CN113679842B

CN113679842B - Preparation method and application of two-phase drug-loaded gel beads

Info

Publication number: CN113679842B
Application number: CN202110693673.XA
Authority: CN
Inventors: 王万能; 张秋寒; 唐一山; 成福
Original assignee: Chongqing University of Technology
Current assignee: Chongqing University of Technology
Priority date: 2021-06-22
Filing date: 2021-06-22
Publication date: 2022-11-15
Anticipated expiration: 2041-06-22
Also published as: CN113679842A

Abstract

The invention relates to a preparation method and application of a two-phase drug-loaded gel bead, belonging to the field of biological medicine materials. The two-phase drug-loaded gel bead comprises an inner-phase drug-loaded gel bead and an outer-phase hydrogel layer, wherein the inner-phase drug-loaded gel bead is prepared by crosslinking sodium alginate, k-carrageenan and drugs through a crosslinking agent and is wrapped by a polyelectrolyte shell consisting of k-CG and epsilon-PL to obtain the two-phase drug-loaded gel bead. The inner phase of the prepared biphase drug-loaded gel beads improves the drug-loaded performance through process optimization, the drug can be protected from degradation of gastric acid and pepsin after the outer phase is coated, the drug can be slowly released in intestinal tracts to achieve the effect of reducing blood sugar, and the biphase drug-loaded gel beads can be used for all drugs which are not resistant to gastric acid and absorbed by the intestinal tracts.

Description

Preparation method and application of two-phase drug-loaded gel beads

Technical Field

The invention belongs to the field of biomedical materials, and relates to a preparation method and application of a two-phase drug-loaded gel bead.

Background

Diabetes is a metabolic disease with chronically increased blood sugar level, and in the last few years, the diabetes patients are growing continuously and become one of the biggest chronic diseases threatening human beings in China. Insulin is one of effective drugs for treating diabetes, is also the first choice drug for patients with insulin-dependent diabetes mellitus, and plays an important role in treating diabetes. However, insulin as a protein drug cannot be directly taken orally, and is easily corroded by gastric acid and degraded and inactivated by digestive enzymes in gastrointestinal fluids, so that insulin is usually administered by injection, but the injection mode has poor acceptance for special people, such as old people and children. In fact, oral insulin first circulates into the liver via the portal vein, similar to the physiological pathway of insulin secretion in non-diabetic patients. In addition, advantages of the method include improved disease management, reduced long-term complications of diabetes. However, the bioavailability of oral insulin is less than 0.5% due to poor gastrointestinal environment and impaired insulin adsorption. In particular, the acidic and enzymatic environment in the stomach is one of the biggest obstacles to targeted intestinal delivery of bioactive molecules.

The hydrogel is a very hydrophilic three-dimensional network structure gel, can be quickly swelled in water and can keep a large volume of water in the swelled state without dissolving, and can be used as a drug-carrying material for drug delivery. In order to overcome the gastrointestinal barrier and avoid the inactivation of proteins by the harsh environment in the stomach, studies based on polypeptides and natural polymer biodegradable pH-sensitive hydrogels have received increasing attention. However, some oral insulin carriers have the problems of nondegradable property, instability in human bodies, release in the stomach and short release time in the intestine, and a single hydrogel is not enough to contain multiple administration functions, so that the gel polymer with multiple components and functions has great prospect. Aiming at various challenges faced by oral insulin carriers, the hydrogel beads are prepared under mild conditions to protect the activity of insulin, and the gel beads are convenient to store and have oral applicability; compared with the common gel beads, the gel beads are added with a layer of network cross-linking phase, and the surface of the gel beads is provided with positive charges so as to be convenient to be close to a mucus layer; the addition of the external phase can completely protect insulin from being damaged by gastric acid and pepsin, prolong the release time of insulin and facilitate the absorption of insulin.

Disclosure of Invention

In view of the above, an object of the present invention is to provide a method for preparing a dual-phase drug-loaded gel bead, a second object of the present invention is to provide a dual-phase drug-loaded gel bead, and a third object of the present invention is to provide an application of the dual-phase drug-loaded gel bead in preparing an oral administration carrier.

