A hierarchical pH-responsive telopeptide oral microcapsule and a preparation method thereof

Abstract

The application discloses a kind of hierarchical pH response telopeptide oral microcapsules and preparation method, belong to biomedicine technical field.A kind of hierarchical pH response microcapsule, comprising: inner phase core, including drug-loaded inorganic nanoparticles and first alginate material;And, outer shell, including the second alginate material wrapped in the outside of the inner phase core;Wherein the drug-loaded inorganic nanoparticles are the complex of polypeptide drug and layered silicate material;The polypeptide drug is telopeptide;The layered silicate material is montmorillonite.Compared with prior art, the pH response microcapsule of the application has good pH response and intestinal targeting release capacity;In intestinal environment, through special ion exchange and pH change, not only can promote the rapid degradation of alginate hydrogel shell, but also can effectively trigger the release of telopeptide from montmorillonite carrier, so as to realize the efficient release and absorption of target site.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a graded pH-responsive oral microcapsule of telpoeptide and its preparation method. Background Technology

[0002] Tirzepatide is a novel glucose-dependent insulinotropic peptide (GIP) and glucagon-like peptide-1 (GLP-1) dual receptor agonist that has demonstrated remarkable clinical efficacy in the treatment of type 2 diabetes and obesity. However, as a large polypeptide drug, tirzepatide is readily degraded by gastric acid and digestive enzymes in the gastrointestinal environment. Furthermore, its large molecular weight and high hydrophilicity result in extremely poor intestinal epithelial permeability. Currently, clinical administration of tirzepatide heavily relies on frequent subcutaneous injections, which not only causes injection pain and local adverse reactions for patients but also severely limits long-term treatment adherence.

[0003] Oral administration is an ideal route for delivering peptide drugs. To address the shortcomings of traditional oral peptide carriers, such as poor protection in gastric acid, easy burst release leading to drug inactivation, and the complexity and protein denaturation inherent in existing preparation processes, this invention provides a graded pH-responsive oral microcapsule of telpoide and its preparation method. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention proposes a graded pH-responsive oral microcapsule of telpoeptide and its preparation method.

[0005] The objective of this invention can be achieved through the following technical solutions: A first aspect of the present invention relates to a graded pH-responsive microcapsule, comprising: The inner phase core comprises drug-loaded inorganic nanoparticles and a first alginate material; And, the outer shell, including a second alginate material wrapped around the outer surface of the inner core; The drug-loaded inorganic nanoparticles are a complex of a polypeptide drug and a layered silicate material; the polypeptide drug is telpoide; and the layered silicate material is montmorillonite.

[0006] Optionally, the viscosity of the first alginate material is 3-5 mPa·s, and the mass-volume concentration in its inner phase core is 5% (w / v).

[0007] Optionally, the viscosity of the second alginate material is 30-90 mPa·s, and the mass-volume concentration in its outer shell is 2% (w / v).

[0008] Optionally, the diameter of the microcapsule is 200~250 μm.

[0009] A second aspect of the present invention relates to a method for preparing the above-described graded pH-responsive microcapsules, comprising the following steps: 1) Preparation of drug-loaded inorganic nanoparticle complex: The polypeptide drug solution and the layered silicate material suspension were mixed in an acidic buffer system, stirred and combined, and then collected by centrifugation and washing to obtain the drug-loaded complex. 2) Preparation of inner and outer phase working solutions: The drug-loaded complex obtained in step 1) is mixed evenly with the first alginate solution as the inner phase; the second alginate solution is used as the outer phase; 3) Microfluidic electrospray molding: Using a coaxial microfluidic device, the inner phase and the outer phase are injected into the inner capillary and the outer capillary respectively, and coaxial droplets are generated under the action of a high voltage electric field; 4) Crosslinking and curing: Collect the generated droplets and cure them in an ion-crosslinking aqueous solution.

[0010] Optionally, the acidic buffer system is prepared using an acetate buffer with a pH of 3.5 to 4.5, so that the polypeptide drug and the layered silicate material have opposite charges on their surfaces; the mass ratio of the polypeptide drug to the layered silicate powder is 1:10.

