin concentration versus insulin loading for different insulin solutions;

FIG. 6 is a graph of different hypotonic times versus insulin loading;

FIG. 7 is a graph of different hypotonic temperatures versus insulin loading;

FIG. 8 is a graph of insulin release in different glucose solutions, i.e., the "on-off" test;

FIG. 9 is a graph showing the effect on blood glucose concentration in diabetic model rabbits following non-ex vivo administration using the present invention.

Detailed Description

The invention is described in detail below with reference to the figures and the specific embodiments.

In order to make those skilled in the art better understand the technical solutions in the present application, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application, and it is obvious that the described embodiments are only a part of the embodiments of the present application, and not all embodiments, and all other embodiments obtained by a person of ordinary skill in the art without making creative efforts based on the embodiments in the present application shall fall within the protection scope of the present application.

Diabetic nephropathy patients need to regularly carry out hemodialysis, poor control of blood sugar is needed to be injected after each meal, diabetic nephropathy is caused, renal failure is caused, and life risks exist if dialysis treatment is not carried out. However, the patient is greatly inconvenienced because the patient needs to carry insulin with him, and once the patient forgets or dose is insufficient, the patient is at a great risk.

The application provides a non-isolated insulin controlled release drug entrapment device, which is used in combination with a hemodialyzer 4 and comprises a red blood cell drug loading section and a drug control switch assembly section, wherein the non-isolated insulin controlled release drug administration carried out by adopting the entrapment device disclosed by the invention as shown in figure 1 specifically comprises the following steps:

the hemodialysis section, namely a hemodialyzer part, comprises a conduit clamp 1, peristaltic pumps 2 and 5, a hemodialyzer 4, a waste liquid tank 7, a dialysis liquid tank 6 and a conduit clamp 3, wherein:

the first end of the peristaltic pump 2 is connected with the artery of the patient, the second end is connected with the hemodialyzer 4, and a conduit clamp 3 is arranged on a conduit connecting the peristaltic pump 2 and the hemodialyzer 4; the first end of the hemodialyzer 4 is connected with the peristaltic pump 2, the second end is connected with the peristaltic pump 5, the third end is connected with the waste liquid tank 7, and the fourth end is connected with the dialysis liquid tank 6; the first end of the peristaltic pump 5 is connected with the hemodialyzer 4, the second end is connected with the vein of the patient, and a conduit clamp 1 is arranged on a conduit connecting the peristaltic pump 5 and the vein of the patient;

the tube clamps 1, 3 are used for controlling whether liquid flows, the peristaltic pumps 2, 5 are used for controlling the liquid flow and the flow direction, the waste liquid cylinder 7 is used for replacing waste liquid, and the dialysate cylinder 6 is used for replacing dialysate.

The red blood cell drug loading section comprises a red blood cell drug loading reactor 9, a low-osmotic liquid reactor 14, a high-osmotic liquid reactor 17, an isotonic liquid reactor 20 and a catheter clamp, wherein the red blood cell drug loading reactor is connected with the low-osmotic liquid reactor;

a semi-permeable bag is arranged in the erythrocyte drug-loaded reactor 9, two ends of the semi-permeable bag extend out of the erythrocyte drug-loaded reactor 9 through a guide tube, one end of the semi-permeable bag is connected with the peristaltic pump 2, a guide tube clamp 8 is arranged on the connecting guide tube, the other end of the connecting guide tube is connected with the GOX reactor 11, and a guide tube clamp 10 is also arranged on the connecting guide tube; the first end of the hypotonic reactor 14, the first end of the hypertonic reactor 17 and the first end of the isotonic reactor 20 are connected with the first end (insulin-coated circulation outlet) of the erythrocyte drug-loaded reactor 9, and a peristaltic pump 22 is arranged on a conduit at the connection part; the second end of the hypotonic reactor 14, the second end of the hypertonic reactor 17 and the second end of the isotonic reactor 20 are connected to the second end (insulin entrapment circulation inlet) of the erythrocyte bioreactor 9. Conduit clamps 15, 13, 18, 16, 21 and 19 are respectively arranged at the conduit positions at the two ends of the hypotonic reactor 14, the hypertonic reactor 17 and the isotonic reactor 20, the circulation flow of the hypotonic solution is controlled by the conduit clamps 15 and 13, the circulation flow of the hypertonic solution is controlled by the conduit clamps 18 and 16, and the circulation flow of the isotonic solution is controlled by the conduit clamps 21 and 19.

Thirdly, drug switch assembly section, including GOx reactor 11, pipe clamp, wherein:

the first end of GOx reactor 11 links to each other with 9 third ends (GOx reaction outlet) of erythrocyte medicine carrying reactor, is equipped with pipe clamp 10 on the connecting tube, and 11 second ends of GOx reactor link to each other with hemodialyzer 4, are equipped with pipe clamp 12 on the connecting tube.

