CN114796091A

CN114796091A - Microneedle array for subcutaneous slow release of calcitonin and preparation method thereof

Info

Publication number: CN114796091A
Application number: CN202210239707.2A
Authority: CN
Inventors: 巨晓洁; 李瑶; 褚良银; 付含; 刘壮; 汪伟; 谢锐
Original assignee: Sichuan University
Current assignee: Sichuan University
Priority date: 2022-03-12
Filing date: 2022-03-12
Publication date: 2022-07-29

Abstract

The invention provides a micro-needle array for subcutaneous sustained release of calcitonin and a preparation method thereof, wherein the micro-needle array consists of a substrate and drug-carrying micro-needles distributed on one side of the substrate in an array manner, the drug-carrying micro-needles are pyramid-shaped and consist of a tip part and a bottom part, the tip part is loaded with calcitonin, the base material of the tip part is different from that of the bottom part, and the base material of the bottom part is the same as that of the substrate; the base materials of the bottom and the substrate are water-soluble biodegradable high polymer materials, and the base material of the tip part is composed of polyethylene glycol and silk fibroin; after the microneedle array is administrated through the skin, the bottom of the drug-carrying microneedle is dissolved firstly, the tip part of the drug-carrying microneedle is separated from the substrate and is reserved in the skin, and the calcitonin loaded on the tip part is slowly released along with the swelling and biodegradation of the base material of the tip part of the drug-carrying microneedle. The invention can realize the slow release of the calcitonin in the body, prolong the action time of the calcitonin in the body, reduce the administration frequency and realize painless self administration of patients.

Description

Microneedle array for subcutaneous slow release of calcitonin and preparation method thereof

Technical Field

The invention belongs to the technical field of transdermal drug delivery, and relates to a microneedle array for calcitonin subcutaneous slow release and a preparation method thereof.

Background

Osteoporosis (OP) is a type of skeletal disease characterized by decreased bone strength and increased risk of fracture. It is common to women in menopause, and is the main cause of systemic bone pain, bone deformation, fracture and even death of the elderly.

Calcitonin (CT) is thirty-two dipeptide secreted by thyroid follicular paracellular cells and containing a disulfide bond, reduces the incidence rate of fracture by inhibiting bone absorption of parathyroid hormone and osteoclast, has strong central nerve analgesic effect, and can relieve systemic bone pain caused by osteoporosis. The structures of calcitonin are similar between different species, the amino acid sequences of calcitonin vary with the species, and calcitonin isolated from vertebrates such as fish has the strongest activity, while calcitonin isolated from mammals has relatively weaker activity. At present, the natural or artificial calcitonin applied to clinical application mainly comprises pig calcitonin (pCT), human calcitonin (hCT), salmon calcitonin (sCT), eel calcitonin (eCT) and the like, wherein the biological activity of the salmon calcitonin can reach 4000 IU/mg-7000 IU/kg, which is equivalent to 30-40 times of the biological activity of the human calcitonin, the action time is longer, and the half-life period is 50-80 min, which is 3-6 times of the human calcitonin. sCT has been widely used in the treatment of early and late postmenopausal osteoporosis and senile osteoporosis.

As a polypeptide, CT has unstable physicochemical properties and low oral bioavailability, and the dosage forms currently used for delivering CT mainly include sterile solutions for intramuscular injection or subcutaneous injection, and nasal sprays. Injections need to be administered once a day for a long period of time, patient compliance is extremely poor, and gastrointestinal side effects such as nausea and abdominal pain, and eruption at the injection site occur in a considerable proportion of clinical cases. Although the nasal spray can reduce adverse reactions, the bioavailability of the administration in the mode is only 3% -5%, the administration is still required every day, the long-acting blood concentration cannot be maintained, and the long-term nasal administration can cause nasal mucosa injuries in different degrees.

Disclosure of Invention

Aiming at the problems of short half-life and long-term frequent administration of the existing calcitonin preparation, the invention provides a microneedle array for subcutaneous slow release of calcitonin and a preparation method thereof, so as to realize slow release of the calcitonin in vivo, prolong the action time of the calcitonin in vivo, reduce the administration frequency and realize painless self administration of patients.

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

the microneedle array is composed of a substrate and drug-loaded microneedles distributed on one side of the substrate in an array manner, wherein the drug-loaded microneedles are in a pyramid shape and composed of a tip part and a bottom part, the tip part is loaded with calcitonin, the tip part is different from a base material of the bottom part, and the bottom part is the same as the base material of the substrate; the base materials of the bottom and the substrate are water-soluble biodegradable high polymer materials, and the base material of the tip part is composed of polyethylene glycol and silk fibroin; after the microneedle array is administrated through the skin, the bottom of the drug-carrying microneedle is dissolved firstly, the tip part of the drug-carrying microneedle is separated from the substrate and is reserved in the skin, and the calcitonin loaded on the tip part is slowly released along with the swelling and biodegradation of the base material of the tip part of the drug-carrying microneedle.