In order to achieve the purpose, the invention provides the following technical scheme:

1. a preparation method of a two-phase drug-loaded gel bead comprises the following steps;

a. dissolving sodium alginate in water of 80 deg.C, adding k-carrageenan, stirring for 30min, cooling, adding medicine, stirring at 100rpm for 30min, ultrasonic degassing for 10-15min, slowly adding the mixture into cross-linking agent, stirring for 30min, washing with distilled water, removing hardener to obtain inner phase medicine-carrying gel beads;

b. and (3) soaking the inner phase drug-loaded gel beads in the k-carrageenan solution for 5-10min, then soaking in an epsilon-polylysine solution with the mass fraction of 1% for 15-20min, repeating for at least 5 times, washing with distilled water, and drying to obtain the biphase drug-loaded gel beads.

As one preferable technical scheme, in the step a, the concentration ratio of the sodium alginate to the k-carrageenan is 3:1.

as one of the preferable technical proposal, in the step a, the cross-linking agent contains epsilon-polylysine and CaCl ₂ The mass fraction of epsilon-polylysine in the solution is 1 percent, and the CaCl is ₂ The concentration of (2) is 0.15mol/L.

As one of preferable technical solutions, in the step b, the mass fraction of the k-carrageenan in the k-carrageenan solution is 1%.

As one of the preferable technical proposal, the pH of the cross-linking agent is 4.

As one of the preferable technical schemes, the medicine is protein, polypeptide medicine and medicine which is not resistant to gastric acid and absorbed by intestinal tract.

In a preferred embodiment, the protein drug is insulin.

2. The biphase drug-loaded gel bead prepared by the preparation method.

As one of the preferable technical proposal, the biphasic medicine-carrying gel beads can not release medicine within 2 hours in gastric juice.

3. The application of the biphasic drug-loaded gel beads in preparing oral administration carriers.

The invention has the beneficial effects that:

the INS-biphase gel bead inner phase is composed of Sodium Alginate (SA), k-carrageenan (k-CG), epsilon-polylysine (epsilon-PL) and Ca ²⁺ And (3) crosslinking to prepare INS-k-inner phase drug-loaded gel beads. Sodium alginate gel solution and Ca ²⁺ Hydrogel beads can be formed under mild pH and temperature conditions. The k-carrageenan (k-CG) carrier has good physical and chemical properties such as electronegativity, viscosity, gelling property and the like, can improve the loading efficiency of simulated gastric juice, and enables the active drug to be stored for a long time. At pH of epsilon-polylysine (. Epsilon. -PL)<9 is strong cation, so epsilon-PL has strong electrostatic interaction with polyanions such as k-CG, alginate and the like. The INS-biphase gel bead outer phase is a polyelectrolyte shell coated outside the inner phase prepared by different experimental methods, wherein the INS-k-inner phase drug-loaded gel beads are coated by k-CG and epsilon-PL coatings, and the INS can be protected from degradation of gastric acid and pepsin after repeating for 5 times or more than 5 times. According to the principle, the INS-biphase gel bead internal phase prepared by the invention improves the drug loading performance through process optimization, and the INS can be protected from degradation of gastric acid and pepsin after being wrapped by the external phase, so that the INS-biphase gel bead internal phase can be slowly released in intestinal tracts to achieve the effect of reducing blood sugar.

Drawings

For the purposes of promoting a better understanding of the objects, aspects and advantages of the invention, reference will now be made to the following detailed description taken in conjunction with the accompanying drawings in which:

FIG. 1 is a diagram of preparation and characterization of INS-k-internal phase drug-loaded gel beads, wherein A is an appearance form and a microscopic form of gel beads prepared under an optimal process, B is an SEM image of the interior of INS-k-internal phase drug-loaded gel beads, C is an FTIR spectrogram, D is the influence of various material factors on drug-loaded performance, and E is an insulin solution standard curve;