This application specifically describes the device in examples 1 and 2, specifically, example 1 is a whole procedure of non-ex vivo administration, and example 2 is a procedure of loading GOx-INS-ER in erythrocytes.

Example 1:

with specific reference to fig. 1, fig. 1 is a schematic structural diagram of the whole administration process of a non-isolated erythrocyte-encapsulated insulin control device provided in example 1 of the present application.

The catheter clamp 8 is opened, the required blood is pumped into a semi-permeable bag arranged in the erythrocyte medicine-carrying reactor 9 through the peristaltic pump 2, and then the catheter clamp 8 is closed. Injecting insulin solution into an insulin injection opening of the semi-permeable bag, and then carrying out hypotonic-hypertonic-isotonic medicine loading process: firstly, reacting with hypotonic solution, opening the pipe clamps 13 and 15 at the two ends of the hypotonic reactor 14, circulating the hypotonic solution into the erythrocyte drug-loaded reactor 9 through the peristaltic pump 22, and continuously circulating to complete the hypotonic reaction under the reaction condition of 4 ℃ (the temperature control is realized by placing the hypotonic reactor in a constant temperature box), controlling the flow rate to be 0.2-0.5L/min by the peristaltic pump 22, and controlling the time to be 2-3 h; after the reaction is finished, the peristaltic pump 22 is closed, the conduit clamps 13 and 15 are closed, then the conduit clamps 16 and 18 at the two ends of the hypertonic reactor 17 are opened, the peristaltic pump 22 is started, the hypertonic solution circularly flows back to the erythrocyte drug-loaded reactor 9, the continuous circulating hypertonic reaction is carried out, the reaction condition is 37 ℃ (the temperature of the hypertonic reactor is controlled by placing the hypertonic reactor in a water bath kettle), the parameter of the peristaltic pump 22 is 0.2-0.5L/min, and the reaction time is 0.5-1 h; after the reaction is finished, closing the peristaltic pump 22, closing the conduit clamps 16 and 18, opening the conduit clamps 19 and 21 at the two ends of the isotonic reactor 20, starting the peristaltic pump 22, and circulating the isotonic solution back to the red blood cell drug-loaded reactor 9 for carrying out isotonic normal-temperature reaction for 10-20 min; after the reaction is finished, closing the peristaltic pump 22, closing the conduit clamps 19 and 21, opening the conduit clamp 10 between the semi-permeable bag and the GOx reactor 11 arranged in the erythrocyte drug-loaded reactor 9, allowing the blood loaded with insulin to flow into the GOx reactor 11 to react with GOx, closing the conduit clamp 10, reacting for 0.2-0.5h at 4 ℃ (the temperature control is realized by placing the GOx reactor in a thermostat), opening the conduit clamp 12 and the conduit clamp 10, injecting 0.9% NaCl physiological saline into an insulin injection port of the semi-permeable bag, injecting the blood modified by the glucolase into the hemodialyzer 4 for conventional hemodialysis, and removing the non-loaded redundant drugs; finally, the tubing clamp 1 between the patient and the hemodialyzer 4 is opened, allowing blood to flow back into the patient's vein.

Example 2:

referring specifically to fig. 2, fig. 2 is a process of the non-isolated erythrocyte-encapsulated insulin control device provided in embodiment 2 of the present application, in which the erythrocyte is encapsulated with insulin and is switched on and off according to GOx.

In this embodiment, the specific steps are as follows: introducing 2-10mL of whole blood from artery, hypotonic treating red blood cells, suspending the red blood cells in an insulin solution with a volume ratio of the red blood cells (whole blood) to the insulin solution of 10:1, suspending the mixed solution of the cells and the insulin in a semipermeable bag arranged in a red blood cell drug-loaded reactor 9, and circulating a hypotonic buffer solution (250mM NaCl, 12.5mM glucose, 12.5mM sodium pyruvate, 12.5mM inosine, 12.5mM NaH) in the red blood cell drug-loaded reactor 9 at 4 DEG C₂PO₄·2H₂O, 0.63mM adenine, 550mOsm/Kg, pH 8), standing for 2-3 h; the erythrocytes were then hypertonically treated so that the hypertonic buffer (250mM NaCl, 12.5mM glucose, 12.5mM sodium pyruvate, 12.5mM inosine, 12.5mM NaH)₂PO₄·2H₂O, 0.63mM adenine at 550mOsm/Kg, pH 8) atThe red blood cell medicine-carrying reactor 9 circularly flows for 0.5 to 1 hour; then carrying out an isotonic reaction, so that isotonic solution (0.9 percent NaCl normal saline) circularly flows in the erythrocyte drug-loaded reactor 9 for 5-10 min. And finally, injecting the solution in the built-in semi-permeable bag of the erythrocyte drug-loaded reactor 9 into the GOx reactor 11, wherein the solution filled in the GOx reactor 11 is as follows: dissolving 10mg of biotin in 1mL of dimethylformamide, adding 5mL of a glucolase solution (2mg/mL) and N, N' -carbonyldiimidazole (10mg), stirring at room temperature for 1 hour, putting the solution into a dialysis bag, and dialyzing at room temperature for 24 hours to obtain the injection solution in the GOx reactor. Modifying insulin-loaded red blood cells with biotin-glucosaccharase for 0.2-0.5 h. And opening the catheter clamp 12, introducing the modified insulin-loaded red blood cells into the hemodialyzer 4, removing the biotin-glucolase which cannot be modified on the surfaces of the red blood cells, and finally introducing the red blood cells into a human body.