[0011] Optionally, the coaxial microfluidic device is coaxially assembled from two glass capillaries with different inner diameters. The inner capillary orifice of the inner phase channel has an inner diameter of 0.2 mm, and the outlet inner diameter of the outer phase capillary has an inner diameter of 0.5 mm.

[0012] Optionally, in step 3), the ratio of internal to external phase flow rates is 1:2 to 1:6; The injection flow rate of the inner phase liquid is 10 μL / min, and the injection flow rate of the outer phase liquid is 50 μL / min; the applied voltage of the high-voltage electric field is 10 kV.

[0013] Optionally, in step 4), the ionic crosslinking aqueous solution is a 2% calcium chloride solution; the droplet collection distance during curing crosslinking is 5 cm.

[0014] A third aspect of the present invention relates to the use of the above-described graded pH-responsive microcapsules in the preparation of oral pharmaceutical formulations for the treatment of type 2 diabetes and / or obesity.

[0015] The beneficial effects of this invention are: (1) This invention prepares microcapsules based on microfluidic technology, which is low in cost and easy to operate. The size of the microcapsules can be adjusted by adjusting the microfluidic parameters to achieve stable mass production.

[0016] (2) The pH-responsive microcapsules prepared by the present invention have good pH responsiveness and intestinal targeted release capability. In the intestinal environment, through special ion exchange and pH change, they can not only promote the rapid degradation of the alginate hydrogel shell, but also effectively trigger the release of tezepatide from the montmorillonite carrier, thereby achieving efficient release and absorption at the target site.

[0017] (3) The pH-responsive microcapsules prepared in this invention can significantly improve the glucose sensitivity of animals in a type 2 diabetic animal model after oral administration, and achieve robust blood glucose control. They are expected to be used as an ideal oral peptide preparation in the clinical treatment of metabolic diseases. Attached Figure Description

[0018] The invention will now be further described with reference to the accompanying drawings.

[0019] Figure 1 This is a schematic diagram of an apparatus for preparing graded pH-responsive microcapsules using a microfluidic-electrospray integrated method; wherein, 1. telpoide solution; 2. montmorillonite suspension; 3. telpoide@montmorillonite complex; 4. inner phase inlet device; 5. outer phase inlet device; 6. inner phase capillary; 7. outer phase capillary.

[0020] Figure 2 Figure 1 shows the preparation of graded pH-responsive microcapsules according to an embodiment of the present invention; wherein, in the figure, a represents the formation process of hydrogel microcapsules, b represents the relationship between the diameter of hydrogel microcapsules and the flow rate of the external phase solution, and c represents the relationship between the diameter of hydrogel microcapsules and the applied voltage.

[0021] Figure 3 The images shown are light micrographs and scanning electron micrographs of the graded pH-responsive microcapsules prepared according to embodiments of the present invention; wherein, a in the figure is a light micrograph and b is a scanning electron micrograph.

[0022] Figure 4 The cumulative drug release curves of the graded pH-responsive microcapsules prepared for embodiments of the present invention in in vitro simulated gastric and intestinal fluid treatments.

[0023] Figure 5 The figure shows the therapeutic effect of oral administration of the graded pH-responsive telpoeptide microcapsules of this invention to type 2 diabetic mice. Detailed Implementation

[0024] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0025] Montmorillonite was purchased from Pioneer Nanomaterials Technology Co., Ltd., model PGW.

[0026] Low-viscosity sodium alginate was purchased from Alfaesa (China) Chemical Co., Ltd., product number B25266.

[0027] The ultra-low viscosity sodium alginate was purchased from Myriel, product number M76389-100G.

