Temperature-stable double-emulsion drug-loaded microspheres and their preparation method and application

Abstract

A temperature-stable double-emulsion droplet drug-loaded microsphere, and its preparation method and application. The present invention belongs to the field of biomedicine technology. The purpose of the present invention is to solve the technical problem that current drug-loaded microspheres are difficult to store for a long time and have poor temperature stability. The method of the present invention comprises: dissolving the drug in deionized water to obtain an inner phase, adding PDMS and its curing agent to white oil to obtain an intermediate phase; dissolving Tween 80 in deionized water to obtain an outer phase; then generating double-emulsion droplets using microfluidics technology; and reintroducing the double-emulsion droplets into the outer phase and curing at room temperature to obtain drug-loaded microspheres. The drug-loaded microspheres of the present invention are mixed with a storage solution composed of propylene glycol, gum arabic, and deionized water and then used as an embolic agent. The embolic agent of the present invention has excellent temperature stability, fluidity, embolic properties, and long-term stable storage. It is also highly versatile, and the effective active ingredients can be replaced and adjusted as needed.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and in particular relates to a temperature-stable double-emulsion droplet drug-loaded microsphere, a preparation method thereof, and an application thereof. Background Art

[0002] Liver cancer is the sixth most common cancer worldwide. Arterial chemoembolization (ARTE) blocks tumor blood vessels while simultaneously delivering active drug ingredients, allowing them to reside within the tumor vessels for a long time, disrupting the tumor's blood supply and reducing the drug's systemic effects. This treatment requires the use of embolic agents, but current embolic agents generally struggle to achieve both fluidity and embolic properties.

[0003] At the same time, the preparation methods of double-emulsion droplet microspheres generally face the problem of temperature and long-term storage stability. Over time and with changes in external temperature, the microspheres will fuse and coalesce, or even break and delaminate. The resulting double-emulsion droplet microspheres are generally not thermodynamically stable and difficult to store for a long time. Therefore, it is of great significance to develop double-emulsion droplet microspheres that combine fluidity, embolic resistance, and temperature stability. Summary of the Invention

[0004] The purpose of the present invention is to solve the technical problems that the current drug-loaded microspheres are difficult to store for a long time and have poor temperature stability, and to provide double-emulsion droplet drug-loaded microspheres with temperature stability and their preparation method and application.

[0005] The technical solutions of the present invention are as follows:

[0006] One of the objectives of the present invention is to provide a method for preparing temperature-stable double-emulsion drug-loaded microspheres, which is carried out by the following steps:

[0007] Step 1: Add the drug to deionized water to obtain the inner phase solution, add PDMS and its curing agent to white oil to obtain the middle phase solution; dissolve Tween 80 in deionized water to obtain the outer phase solution;

[0008] Step 2: Fill each channel of the microfluidic glass capillary microfluidic chip with deionized water, and then inject the inner phase solution, the middle phase solution, and the outer phase solution into the corresponding inlets of the glass capillary microfluidic chip to generate double emulsion droplets;

[0009] Step 3: The double emulsion droplets are reintroduced into the external phase solution described in step 1 and solidified at room temperature to obtain temperature-stable double emulsion droplet drug-loaded microspheres.

[0010] It is further defined that the concentration of the inner phase solution in step 1 is 0.6-1 mg / mL, the mass ratio of PDMS, curing agent and white oil in the intermediate phase solution is (3-5):1:(5-7), and the concentration of Tween 80 in the outer phase solution is 0.5-1.5 wt%.

[0011] It is further defined that in step 2, the injection flow rate of the inner phase solution is 500-900 μL / h, the injection flow rate of the intermediate phase solution is 500-700 μL / h, and the injection flow rate of the outer phase solution is 25-30 mL / h.

[0012] It is further defined that in step 3, the curing temperature is 20-37° C. and the curing time is 24-48 hours.

[0013] The second object of the present invention is to provide a temperature-stable double-emulsion drug-loaded microsphere prepared by the above method, wherein the size of the microsphere is 150-200 μm.