In the technical scheme of the microneedle array for the subcutaneous slow release of calcitonin, the silk fibroin has excellent mechanical property and can be processed in a full water environment to realize the controllable adjustment of biodegradability. The polyethylene glycol can induce the secondary structure of the silk fibroin to be transformed by influencing the structure and the property of water around the silk fibroin, so that the crystal form of the silk fibroin is changed, and the release rate of the calcitonin loaded on the microneedle tip part is further influenced. The release rate of the calcitonin loaded on the micro-needle tip part can be adjusted by selecting polyethylene glycol with different molecular weights or/and changing the mass ratio of the polyethylene glycol to the silk fibroin according to the requirements on the administration speed or the drug release rate in practical application.

We found through experiments that, in a certain range, generally, the larger the molecular weight and the higher the content of the polyethylene glycol, the slower the release rate of salmon calcitonin loaded to the tip portion of the microneedle is. Preferably, the base material of the microneedle tip part is composed of polyethylene glycol and silk fibroin according to the mass ratio of (10-40): 100. Further preferably, the molecular weight of the polyethylene glycol is 800-20000.

In the technical scheme of the microneedle array for subcutaneous slow release of salmon calcitonin, different base materials are adopted at the tip part and the bottom of the drug-carrying microneedle, the bottom of the drug-carrying microneedle is the same as the base material of the substrate, the base materials of the bottom of the drug-carrying microneedle and the substrate are designed into water-soluble biodegradable high polymer materials, the base material of the tip part of the drug-carrying microneedle is designed to be composed of polyethylene glycol and silk fibroin, and the microneedle array has the main effects of intensively loading the drug calcitonin on the tip part of the drug-carrying microneedle, preventing the drug from diffusing to the substrate and reducing the drug waste, and the substrate only provides mechanical support for the drug-carrying microneedle distributed on the substrate, so that the drug-carrying microneedle can be pressed and embedded in the skin when in use.

In the technical scheme of the microneedle array for the subcutaneous sustained release of calcitonin, the base materials of the bottom and the substrate of the drug-carrying microneedle are designed into water-soluble biodegradable high polymer materials, so that the drug-carrying microneedle of the microneedle array can dissolve the bottom of the drug-carrying microneedle by using the aqueous environment of the skin after penetrating into the skin to separate the tip part and the bottom of the drug-carrying microneedle and keep the tip part in the skin, and of course, the substrate remained on the surface of the skin after the tip part and the bottom of the drug-carrying microneedle are separated can be removed by means of aqueous solvent cleaning. The water-soluble biodegradable polymer material is not particularly required, and examples of the water-soluble biodegradable polymer material include hyaluronic acid, polyvinyl alcohol, polyacrylic acid, polyvinylpyrrolidone, sodium carboxymethylcellulose, and the like. The better the water solubility of the water-soluble biodegradable high polymer material is, the faster the separation speed of the tip part and the bottom of the drug-loaded microneedle is, and the drug-loaded microneedle can be selected according to the actual application requirements. For example, hyaluronic acid with the molecular weight of 10-100 kDa is selected as a water-soluble biodegradable high polymer material, which is beneficial to the rapid separation of the tip part and the bottom part of the drug-carrying microneedle.

In the technical scheme of the microneedle array for subcutaneous slow release of calcitonin, parameters such as the shape of the drug-carrying microneedles, the distance between the tip parts of the drug-carrying microneedles distributed in the array, the height of the drug-carrying microneedles and the like can be adjusted by adjusting the size of the microneedle mould according to actual application requirements. In order to ensure good mechanical strength, the drug-carrying microneedle is preferably in a regular quadrangular pyramid shape, and the ratio of the bottom side length of the drug-carrying microneedle to the height of the drug-carrying microneedle is preferably 1 (2-3). The height of the drug-carrying micro-needle is required to ensure that the skin stratum corneum is punctured without touching nerves, and the preferred height of the micro-needle is 400-800 μm in consideration of the elasticity of the skin.

In the technical scheme of the microneedle array for subcutaneous slow release of calcitonin, the calcitonin is natural calcitonin or artificially synthesized calcitonin which is currently clinically applied, and mainly comprises porcine calcitonin, human calcitonin, salmon calcitonin, eel calcitonin and the like.