FIG. 2 is a comparison representation of INS-k-internal phase drug loaded gel beads and INS-dual phase gel beads, wherein A is a 40X microscopic comparison representation of single and dual phase gel beads, B is a SEM comparison representation of the interior of single and dual phase gel beads, and C is a single and dual phase FTIR spectrum;

fig. 3 is a study of the transgastric performance of gel beads with different numbers of prepared layers of external phase, a is the difference of the particle sizes of the internal and external phases of the gel beads with different numbers of prepared layers, B is the swelling performance of the gel beads with different numbers of prepared layers (a =2,b = 5) in SGF, C is the release and transgastric conditions with different numbers of prepared layers (a =2,b = 5) in SGF, D is the swelling performance of the gel beads with different numbers of prepared layers (a =2,b = 5) in SIF, and E is the release profile of the gel beads with different numbers of prepared layers (a =2,b = 5) in SIF;

FIG. 4 is a graph comparing the release characteristics of single-phase and dual-phase gel beads, wherein A is the swelling ratio of single-phase and dual-phase gel beads in gastrointestinal medium; b is the release rate comparison of the single-phase and double-phase gel beads in the gastrointestinal tract medium.

FIG. 5 is the experimental study of blood sugar reduction in rats, wherein a is pathological changes of pancreas and kidney (10X 40) in normal control group and model group, A is normal control group of pancreas, B1-B2 are normal control group of pancreas, C is normal control group of kidney, and D1-D2 are normal control group of kidney; and b, comparing the blood sugar reducing curative effects of the experimental groups.

Detailed Description

The preparation method of the present invention and the in vitro and in vivo effects of the insulin-loaded biphasic gel beads prepared by the method are described below by preparing insulin-loaded biphasic gel beads, and other advantages and effects of the present invention will be readily apparent to those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.

Example 1

Preparation method of INS-biphase gel beads

(1) Preparation and optimization of INS-k-internal phase drug-loaded gel beads

SA is weighed and dissolved in distilled water, stirred for 3h at room temperature for standby, and INS is dissolved in phosphate buffer for standby. Stirring SA in 80 deg.C water, slowly adding k-CG, stirring for 30min, and covering the beaker with tinfoil paper to prevent water evaporation. Naturally cooling to room temperature, adding INS solution, stirring at 100rpm for 30min, and ultrasonic degassing for 15min. The mixture was slowly extruded through a 2.5mL syringe to contain ε -PL and 0.15mol/LCaCl ₂ In the crosslinker solution of (1). After stirring gently for 30min, the mixture was washed three times with distilled water to remove the hardener. And observing the appearance and the internal structure by using an optical microscope and a scanning electron microscope, and performing component analysis by using Fourier transform infrared spectroscopy.

Measuring insulin content by high performance liquid chromatography; based on a basic process formula and other factors are unchanged, the influence of the concentration of sodium alginate, the ratio of the concentration of sodium alginate to the concentration of k-carrageenan, the concentration of epsilon-polylysine and the pH of a cross-linking agent on the embedding rate, the drug loading rate and the particle size of insulin is respectively inspected.

Collecting CaCl obtained by filtering and washing gel beads ₂ And combining the crosslinking solution and the cleaning solution, taking 1mL of mixed solution, and determining the content of the insulin by using a high performance liquid chromatography. The envelope rate calculation formula (1.1) is as follows:

in the formula: EE represents the embedding rate,%; wa is the content of added INS in mg; wb is INS content in the crosslinking solution, mg.

Fresh gel beads were dried at 50 ℃ for 3 hours and weighed on an electronic balance. The drug loading formula (1.2) is as follows:

in the formula: i is drug loading,%; wa is the content of added INS in mg; wb is INS content in the crosslinking liquid, mg; w is the dry weight of the hydrogel beads, mg.

Randomly selecting 20 freshly prepared gel beads from each batch of samples, closely arranging the gel beads in a row, measuring the total diameter of the gel beads by using a micrometer screw, calculating the average particle size, and repeating the test for three times.