[0014] The third object of the present invention is to provide a storage solution for temperature-stable double-emulsion drug-loaded microspheres prepared by the above method, wherein the storage solution is composed of propylene glycol, gum arabic and deionized water.

[0015] It is further defined that the concentration of propylene glycol in the storage solution is 1-3 wt %, and the concentration of gum arabic is 0.5-1 wt %.

[0016] A fourth object of the present invention is to provide a method for preparing the above-mentioned storage solution, wherein:

[0017] Gum arabic was dissolved in deionized water, and then gum arabic was added while magnetically stirring to obtain a stock solution.

[0018] A fifth object of the present invention is to provide a method for using the drug-loaded microspheres mixed with the storage solution as an embolic agent.

[0019] A sixth object of the present invention is to provide a liver vascular embolic agent having both fluidity and embolic properties, wherein the embolic agent comprises the above-mentioned drug-loaded microspheres and the above-mentioned storage solution.

[0020] It is further defined that the active drug in the drug-loaded microspheres is a microalgae suspension at 3-5 mg / mL.

[0021] Compared with the prior art, the present invention has the following advantages:

[0022] (1) The double-emulsion drug-loaded microspheres prepared by the present invention have excellent temperature storage stability when used as embolic agents and can adapt to changes in external temperature from -8°C to 40°C.

[0023] (2) When the double-emulsion drug-loaded microspheres prepared by the present invention are used as embolic agents, both fluidity and embolic properties are taken into consideration.

[0024] (3) The double-emulsion drug-loaded microspheres prepared by the present invention can be stored stably for a long time for more than two months.

[0025] (4) The preparation method of the double emulsion droplet embolic agent of the present invention has high throughput and high uniformity.

[0026] (5) The double-emulsion drug-loaded microsphere embolic agent of the present invention is universal, and the type and concentration of the active ingredients in the core can be changed and adjusted as needed. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Schematic diagram of the structure of the microfluidic glass capillary microfluidic chip of the present invention;

[0028] Figure 2 This is an optical morphology image of a double emulsion droplet encapsulating collagen peptides according to the present invention;

[0029] Figure 3 This is the optical morphology of the double-emulsion droplet drug-loaded microsphere embolic preparation prepared in Example 1;

[0030] Figure 4 This is the particle size distribution diagram of the double-emulsion drug-loaded microsphere embolic preparation prepared in Example 1;

[0031] Figure 5 This is an optical morphology image of the double-emulsion drug-loaded microsphere embolic agent prepared in Example 1 after heat treatment;

[0032] Figure 6 This is an optical morphology image of the double-emulsion drug-loaded microsphere embolic agent prepared in Example 1 after refrigeration treatment;

[0033] Figure 7 This is the optical morphology of the double-emulsion drug-loaded microsphere embolic preparation prepared in Example 1 after long-term storage;

[0034] Figure 8 This is the optical morphology of the double-emulsion drug-loaded microsphere embolic preparation prepared in Example 1 after osmotic pressure treatment;

[0035] Figure 9 Schematic diagram of a device for using the double-emulsion drug-loaded microsphere embolic agent prepared in Example 1 as a vascular embolic agent;

[0036] Figure 10 This is an optical morphology image of the double-emulsion droplet drug-loaded microsphere embolic agent prepared in Example 1 blocking the microchannel. DETAILED DESCRIPTION

[0037] The present invention provides a specific embodiment for preparing a liver vascular embolic agent having both fluidity and embolic properties based on temperature-stable double-emulsion drug-loaded microspheres. The specific process is as follows:

[0038] (1) Dilute the microalgae suspension with a concentration of 3-5 mg / mL with deionized water 5 times and mix well to obtain an internal phase solution;

[0039] (2) PDMS, curing agent, and white oil (viscosity 10 cs) were stirred and mixed in a mass ratio of (3-5):1:(5-7), and after mixing, vacuum treatment was performed to remove bubbles to obtain an intermediate phase solution;