The invention also provides a preparation method of the microneedle array for the subcutaneous sustained release of the calcitonin, which comprises the following steps:

(1) preparing solution

Dissolving silk fibroin and polyethylene glycol in water to form a tip base material solution, fully mixing the calcitonin aqueous solution with the tip base material solution, and removing bubbles to obtain a drug-loaded needle tip solution;

dissolving a water-soluble substrate base material in water, and removing bubbles to obtain a substrate solution;

(2) microneedle mould pretreatment

Carrying out oxygen plasma treatment on the surface of a microneedle mould to improve the surface wettability of the microneedle mould, wherein the microneedle mould comprises needle point cavities distributed in an array manner and a substrate cavity communicated with the open ends of the needle point cavities;

(3) preparation of microneedle arrays

Adding the drug-carrying needle point solution into a microneedle mould, fully centrifuging at 1-4 ℃ to enable the drug-carrying needle point solution to fill the needle point cavity, recovering the drug-carrying needle point solution which does not enter the needle point cavity, and drying and forming under the conditions of constant temperature and constant humidity to form the tip part of the drug-carrying microneedle; and then adding the substrate solution into the microneedle mould, fully centrifuging at 1-4 ℃ to enable the substrate solution to fill the microneedle mould, drying and forming under the conditions of constant temperature and constant humidity, and demoulding to obtain the microneedle array for subcutaneous slow release of calcitonin.

In the technical scheme of the preparation method, the concentration of the silk fibroin in the drug-carrying needle tip solution in the step (1) is preferably 10-20 wt.%, and the concentration of the substrate base material in the substrate solution is preferably 100-200 mg/mL.

In the technical scheme of the preparation method, in the step (3), when the drying operation is carried out, the drying temperature is 20-30 ℃, preferably 20-25 ℃, and the relative humidity is 70-85%, preferably 70-80%. The drying time is usually 2-12 h.

In the technical scheme of the preparation method, the material of the microneedle mould in the step (2) is polydimethylsiloxane, and the time for carrying out oxygen plasma treatment on the surface of the microneedle mould in the step (2) is 30-120 s. The surface of the microneedle mould is subjected to oxygen plasma treatment, so that the surface wettability of the microneedle mould can be improved, and then the drug-carrying needle point solution can enter the microneedle mould and be demoulded subsequently.

In the technical scheme of the preparation method, the centrifugal rotating speed of the centrifugal operation in the step (3) is only required to ensure that the drug-carrying needle tip solution can fill the needle tip cavity and the substrate solution can fill the microneedle mould, and the centrifugal rotating speeds adopted along with the size difference of the microneedle mould are different, and generally, the centrifugal rotating speed of 1000-5000 rpm can be adopted for centrifugation for 10-30 min.

Compared with the prior art, the technical scheme provided by the invention has the following beneficial technical effects:

1. the invention provides a microneedle array for subcutaneous slow release of calcitonin, which comprises a substrate and drug-carrying microneedles arrayed on one side of the substrate, wherein the drug-carrying microneedles are pyramid-shaped, the drug-carrying microneedles and the substrate are tightly combined before being applied to skin, and after the drug-carrying microneedles are pressed and applied to the skin, the tip parts of the drug-carrying microneedles can be quickly separated from the substrate due to the dissolution of the drug-carrying microneedles in the aqueous environment of the skin. The tip part of the drug-loaded micro-needle realizes the slow release of the loaded calcitonin in the body through the matching of biodegradable silk fibroin and polyethylene glycol. The invention can solve the problems of short half-life and long-term frequent administration requirement of the existing calcitonin preparation and the problem of gastrointestinal and skin side effects easily caused by long-term frequent administration. Meanwhile, the microneedle array provided by the invention is simple in application mode, can realize painless self-administration of a patient, and can effectively improve the compliance of the patient compared with an injection administration mode adopted by the existing calcitonin preparation.

2. The drug-carrying microneedle of the microneedle array for subcutaneous slow release of calcitonin and the substrate are made of biocompatible high polymer materials, and the preparation process of the microneedle array is all aqueous phase preparation, so that the microneedle array has good biocompatibility, no irritant substance is generated in the process of loading and releasing calcitonin, the bioactivity of the drug is not influenced, or skin inflammation is not caused, and the administration safety is high.

3. Experiments prove that the microneedle array provided by the invention has good mechanical strength, can quickly puncture the skin, realizes self-administration, and can avoid the problems of pain of patients, induration of injection parts, rubella and the like caused by long-term frequent injection administration.

4. The invention also provides a preparation method of the microneedle array for the subcutaneous slow release of the calcitonin, which has the advantages of simple operation, no need of using complex instruments, mild preparation conditions, no need of high-temperature operation and contribution to protecting the bioactivity of the loaded calcitonin.

Drawings

Fig. 1 is a schematic diagram of a preparation process of the drug-loaded microneedle array of the present invention, wherein a is a microneedle mould, b is a tip of a drug-loaded microneedle formed by adding a drug-loaded needle point solution into the microneedle mould, centrifuging, recovering the drug-loaded needle point solution which does not enter a needle point cavity, and drying and molding, c is a product formed by adding a substrate solution into the microneedle mould, centrifuging, and drying and molding, and d is the microneedle array obtained by demoulding.

Fig. 2 is an optical photograph (a, b) and a confocal laser microscope photograph (c-e) of the distribution of the drug of the microneedle array prepared in example 1.

Fig. 3 is a scanning electron microscope image of the microneedle array prepared in example 2 at different viewing angles.