As a result, as shown in FIGS. 1A to E, an Insulin (INS) measurement method (FIG. 1E) was established; the optimal preparation process of the INS-k-internal phase drug-loaded gel bead is determined as follows: SA with the mass fraction of 1.5%, k-CG =3:1, epsilon-PL with the mass fraction of 1%, and cross-linking agent pH =4, wherein the evaluation index embedding rate is 66.7%, the drug loading is 0.45mg/mg, and the particle size is 1.57 +/-0.11 mm (D in figure 1); macroscopic observation shows that INS-k-inner phase drug-loaded gel beads are round, and the surface of the INS-k-inner phase drug-loaded gel beads is coated with epsilon-PL and is white. Optical microscopy 40 x showed that the surface was slightly rough (a in fig. 1); scanning Electron Microscopy (SEM) showed surface roughness with wrinkles; the outer layer is provided with a layer of compact shell, the interior is porous, and the outer layer is mostly in a grid shape (B in figure 1); fourier transform infrared spectrum results show that INS-k-inner phase drug-loaded gel beads are successfully prepared (C in figure 1).

(2) Encapsulation of external phase solutions

Dissolving k-CG in hot water of 60 ℃, dissolving epsilon-PL at room temperature, preparing INS-k-internal phase drug-loaded gel beads according to the optimal process conditions in the step (1), soaking the INS-k-internal phase drug-loaded gel beads in a k-carrageenan solution with the mass fraction of 1% for 5min, then soaking the INS-k-internal phase drug-loaded gel beads in an epsilon-polylysine solution with the mass fraction of 1% for 15min, repeating the steps for 5 times, washing the solution for 3 times by using distilled water, and carrying out vacuum filtration for 10min. Then stored at 4 ℃. And characterized by scanning electron microscopy and fourier transform infrared spectroscopy.

As shown in A-C in FIG. 2, the INS-k-inner phase drug-loaded gel beads and the INS-dual phase gel beads are white in appearance color, and the INS-dual phase gel beads are bright and opaque white compared with the INS-k-inner phase drug-loaded gel beads. The surface of the INS-biphasic gel beads was smoother than the surface of the INS-k-internal phase loaded gel beads (a in figure 2). Observing the SEM picture inside the gel beads to obtain INS-k-inner phase drug-loaded gel beads with large inner pore diameter, grid shape and a thin shell outside; the INS-biphasic gel beads have small internal pore size, a sharp boundary between the external and internal phases, and the external phase is a thick polyelectrolyte (B in FIG. 2). The presence of INS-biphasic gel beads with the same infrared signature as INS-k-internal phase loaded gel beads, the difference in absorption peak intensities indicates that the two gel beads differ in structure (C in fig. 2).

Example 2

In vitro detection of transgastric release Properties of INS-biphasic gel beads

Swelling experiments: soaking the single-phase two-phase hydrogel beads in 50mL of release media with different pH values respectively, shaking in NaCI-HCI buffer solution (pH 1.2) for 2h, and shaking in phosphate buffer solution (pH 7.4). Thereafter, the beads were removed from the swelling medium, and excess water on the surface was blotted off with absorbent paper and then weighed. The Swelling Ratio (SR) is calculated from the dynamic weight change of the gel beads with respect to pH, and is calculated according to the following equation (2.1):

where Ws is the weight of the beads in the expanded state and Wd is the initial weight of the dried beads. Each experiment was repeated three times.

In vitro release experiments: the in vitro release studies of loaded gel beads were performed in different release media with pH 1.2, 7.4, respectively according to the swelling experiments. The samples were incubated at 37 ℃ with constant shaking at 100 r/min. At predetermined time intervals, 1ml samples were collected from the release medium and replaced with an equal amount of fresh medium. The collected samples were centrifuged (15000, 5 min) and the insulin content of the supernatant determined by high performance liquid chromatography. Cumulative release rate (CR) calculation formula (2.2) is as follows:

in the formula, CR represents the INS cumulative release rate,%; cn represents the concentration of INS measured in the nth time, mg/mL; v represents the total volume of release medium, mL; cn-1 denotes the n-1 measured INS concentration, mg/mL, vi denotes the n-1 sample volume, here 1mL; w0 represents the initial mass of the gel beads before swelling, g; LC% is drug loading, mg/100mg gel beads.