[0040] (3) Tween 80 was dissolved in deionized water, and then impurities were removed using a 0.45 μm filter to obtain an external phase with a concentration of 0.5-1.5 wt%;

[0041] (4) Each channel of the microfluidic glass capillary microfluidic chip used for double emulsion droplet preparation (structure as Figure 1 The inner portion of the water phase channel is filled with deionized water; the glass capillary tube used in the inner water phase channel is circular, with an inner diameter of 300 μm, an outer diameter of 400 μm, and a channel diameter of 50 μm; the glass capillary tube used in the middle oil phase channel is circular, with an inner diameter of 580 μm, an outer diameter of 1000 μm, and a channel diameter of 90 μm; the glass capillary tube used in the outer phase channel is square, with an inner diameter of 1050 μm; the collection channel has a diameter of 240 μm; the glass capillaries are made of borosilicate glass;

[0042] (5) injecting the three-phase solutions obtained in steps (1) to (3) into the corresponding inlets of the glass capillary microfluidic chip in step (4) to generate double emulsion droplets; wherein the inner aqueous phase injection flow rate is 500-900 μL / h, the middle oil phase injection flow rate is 500-700 μL / h, and the outer phase injection flow rate is 25-30 mL / h;

[0043] (6) re-introducing the double emulsion droplets obtained in step (5) into the external phase solution described in step (3) and curing at 20-37° C. for 24-48 hours to obtain temperature-stable double emulsion droplet drug-loaded microspheres;

[0044] (7) dissolving gum arabic in deionized water, and then adding gum arabic while magnetically stirring to obtain a storage solution; wherein the concentration of propylene glycol in the storage solution is 1-3 wt %, the concentration of gum arabic is 0.5-1 wt %, the rotation speed is 400-600 rpm, and the time is 10-20 min;

[0045] (8) Add the microspheres obtained in step (6) to the storage solution in step (7) to obtain an embolic agent with a concentration of 20-30 wt%.

[0046] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0047] The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, methods, and instruments used are conventional in the art and can be obtained commercially by those skilled in the art unless otherwise specified.

[0048] As used in the following examples, the terms "comprising," "including," "having," "containing," or any other variations thereof, are intended to cover a non-exclusive inclusion. For example, a composition, process, method, article, or apparatus that comprises the listed elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, process, method, article, or apparatus.

[0049] When amount, concentration or other value or parameter are represented with the range of scope, preferred range or a series of upper preferred value and lower preferred value limit, this should be understood as specifically disclosing all ranges formed by any pairing of any range upper limit or preferred value and any range lower limit or preferred value, and no matter whether this scope is disclosed separately.For example, when disclosing scope "1 to 5", described scope should be interpreted as including scope "1 to 4", "1 to 3", "1 to 2", "1 to 2 and 4 to 5", "1 to 3 and 5" etc.When numerical range is described in this article, unless otherwise stated, otherwise this scope is intended to include its end value and all integers and fractions within the scope.In this application specification and claims, range limitation can be combined and / or interchanged, and if these ranges are not otherwise stated, include all subranges contained therein.

[0050] The endpoints of the ranges and any values ​​disclosed in the present invention are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be considered to be specifically disclosed herein.

[0051] Example 1: The preparation process of the liver vascular embolic agent with both fluidity and embolic properties based on temperature-stable double-emulsion drug-loaded microspheres is as follows:

[0052] (1) Dilute a 4 mg / mL microalgae suspension 5 times with deionized water, wherein the microalgae is Chlorella polysaccharide, and mix well to obtain an internal phase solution;

[0053] (2) PDMS, curing agent (Dow Corning SYLGARD 184) and white oil (Kaswe lubricating oil, viscosity 10 cs) were stirred and mixed in a mass ratio of 4:1:6, and then vacuum treated to remove bubbles to obtain an intermediate phase solution;

[0054] (3) Tween 80 was dissolved in deionized water, and then impurities were removed using a 0.45 μm filter to obtain an external phase with a concentration of 1 wt%;