Fig. 4 is a schematic diagram of a process for preparing a blank SF microneedle array, wherein a is a microneedle mold, b is a product obtained by adding an SF aqueous solution into the microneedle mold, centrifuging and drying the product, and c is the blank SF microneedle array obtained by demolding.

FIG. 5 is an optical image (a-d diagram) and a mechanical property test result (e diagram) of the microneedle array prepared in comparative examples 1 to 4.

FIG. 6 shows the results of mechanical property tests of microneedle arrays prepared in comparative example 5 and examples 2 to 4.

Fig. 7 shows the results of drug loading measurement of the microneedle array prepared in example 1.

Fig. 8 is a test result of in vitro drug release behavior of the drug-loaded microneedle arrays prepared in examples 1, 5, 6 and comparative example 6.

Fig. 9 is a photograph of an in vitro puncture of the microneedle array prepared in example 2 (a panels) and sections of the excised rat skin before and after microneedle application (b, c panels).

Detailed Description

The microneedle array for the subcutaneous sustained release of calcitonin and the preparation method thereof provided by the present invention are further illustrated by the following examples. It should be noted that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention, and those skilled in the art can make some insubstantial modifications and adjustments to the present invention based on the above disclosure and still fall within the scope of the present invention.

In the following examples and comparative examples:

the adopted micro-needle mould comprises needle point cavities distributed in an array manner and a substrate cavity communicated with the opening end of the needle point cavity, the needle point cavity is in a regular quadrangular pyramid shape, the substrate cavity is a cuboid cavity with a square bottom surface, the size of the bottom surface of the substrate cavity is 0.5cm multiplied by 0.5cm, 100 needle point cavities are distributed in the micro-needle mould in an array manner (10 multiplied by 10 matrix), and the micro-needle mould is made of Polydimethylsiloxane (PDMS).

The equipment used for the centrifugation is a plate centrifuge. The equipment adopted for oxygen plasma treatment is a plasma surface treatment instrument.

The molecular weight of Hyaluronic Acid (HA) is 10-100 kDa.

Example 1

In this example, a subcutaneous sustained-release microneedle array loaded with fluorescence-labeled salmon calcitonin (FITC-sCT) was prepared, and the preparation process thereof is shown in fig. 1, and the steps thereof are as follows:

(1) preparing solution

Dissolving Silk Fibroin (SF) in deionized water to obtain an SF solution, and dissolving polyethylene glycol (PEG) with the molecular weight of 4000 in the SF solution to obtain a tip base material solution; dissolving FITC-sCT in a proper amount of deionized water to form a FITC-sCT solution; and (3) fully mixing the FITC-sCT solution with the tip base material solution, standing to remove bubbles, and thus obtaining the drug-loaded needle tip solution. In the drug-loaded needle tip solution, the concentration of SF is 15 wt.%, the mass ratio of PEG to SF is 20:100, and the concentration of FITC-sCT is 5 mg/mL.

Dissolving HA with the molecular weight of 10-100 kDa in deionized water, standing and removing bubbles to obtain a substrate solution; the HA concentration in the base solution was 200 mg/mL.

(2) Microneedle mould pretreatment

And performing oxygen plasma treatment on the surface of the microneedle mould by using a plasma surface treatment instrument to improve the surface wettability of the microneedle mould, so that a water-based drug-loaded needle point solution and a substrate solution can enter the microneedle mould and subsequent demoulding is facilitated, and the time for the oxygen plasma treatment is 30 s.

(3) Preparation of microneedle arrays

Dripping the drug-carrying needle point solution into a microneedle mould, centrifuging at the rotation speed of 4000rpm at 4 ℃ for 20min to enable the drug-carrying needle point solution to fill the needle point cavity, recovering the drug-carrying needle point solution which does not enter the needle point cavity, and drying for 2h under the conditions of 25 ℃ and 75% of relative humidity to form the tip part of the drug-carrying microneedle; and then, dropwise adding the substrate solution into the microneedle mould, centrifuging at the rotation speed of 4000rpm for 20min at the temperature of 4 ℃ to enable the substrate solution to fill the microneedle mould, drying for 12h at the temperature of 25 ℃ and the relative humidity of 75% to realize molding, demolding to obtain the FITC-sCT-loaded subcutaneous slow-release microneedle array, and placing the array in a drying cabinet for storage for later use.

The appearance of the FITC-sCT-loaded subcutaneous sustained-release microneedle array prepared in this example was characterized, and the drug distribution in the microneedle array was observed using an optical microscope and a fluorescence confocal microscope, with the result shown in fig. 2. Fig. 2, panels a-e, show the distribution of the drug on the microneedle array, with FITC-sCT uniformly distributed within the microneedles without diffusing to the substrate.

Example 2

In this example, a salmon calcitonin (sCT) -loaded subcutaneous sustained-release microneedle array was prepared, and the operation was substantially the same as in example 1, except that the drug contained in the drug-loaded needle tip solution was sCT, and the concentration of sCT in the drug-loaded needle tip solution was 5 mg/mL.