[0028] Example 1 A graded pH-responsive telpoeptide oral microcapsule includes the following steps: (1) Preparation of telpoeptide@montmorillonite complex: Thiopeptide powder was dissolved in acetate buffer at pH 4.0 and shaken to ensure complete dissolution, yielding a 1 mg / mL thiopeptide solution. 10 mg of montmorillonite powder was weighed and dispersed in deionized water, and stirred continuously at room temperature for 24 h to obtain a homogeneous montmorillonite suspension. The montmorillonite suspension was centrifuged, and the bottom montmorillonite precipitate was collected and resuspended in 1 mL of acetate buffer at pH 4.0. 1 mL of the 1 mg / mL thiopeptide solution was added dropwise to the resuspended montmorillonite suspension, and the reaction was stirred at room temperature for 2 h. After the reaction, the bottom precipitate was collected by centrifugation and washed three times with deionized water to remove unbound free drug, yielding the thiopeptide@montmorillonite complex.

[0029] (2) Design of microfluidic chips: The microfluidic device is assembled from two glass capillaries with different inner diameters. A capillary glass tube with a diameter of 0.75 mm is selected as the outer phase capillary, and the outlet is adjusted to 0.5 mm by pulling the tube. A capillary glass tube with an inner diameter of 0.2 mm is selected as the inner phase capillary. Before assembly, the glass capillaries are treated with a hydrophobic surface. The two capillaries are nested together using a coaxial method to form a capillary channel with a coaxial outlet, and then fixed to a glass slide using adhesive.

[0030] (3) Preparation of microcapsules: The telpoeptide@montmorillonite complex prepared in step (1) was mixed uniformly with a 5% (w / v) ultra-low viscosity sodium alginate solution as the inner phase liquid; wherein the viscosity of the ultra-low viscosity sodium alginate in a 1% aqueous solution is 3-5 mPa·s; a 2% (w / v) low viscosity sodium alginate solution was prepared as the outer phase liquid; wherein the viscosity of the low viscosity sodium alginate in a 1% aqueous solution is 30-90 mPa·s; the inner and outer phase liquids were coaxially injected into the microfluidic device using a precision injection pump; the outer phase flow rate was set to 50 μL / min and the inner phase flow rate to 10 μL / min; a voltage of 10 kV was applied at the nozzle of the microfluidic device to generate droplets under the action of the electric field; a collection liquid, which is a 2% (w / v) CaCl2 solution, was set 5 cm away from the nozzle.

[0031] By adjusting the parameters of the microfluidic system, the diameter of the microcapsules can be precisely controlled, such as... Figure 2 As shown, the diameter of the microcapsule is directly proportional to the flow rate of the external phase solution and inversely proportional to the applied voltage.

[0032] To observe the uniformity and microstructure of the microcapsules, they were characterized using optical microscopy and scanning electron microscopy, such as... Figure 3 As shown in the figure, the microcapsules have a core-shell structure and are uniform in size.

[0033] Example 2 In this embodiment, relevant experiments were conducted to verify the graded pH-responsive release characteristics of the microcapsules prepared in Example 1 in a simulated gastrointestinal environment.

[0034] Equal volumes of the drug-loaded microcapsules from Example 1 were placed in 3 mL of HCl buffer (pH 1.2) and phosphate buffer (PBS, pH 7.4), respectively, and incubated at 37°C and 150 rpm. At predetermined time intervals, 1 mL of the supernatant was collected and immediately replaced with an equal volume of the same fresh buffer; the released drug concentration was determined; as... Figure 4 As shown, the microcapsules prepared in Example 1 exhibited a cumulative drug release rate of only about 5% after incubation in simulated gastric fluid (pH 1.2) for 2 hours, demonstrating excellent resistance to gastric acid degradation. After incubation in simulated intestinal fluid (pH 7.4), the microcapsules gradually degraded and released the drug, with a cumulative drug release rate of about 98% within 24 hours. This indicates that the microcapsule structure provided by the present invention can effectively avoid premature drug release in the gastric fluid environment and achieve stable and long-lasting release in the intestinal fluid environment.

[0035] Example 3 In this embodiment, the microcapsules prepared in Example 1 were administered to type 2 diabetic mice by gavage to improve blood glucose and body weight.