The results of the transgastric performance of INS-biphasic gel beads are shown in FIGS. 3A-E, where the outer phase is wrapped 2 times, 30% INS is released in 30min and 40% INS is released in 2 h. When the external phase is wrapped for 5 times, the cumulative release rate in the simulated gastric juice is 0, so that the insulin can be protected in the stomach by overlapping the wrapping layers of the external phase. Respectively preparing INS-biphase gel beads with more than 5 layers of external phases, and obtaining that the accumulated release rate of INS in simulated gastric juice is 0 when the number of the wrapped external phase layers is more than 5, so that insulin can be protected in the stomach. Therefore, it can be concluded that when the wrapping times are more than 5 times, the INS-biphasic gel beads have transgastric performance (C in fig. 3), and D, E in fig. 3 shows the pH responsiveness of the INS-biphasic gel beads to swell in an alkaline environment, so that insulin is protected by the gel beads in the stomach and targeted drug release is performed in the intestine; the gel beads have mild and sustained drug release properties, suggesting that they have the potential for sustained drug release in the intestinal tract. The drug release characteristics of the INS-k-inner phase drug-loaded gel beads and the INS-dual-phase gel beads are shown in figure 4, wherein A in figure 4 shows that the INS-dual-phase gel beads have large shrinkage in Simulated Gastric Fluid (SGF), are favorable for protecting INS activity, have small swelling amplitude in Simulated Intestinal Fluid (SIF), and are favorable for slow release of INS in intestinal tract targeting; it has potential as an oral INS vector compared with INS-k-inner phase drug-loaded gel beads; panel B shows that INS-biphasic gel beads have a protective effect on INS in gastric juice and are slowly and continuously released in a simulated small intestine environment.

Example 3

Study of blood glucose lowering experiments with INS-biphasic gel beads

(1) Establishment of diabetic rat model

And (3) preparing a citric acid buffer solution, namely adding 2.1g of citric acid into 100mL of double distilled water to prepare solution A, adding 2.94g of sodium citrate into 100mL of double distilled water to prepare solution B, mixing the A, B solutions according to a ratio of 1.32 when in use, and adjusting the pH = 4.2-4.5, namely preparing the 0.1mol/L citric acid buffer solution required to be prepared.

After overnight fasting, the weight of the rat is weighed, streptozotocin (STZ) is weighed according to the total weight ratio of the rat, the rat is placed in a dry sterilization bottle, and the STZ is dissolved in groups after being wrapped by tinfoil. The injection is performed by dissolving STZ in citric acid buffer solution at 10mg/mL, injecting corresponding STZ with fasting body weight, and completing injection within 30 min. The rats in the model group were injected intraperitoneally with STZ at a dose of 60mg/kg, and the control group was given the same dose of sodium citrate buffer. Fasting blood glucose was measured 3 days after STZ injection.

(2) Histopathological examination sample processing

Fixing: removing neck of diabetic rat to death, collecting fresh kidney and pancreas, washing with PBS for 2-3 times, and fixing in paraformaldehyde for 24 hr;

and (3) dehydrating: taking out the tissue, putting the tissue into an embedding box, washing the tissue for a period of time by PBS, and then soaking and dehydrating the tissue by 30%, 50%, 70%, 80%, 95% and 100% ethanol and the like respectively;

and (3) transparency: then placing the tissue block in a transparent agent xylene which is soluble in alcohol and paraffin for transparency, and replacing the alcohol in the tissue block with xylene to soak the wax for embedding;

embedding: the transparent tissue block is placed in melted paraffin and put into a thermostat at 65 ℃. And after the paraffin is completely immersed into the tissue block, embedding, cooling and solidifying into blocks.

Slicing: the embedded wax blocks were fixed on a microtome and cut into 5 μm slices.

Baking slices: and (3) ironing the cut wrinkle slices in a water bath kettle at 43 ℃, pasting the wrinkle slices on a glass slide, transversely baking the glass slide for 2 hours in a thermostat at 60 ℃, standing the glass slide for overnight baking, and then putting the glass slide into a box for storage.