The microneedle array prepared in this example was fixed on a sample stage, nitrogen purging was performed before and after gold spraying, the morphology was observed using a scanning electron microscope, and the relevant dimensions were measured, with the results shown in fig. 3. As can be seen from diagrams a to e of FIG. 3: 100 microneedles are arranged in an array (10 × 10 matrix) on a substrate, and each microneedle is in a solid regular quadrangular prism shape with a uniform and complete structure and a sharp needle shape. The height of the microneedle is about 450 μm, the bottom of the microneedle is square, the side length is about 230 μm, and the distance between the tips of the adjacent microneedles in the same transverse row or the same longitudinal row is about 465 μm.

Example 3

In this example, the operation of preparing the sCT-loaded subcutaneous sustained-release microneedle array is substantially the same as that in example 1, except that the drug contained in the drug-loaded needlepoint solution is sCT, and the concentration of sCT in the drug-loaded needlepoint solution is 10 mg/mL.

Example 4

In this example, the operation of preparing the sCT-loaded subcutaneous sustained-release microneedle array is substantially the same as that in example 1, except that the drug contained in the drug-loaded needlepoint solution is sCT, and the concentration of sCT in the drug-loaded needlepoint solution is 20 mg/mL.

Example 5

In the embodiment, the operation of preparing the FITC-sCT-loaded subcutaneous sustained-release microneedle array is basically the same as that in the embodiment 1, except that the mass ratio of PEG to SF in the drug-loaded needlepoint solution is 10: 100.

Example 6

In the embodiment, the operation of preparing the FITC-sCT-loaded subcutaneous sustained-release microneedle array is basically the same as that in the embodiment 1, except that the mass ratio of PEG to SF in the drug-loaded needle tip solution is 40: 100.

Comparative example 1

In this comparative example, a blank SF microneedle array was prepared, the process of which is shown in fig. 4, and the steps are as follows:

(1) preparing solution

After degumming, dissolution, dialysis and centrifugation, the silkworm cocoons are concentrated by 15% (w/w) PEG2000 and kept stand to remove air bubbles, so that an SF aqueous solution with the concentration of 5 wt.% is obtained.

(2) Microneedle mould pretreatment

(3) Preparation of blank SF microneedle array

And adding the SF aqueous solution into a microneedle mould, filling the microneedle mould, centrifuging at the rotation speed of 4000rpm for 20min at the temperature of 4 ℃, drying for 12h at the temperature of 25 ℃ and the relative humidity of 75% to realize molding, demolding to obtain a blank SF microneedle array, and placing the blank SF microneedle array in a drying cabinet for storage and standby.

Comparative example 2

In this comparative example, a blank SF microneedle array was prepared, and the operation was substantially the same as in comparative example 1 except that the concentration of SF in the SF aqueous solution was 10 wt.%.

Comparative example 3

In this comparative example, a blank SF microneedle array was prepared, and the operation was substantially the same as in comparative example 1 except that the concentration of SF in the SF aqueous solution was 15 wt.%.

Comparative example 4

In this comparative example, a blank SF microneedle array was prepared, and the operation was substantially the same as in comparative example 1 except that the concentration of SF in the SF aqueous solution was 20 wt.%.

Example 7

In this example, the molding effect and mechanical properties of the blank SF microneedle arrays prepared in comparative examples 1 to 4 were examined.

The appearance of the blank SF microneedle arrays prepared in the comparative examples 1-4 is observed by using an optical microscope, and the results are shown in a graph a-d of fig. 5, the blank SF microneedles prepared in the comparative examples 1-4 can be molded, when the concentration of the adopted SF aqueous solution is low, the hollow microneedle array is prepared, and the appearance of the microneedles is gradually changed into a solid structure along with the increase of the concentration of the SF aqueous solution.

The mechanical strength of the blank SF microneedle arrays prepared in comparative examples 1 to 4 was measured by a universal tensile testing machine. And placing the blank SF micro-needle array on a stainless steel platform in a direction that the needle point is vertically upward, vertically moving downwards at a constant speed of 10 mu m/s by using a 100N pressure sensor, applying continuous pressure to the needle point of the micro-needle array on the stainless steel platform, and automatically stopping applying pressure after the instrument reaches a preset load of 80N. And recording the compression displacement of the microneedle tip and the change of the pressure borne by the microneedle in the compression process, wherein the displacement-pressure test result is shown in a graph e of fig. 5. The blank SF microneedle array prepared in the comparative example 1 has poor mechanical properties, the possibility of needle body collapse exists, the compression force borne by the blank SF microneedle array prepared in the comparative examples 2-4 is increased along with the increase of the deformation amount of the needle point of the microneedle, the blank SF microneedle array is continuously deformed without an obvious turning point, and the mechanical properties are enhanced along with the increase of the SF concentration.