[0036] Male db / db mice aged 6-8 weeks were used as a type 2 diabetes mellitus (T2DM) model, with fasting blood glucose (FBG) levels all exceeding 11.1 mmol / L. The db / db type 2 diabetic mice were randomly divided into a control group and an experimental group, with 5 mice in each group. The control group was orally administered an equal volume of physiological saline; the experimental group was orally administered microcapsules loaded with telpolide (microcapsules prepared in Example 1, containing 100 nmol / kg of telpolide) once daily. Fasting blood glucose levels and body weight changes were monitored and recorded regularly. The results of blood glucose and body weight monitoring are as follows: Figure 5 As shown, it demonstrates good blood glucose and weight control effects (arrows indicate the microcapsule group); indicating that the graded pH-responsive microcapsules prepared in this invention can achieve effective oral delivery of telpolide and exert good hypoglycemic and weight-loss effects in vivo.

[0037] In the description of this specification, references to terms such as "an embodiment," "example," "specific example," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0038] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the claimed invention.

Claims

1. A graded pH-responsive microcapsule, characterized in that, include: The inner phase core comprises drug-loaded inorganic nanoparticles and a first alginate material; And, the outer shell, including a second alginate material wrapped around the outer surface of the inner core; The drug-loaded inorganic nanoparticles are a complex of a polypeptide drug and a layered silicate material; the polypeptide drug is telpoide; and the layered silicate material is montmorillonite.

2. The graded pH-responsive microcapsule according to claim 1, characterized in that, The viscosity of the first alginate material is 3-5 mPa·s, and the mass volume concentration in its inner phase core is 5% (w / v).

3. The graded pH-responsive microcapsule according to claim 1, characterized in that, The viscosity of the second alginate material is 30-90 mPa·s, and the mass-volume concentration in its outer shell is 2% (w / v).

4. The graded pH-responsive microcapsules according to any one of claims 1-3, characterized in that, The microcapsules have a diameter of 200~250 μm.

5. A method for preparing graded pH-responsive microcapsules as described in any one of claims 1-4, characterized in that, Includes the following steps: 1) Preparation of drug-loaded inorganic nanoparticle complex: The polypeptide drug solution and the layered silicate material suspension were mixed in an acidic buffer system, stirred and combined, and then collected by centrifugation and washing to obtain the drug-loaded complex. 2) Preparation of inner and outer phase working solutions: The drug-loaded complex obtained in step 1) is mixed evenly with the first alginate solution to form the inner phase; The second alginate solution was used as the external phase; 3) Microfluidic electrospray molding: Using a coaxial microfluidic device, the inner phase and the outer phase are injected into the inner capillary and the outer capillary respectively, and coaxial droplets are generated under the action of a high voltage electric field; 4) Crosslinking and curing: Collect the generated droplets and cure them in an ion-crosslinking aqueous solution.

6. The preparation method according to claim 5, characterized in that, The acidic buffer system is prepared using an acetate buffer solution with a pH of 3.5 to 4.5, so that the polypeptide drug and the layered silicate material have opposite charges on their surfaces; the mass ratio of the polypeptide drug to the layered silicate powder is 1:

10.

7. The preparation method according to claim 5, characterized in that, The coaxial microfluidic device is assembled coaxially from two glass capillaries with different inner diameters. The inner capillary orifice of the inner phase channel has an inner diameter of 0.2 mm, and the outlet inner diameter of the outer phase capillary has an inner diameter of 0.5 mm.

8. The preparation method according to claim 5, characterized in that, In step 3), the ratio of internal to external phase flow rates is 1:2 to 1:6; The injection flow rate of the inner phase liquid is 10 μL / min, and the injection flow rate of the outer phase liquid is 50 μL / min; the applied voltage of the high-voltage electric field is 10 kV.

9. The preparation method according to claim 5, characterized in that, In step 4), the ion crosslinking aqueous solution is a 2% calcium chloride solution; the droplet collection distance during curing crosslinking is 5 cm.

10. The use of a graded pH-responsive microcapsule as described in any one of claims 1-4 in the preparation of an oral pharmaceutical formulation for the treatment of type 2 diabetes and / or obesity.

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