Example 3

Characterization of the prepared erythrocyte-entrapped insulin:

fig. 3 is a Scanning Electron Microscope (SEM) image of the non-isolated insulin controlled release drug prepared in example 2, and it can be seen that there is no significant change in cell morphology after the red blood cells are entrapped. FIG. 4 is a representation of the controlled release drug by confocal laser scanning microscopy, where FITC-labeled insulin fluoresces green and rhodamine B-labeled glucosidase fluoresces red, indicating that erythrocytes have successfully encapsulated insulin and GOx.

Example 4

Effect of initial concentration of insulin on the loading of insulin by erythrocytes:

the method of example 2 was used to encapsulate the non-ex vivo insulin controlled release drug, wherein 10mL of whole blood was withdrawn, the volume of the injected insulin solution was 1mL, the hypotonic temperature was 4 ℃ and the hypotonic reaction time was 1 h.

The insulin solution infusion ratio at different concentrations was compared to the amount of insulin finally entrapped. As can be seen from FIG. 5, the insulin entrapment amount of the erythrocytes is related to the initial concentration of insulin, and the insulin entrapment amount is increased with the increase of the insulin concentration, and is in positive correlation, and when the concentration is increased to 150U/mL, the entrapment amount is not obviously increased.

In view of the loading amount, a concentration of 150U/mL in the insulin solution is preferred. Or directly takes the concentration of the insulin injection drug which is commonly on the market and is 100U/mL as the injection concentration in consideration of cost effectiveness.

Example 5

The effect of optimization of the conditions of the hypotonic pre-expansion method on the insulin loading:

the method of example 2 was used to encapsulate a non-ex vivo insulin controlled release drug, wherein 10mL of whole blood was withdrawn, the volume of the injected 100U/mL insulin solution was 1mL, and the hypotonic temperature was 4 ℃.

The final insulin loading was compared at different hypotonic temperatures. As can be seen in fig. 6, the insulin loading by red blood cells is related to the hypotonic time, the longer the hypotonic time, the greater the insulin loading by red blood cells. The equilibrium is reached after 3h of hypotonic time, and the increase of the loading amount is not obvious. And the time for the patient to perform hemodialysis is not suitable to be too long, so the hypotonic time is controlled to be 2 hours.

The hypotonic temperature comparison is carried out in the same entrapment mode, and the hypotonic time is 2 h.

As can be seen in fig. 7, the loading of INS by erythrocytes is related to the temperature at hypotonic conditions, with the loading being greatest at a hypotonic temperature of 4 ℃.

Therefore, considering the comprehensive consideration, the conditions of the hypotonic pre-expansion method are 2h and the temperature is 4 ℃.

Example 6

Release effect of GOx switch on release of insulin:

as can be seen from FIG. 8, the on-off release effect of GOx on insulin release was tested by "activating" the insulin controlled-release drugs obtained in the present invention in glucose solutions of different concentrations. With increasing glucose concentration, insulin release gradually increases. This indicates that the insulin-encapsulated erythrocytes constructed in this study have the property of releasing insulin in response to glucose, and the release effect is exerted by GOx on the surface of the erythrocytes, so as to achieve the effect of releasing insulin.

Example 7

The sustained release effect of the controlled release drug of the invention

Establishing a diabetic model rabbit: after fasting for 8h, the alloxan is prepared into a 5% solution by normal saline, the solution is injected into the rabbit body from the ear edge vein within half a minute according to 150mg/Kg, and the molding success is realized if the blood sugar is higher than 16mmol/L after 72 h.

Hemodialysis in combination with non-ex vivo insulin administration: non-isolated insulin controlled release drug entrapment was performed in example 2, and non-isolated administration was performed on the diabetic model rabbits in the administration manner of example 1, and the blood glucose values of the diabetic model rabbits were varied as shown in fig. 9.

As can be seen from figure 9, the non-isolated insulin controlled release drug administration mode of the invention can realize the control of blood sugar for a long time, and after the non-isolated insulin administration is performed through the hemodialysis, the blood sugar of the diabetic model rabbit is reduced to below 6.1mmol/L and is kept for 48 hours.

Other embodiments of the present application will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. This application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the application being indicated by the following claims.

The above-described embodiments of the present application do not limit the scope of the present application.