Comparative example 5

In this comparative example, a blank microneedle array was prepared by the following steps:

(1) preparing solution

Dissolving SF in deionized water to obtain SF solution, and dissolving PEG with molecular weight of 4000 in the SF solution to obtain the tip base material solution. In the solution of the base material of the tip, the concentration of SF was 15 wt.%, and the mass ratio of PEG to SF was 20: 100.

(2) Microneedle mould pretreatment

(3) Preparation of blank microneedle array

Dripping the base material solution of the tip part into a microneedle mould, centrifuging at the rotation speed of 4000rpm at 4 ℃ for 20min to enable the base material solution of the tip part to fill the cavity of the needlepoint, recovering the base material solution of the tip part which does not enter the cavity of the needlepoint, and drying for 2h under the conditions of 25 ℃ and 75% of relative humidity to form the tip part of the microneedle; and then dripping the substrate solution into the microneedle mould, centrifuging at the rotation speed of 4000rpm for 20min at the temperature of 4 ℃ to enable the substrate solution to fill the microneedle mould, drying for 12h at the temperature of 25 ℃ and the relative humidity of 75% to realize molding, demolding to obtain a blank microneedle array, and placing the blank microneedle array in a drying cabinet for storage and standby.

Example 8

In this example, the mechanical properties of the microneedle arrays prepared in comparative example 5 and examples 2 to 4 were examined.

Mechanical performance tests were performed on the microneedles prepared in examples 2-4 and comparative example 5 by a universal tensile testing machine. The microneedle arrays prepared in comparative example 5 and examples 2 to 4 were placed on a stainless steel platform with the tips facing vertically upward, and were moved vertically downward at a constant speed of 10 μm/s using 100N pressure sensors, respectively, to apply continuous pressure to the tips of the microneedle arrays on the stainless steel platform, and the instrument was stopped automatically after reaching a preset load of 80N. The compressive displacement of the needle point of the microneedle and the change of the pressure born by the microneedle in the compression process are recorded, a displacement-pressure relation graph is shown in fig. 6, the compressive force born by the four microneedles is increased along with the increase of the deformation of the needle point of the microneedle, the microneedles continuously deform without obvious turning points, and the mechanical strength of the microneedle loaded with the drug is slightly weaker than that of a blank microneedle.

Example 9

In this example, the content of FITC-sCT in the microneedle array prepared in example 1 was measured by a fluorescence spectrophotometry, which includes the following steps:

(1) and (3) standard curve determination: dissolving FITC-sCT in anhydrous formic acid, and preparing a FITC-sCT solution with the concentration of 0.1-1 mu g/mL according to a gradient proportion; and (2) determining an excitation wavelength and an emission wavelength by using a fluorescence spectrophotometer, reading fluorescence intensity under the emission wavelength after scanning under the excitation wavelength, and drawing a fluorescence-concentration standard curve according to the relation between concentration and fluorescence intensity, wherein as shown in a diagram of fig. 7, the R value is more than 0.99, and the linear relation is good.

(2) And (3) measuring the drug loading capacity: randomly taking 9 pieces of the microneedle arrays prepared in example 1, completely dissolving each microneedle array in 10mL of anhydrous formic acid, scanning at an excitation wavelength, reading fluorescence intensity at an emission wavelength, and calculating the content of FITC-sCT according to the standard curve determined in the step (1).

Example 1 in the preparation of a microneedle array, the concentration of FITC-sCT in the drug-loaded needle tip solution was 5 mg/mL. The results of drug loading measurement in this example are shown in the b-chart of fig. 7, where the average drug loading of the microneedle array was about 11.45 μ g, the drug loading variation between each microneedle array was small, and the drug loading of the microneedles was fluctuated but was stable as a whole. At present, the clinical administration amount of sCT is about 40 mug per week, and the area of the drug-loaded microneedle array prepared in example 1 is only 0.25cm ² The clinical medication requirement can be met by enlarging the area of the microneedle array.

Comparative example 6

In this comparative example, a PEG-free fluorescence-labeled salmon calcitonin-loaded microneedle array was prepared in substantially the same operation as in example 1, except that no PEG was added to the drug-loaded needle tip solution.

Example 10

In this example, the in vitro drug release curves of the microneedle arrays prepared in examples 1, 5, and 6 and comparative example 6 were measured by fluorescence spectrophotometry, and the procedure was as follows:

(1) and (3) standard curve determination: dissolving FITC-sCT in a PBS buffer solution, proportioning FITC-sCT solution with gradient concentration within the range of 0.1-0.5 mu g/mL, determining excitation wavelength and emission wavelength by using a fluorescence spectrophotometer, scanning at the excitation wavelength, reading fluorescence intensity at the emission wavelength, and drawing a fluorescence-concentration standard curve according to the relation between the concentration and the fluorescence intensity, wherein as shown in a diagram of figure 8, the R value is greater than 0.99, and the linear relation is good.