(3) H & E staining

Before staining, paraffin in the sections is removed by xylene, and the tissues are then placed in 100% ethanol for 5min,90%,80%, 80% ethanol for 2min, and distilled water for 2min. Wiping off water outside the section, performing hematoxylin staining, covering the sample with hematoxylin staining solution for 8-10 min, washing with distilled water, then placing the section into 70% alcohol differentiation solution containing 1% hydrochloric acid for differentiation for 5s, and washing with tap water for 10min. Wiping off excessive water in the section, taking eosin staining solution to stain the sample for 1min, and soaking the sample in tap water for 1-2 min to wash off the excessive staining solution. The dyed slices are dehydrated by pure alcohol and then are transparent by xylene. And (4) dripping gum on the transparent section, covering a cover glass, sealing, attaching a label, and observing by using a microscope.

(4) Hypoglycemic experiments with oral INS-biphasic gel beads

15 successfully modeled diabetic rats were selected and divided into 3 groups, and 5 rats in each group were used as parallel experiments. Group 1 was blank and was not treated with INS, group 2 was oral insulin solution group, group 3 was oral insulin hydrogel beads group, and the dosage of oral INS was 50IU/kg. Mixing INS hydrogel beads with small amount of edible oil, and performing intragastric administration with INS-biphase gel beads with corresponding dosage to diabetic rats according to different body weights. After administration, blood was taken from the tail apex at 0.5h,1h,2h,3h,4h,6h,8h, 10h, and 12h, respectively, and the blood glucose level was measured by an automatic blood glucose meter and the relative blood glucose level was calculated according to the calculation formula (3.1).

The results are shown in a and b in fig. 5 and tables 1 to 3, wherein a in fig. 5 shows that the model group presents the pathological features of diabetes, the successful establishment of the diabetic rat model is verified, and the model establishment is also shown by the significant difference between the body weight and the blood sugar of the model group and the control group in tables 1 and 2. In fig. 5, b shows that the blood sugar value of the diabetic rats orally taking INS-biphasic gel beads is reduced, a slow blood sugar reducing effect is shown, and the blood sugar of the diabetic rats directly orally taking insulin solution has no obvious difference compared with the control group.

TABLE 1 mean body weight change (g) in the hypoglycemic experiments in the control and model rats

TABLE 2 blood glucose Change (mmol/L) in rats of control group and model group in hypoglycemic experiments

TABLE 3 results of the hypoglycemic experiments

Finally, the above embodiments are only intended to illustrate the technical solutions of the present invention and not to limit the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it will be understood by those skilled in the art that modifications or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions, and all of them should be covered by the claims of the present invention.

Claims

1. A preparation method of a two-phase medicine-carrying gel bead is characterized by comprising the following steps of;

b. and (3) soaking the inner-phase drug-loaded gel beads in a k-carrageenan solution for 5-10min, then soaking in an epsilon-polylysine solution with the mass fraction of 1% for 15-20min, repeating for at least 5 times, washing with distilled water, and drying to obtain the biphase drug-loaded gel beads.

2. The method of claim 1, wherein in step a, the ratio of the concentrations of sodium alginate to k-carrageenan is 3:1.

3. the method of claim 1, wherein in step a, the cross-linking agent comprises epsilon-polylysine and CaCl ₂ The mass fraction of epsilon-polylysine in the solution is 1 percent, and the CaCl is added ₂ The concentration of (2) is 0.15mol/L.

4. The method according to claim 1, wherein in the step b, the mass fraction of the k-carrageenan in the k-carrageenan solution is 1%.

5. The method of claim 1, wherein the crosslinking agent has a pH of 4.

6. The method according to any one of claims 1 to 4, wherein the drug is a protein or polypeptide drug.

7. The method of claim 6, wherein the proteinaceous agent is insulin.

8. Biphasic drug-loaded gel beads prepared by the preparation method of any one of claims 1 to 7.

9. The dual phase drug loaded gel bead of claim 8, wherein the dual phase drug loaded gel bead does not release drug within 2 hours in gastric fluid.

10. Use of the biphasic drug loaded gel beads of claim 8 in the preparation of an oral delivery vehicle.