(2) In vitro drug release: PBS buffer was used to mimic the release of sCT in body fluids. The microneedle arrays prepared in examples 1, 5, 6 and comparative example 6 were respectively immersed in 10mL of PBS buffer (pH 7.4,37 ℃) and shaken at 50rpm, four samples were set for each set, 1mL of the solution was taken out at intervals and then fresh PBS buffer preheated to 37 ℃ was added, and then the in vitro release of the drug loaded in the microneedle arrays was measured by fluorescence spectroscopy.

In vitro drug release curves of the microneedle arrays prepared in examples 1, 5, and 6 and comparative example 6 are shown in the b-diagram of fig. 8, and for the microneedle array prepared in comparative example 6, as the sCT is dissolved, the acting force is gradually reduced to accelerate the dissolution rate of the sCT. The rate of degradation of the microneedles can be slowed down by mixing PEG into the base material at the tips of the microneedles, so that the burst release of sCT is reduced, for example, the microneedle arrays prepared in examples 1, 5 and 6 release at a constant speed within 7 days, and the release amount of FITC-sCT in the first 3 days is reduced from 57.75% to 37.19% along with the increase of the added amount of PEG. In vitro release experiment results prove that SF and PEG are matched in a proper proportion to be used as base materials of the tip part of the microneedle together, the release rate of sCT can be slowed down, the controllable adjustment of the release rate of sCT is realized along with the change of the proportional relation of PEG and SF, and the microneedle array provided by the invention has the capability of long-acting slow release of sCT.

Example 11

In this example, the capability of the microneedle array prepared in example 2 to penetrate the ex vivo skin was studied.

The microneedle array prepared in example 2 was inserted into the skin of SD rats ex vivo, held for 30 seconds by pressing, and removed. The puncture point of the microneedle array was coated with trypan blue (trypan blue) solution, the solution was stained for 1min and washed away with physiological saline, and the SD rat excised skin was observed with a microscope to calculate the microneedle puncture rate. Then, the microtome was used to cut the microtome into thin sections of 25 to 30 μm, the sections were placed on a glass slide, and the depth of penetration and deformation of the microneedles were observed under a high power microscope, and the results are shown in FIG. 9.

From the dyeing results shown in the a-graph of fig. 9, it can be seen that the microneedle penetration rate of the microneedle array prepared in example 2 was > 95%. Fig. 9 b and c show frozen tissue sections of the microneedle array before and after the penetration into the rat, and it can be seen from comparison of fig. 9 b and c that the microneedles successfully penetrated the skin to a depth of about 250 ± 50 μm, which is sufficient to penetrate the stratum corneum layer (stratum corneum layer thickness is 10-20 μm) of the skin to reach the dermis layer. The depth of penetration of the microneedles is less than the height of the microneedles (450 μm) due to the inability of the microneedles to fully penetrate the tissue interior due to skin elasticity and deformation of the tips of the microneedles during compression.

Example 12

In this example, an eel calcitonin (ecct) -loaded subcutaneous sustained-release microneedle array was prepared, including the following steps:

(1) preparing solution

Dissolving SF in deionized water to obtain SF solution, and dissolving PEG with molecular weight of 20000 in the SF solution to obtain tip matrix material solution; dissolving eCT in proper amount of deionized water to form eCT solution; and fully mixing the eCT solution with the base material solution of the tip part, standing to remove bubbles, and thus obtaining the drug-loaded needle tip solution. In the drug-loaded needle tip solution, the concentration of SF is 20 wt.%, the mass ratio of PEG to SF is 10:100, and the concentration of eCT is 15 mg/mL.

Dissolving polyvinyl alcohol in deionized water, standing to remove bubbles to obtain a substrate solution; the concentration of polyvinyl alcohol in the base solution was 100 mg/mL.

(2) Microneedle mould pretreatment

And carrying out oxygen plasma treatment on the surface of the microneedle mould by using a plasma surface treatment instrument so as to improve the surface wettability of the microneedle mould, and be beneficial to the entry of aqueous medicine-carrying needle point solution and substrate solution into the microneedle mould and the subsequent demoulding, wherein the time of the oxygen plasma treatment is 30 s.

(3) Preparation of microneedle arrays

Dripping the drug-carrying needle point solution into a microneedle mould, centrifuging at the rotation speed of 5000rpm at 4 ℃ for 10min to enable the drug-carrying needle point solution to fill the needle point cavity, recovering the drug-carrying needle point solution which does not enter the needle point cavity, and drying for 2h under the conditions of 30 ℃ and 85% of relative humidity to form the tip part of the drug-carrying microneedle; and then dripping the substrate solution into the microneedle mould, centrifuging at the rotation speed of 5000rpm at 4 ℃ for 10min to enable the substrate solution to fill the microneedle mould, drying at the temperature of 30 ℃ for 12h under the condition that the relative humidity is 85%, and then realizing molding and demoulding to obtain the subcutaneous sustained-release microneedle array loaded with the eCT.

Example 13

In this example, an ept-loaded subcutaneous sustained release microneedle array was prepared by the following steps:

(1) preparing solution

Dissolving SF in deionized water to obtain an SF solution, and dissolving PEG with the molecular weight of 800 in the SF solution to obtain a tip base material solution; dissolving eCT in proper amount of deionized water to form eCT solution; and fully mixing the eCT solution with the base material solution of the tip part, standing to remove bubbles, and thus obtaining the drug-loaded needle tip solution. In the drug-loaded needle tip solution, the concentration of SF is 10 wt.%, the mass ratio of PEG to SF is 40:100, and the concentration of eCT is 10 mg/mL.

Dissolving polyacrylic acid in deionized water, standing to remove bubbles to obtain a substrate solution; the concentration of polyvinyl alcohol in the base solution was 150 mg/mL.

(2) Microneedle mould pretreatment

And performing oxygen plasma treatment on the surface of the microneedle mould by using a plasma surface treatment instrument to improve the surface wettability of the microneedle mould, so that a water-based drug-loaded needle point solution and a substrate solution can enter the microneedle mould and subsequent demoulding is facilitated, and the time of the oxygen plasma treatment is 60 s.

(3) Preparation of microneedle arrays

Dripping the drug-carrying needle point solution into a microneedle mould, centrifuging at the rotation speed of 1000rpm at 4 ℃ for 30min to enable the drug-carrying needle point solution to fill the needle point cavity, recovering the drug-carrying needle point solution which does not enter the needle point cavity, and drying for 4h at the temperature of 20 ℃ and the relative humidity of 70%, so as to form the tip part of the drug-carrying microneedle; and then, dripping a substrate solution into the microneedle mould, centrifuging at the rotating speed of 1000rpm for 30min at the temperature of 4 ℃ to enable the substrate solution to fill the microneedle mould, drying for 12h at the temperature of 20 ℃ and the relative humidity of 70% to realize molding, and demolding to obtain the subcutaneous slow-release microneedle array loaded with the eCT.

Claims

1. The micro-needle array for the subcutaneous sustained release of calcitonin is characterized by consisting of a substrate and drug-carrying micro-needles distributed on one side of the substrate in an array manner, wherein the drug-carrying micro-needles are pyramid-shaped and consist of a tip part and a bottom part, the tip part is loaded with calcitonin, the base material of the tip part is different from that of the bottom part, and the base material of the bottom part is the same as that of the substrate; the base materials of the bottom and the substrate are water-soluble biodegradable high polymer materials, and the base material of the tip part is composed of polyethylene glycol and silk fibroin; after the microneedle array is administrated through the skin, the bottom of the drug-carrying microneedle is dissolved firstly, the tip part of the drug-carrying microneedle is separated from the substrate and is reserved in the skin, and the calcitonin loaded on the tip part is slowly released along with the swelling and biodegradation of the base material of the tip part of the drug-carrying microneedle.

2. The microneedle array for the subcutaneous sustained release of calcitonin according to claim 1, wherein the base material of the tip portion is composed of polyethylene glycol and silk fibroin in a mass ratio of (10-40): 100.

3. The microneedle array for the subcutaneous sustained release of calcitonin according to claim 1, wherein a molecular weight of the polyethylene glycol is 800 to 20000.

4. The microneedle array for the subcutaneous sustained release of calcitonin according to claim 1, wherein the water-soluble biodegradable high molecular material is hyaluronic acid, polyvinyl alcohol, polyacrylic acid, polyvinylpyrrolidone or sodium carboxymethylcellulose.

5. The microneedle array for the subcutaneous sustained release of calcitonin according to any one of claims 1 to 4, wherein the drug-loaded microneedles are in the shape of a regular quadrangular pyramid.

6. The microneedle array for the subcutaneous sustained release of calcitonin according to claim 5, wherein the ratio of the bottom surface side length of the drug-loaded microneedle to the height of the drug-loaded microneedle is 1 (2-3).

7. The microneedle array for the subcutaneous sustained release of calcitonin according to claim 1, wherein the height of the drug-loaded microneedle is 400 to 800 μm.

8. A method of preparing a microneedle array for the subcutaneous sustained release of calcitonin according to any one of claims 1 to 7, comprising the steps of:

(1) preparing solution

(2) microneedle mould pretreatment

(3) preparation of microneedle arrays

9. The method for preparing a microneedle array for the subcutaneous slow release of calcitonin according to claim 8, wherein the concentration of silk fibroin in the drug-loaded needle tip solution in step (1) is 10 wt.% to 20 wt.%, and the concentration of the substrate base material in the substrate solution is 100mg/mL to 200 mg/mL.

10. A method of preparing a microneedle array for the subcutaneous sustained release of calcitonin according to claim 8 or 9, wherein in the step (3), the drying temperature is 20 to 30 ℃ and the relative humidity is 70 to 85% during the drying operation.