[0055] (4) Each channel of the microfluidic glass capillary microfluidic chip used for double emulsion droplet preparation (structure as Figure 1 (as shown) the interior is filled with deionized water;

[0056] (5) injecting the three-phase solutions obtained in steps (1) to (3) into the corresponding inlets of the glass capillary microfluidic chip in step (4) to generate double emulsion droplets; wherein the inner aqueous phase injection flow rate is 800 μL / h, the middle oil phase injection flow rate is 500 μL / h, and the outer phase injection flow rate is 25 mL / h;

[0057] (6) The double emulsion droplets obtained in step (5) were reintroduced into the external phase solution described in step (3) and solidified at 23° C. for 24 h to obtain double emulsion droplet drug-loaded microspheres with a particle size of 150-200 μm;

[0058] (7) dissolving gum arabic in deionized water, and then adding gum arabic while magnetically stirring to obtain a storage solution; wherein the concentration of propylene glycol in the storage solution is 2 wt %, the concentration of gum arabic is 0.6 wt %, the rotation speed is 500 rpm, and the stirring time is 15 min;

[0059] (8) The microspheres obtained in step (6) are added to the storage solution in step (7) to obtain an embolic agent with a concentration of 25 wt %.

[0060] like Figure 2 As shown, in the process of generating drug-loaded microspheres in step (6) of Example 1, the droplet size is uniform and the throughput is high, and the production effect is high.

[0061] like Figure 3 As shown, this is the optical morphology of the double-emulsion drug-loaded microspheres obtained in step (6) of Example 1 under a microscope. The size of the Clostridium butyricum double microcapsules prepared under this condition is uniform.

[0062] like Figure 4 As shown, it is a size distribution diagram of the double-emulsion droplet drug-loaded microspheres obtained in step (6) of Example 1. It can be seen that the size of the prepared double-emulsion droplet drug-loaded microspheres is 150-200 μm.

[0063] Test example 1: High temperature stability test.

[0064] The double-emulsion drug-loaded microsphere embolic agent sample prepared in Example 1 was placed in a metal bath at 40° C. and heated at a constant temperature. The sample was taken out after 24 hours and observed to see if oil-water separation occurred.

[0065] like Figure 5 As shown in FIG, the microsphere embolic agent is subjected to a microscopic morphology test after being subjected to a heating temperature stability test. It is found that the drug-loaded microsphere embolic agent does not break or fuse, and has a certain high-temperature stability.

[0066] Test example 2: low temperature stability test.

[0067] The double-emulsion drug-loaded microsphere embolic agent sample prepared in Example 1 was placed in a constant temperature refrigeration environment at -8°C and taken out after 24 hours to observe whether oil-water separation occurred and whether the embolic agent solution was frozen.

[0068] like Figure 6 As shown in FIG, the microsphere embolic agent is subjected to a refrigeration temperature stability test under a microscope. It is found that the drug-loaded microsphere embolic agent does not break or fuse, and has a certain low-temperature stability.

[0069] Test Example 3: Long-term storage stability test.

[0070] The double-emulsion droplet drug-loaded microsphere embolic preparation prepared in Example 1 was stored at room temperature. After 2 months, the sample was observed under a microscope to evaluate its morphology to determine whether the drug-loaded microsphere embolic preparation had the ability to be stored stably for a long time.

[0071] like Figure 7 As shown in FIG, the microscopic morphology of the microsphere embolic agent after long-term storage stability testing, it was found that the drug-loaded microsphere embolic agent did not break or fuse, and had a storage stability of at least 2 months.

[0072] Test Example 4: Internal phase release test.

[0073] The double-emulsion drug-loaded microsphere embolic agent sample prepared in Example 1 was placed in a 1 mol / L KCl solution, and the drug-loaded microsphere embolic agent was observed under a microscope after 10 minutes to observe its morphology.

[0074] like Figure 8 As shown in FIG, the microscopic morphology of the microsphere embolic agent after osmotic pressure treatment is shown. It is found that the drug in the inner core of the drug-loaded microsphere embolic agent is released, leaving only the outer oil shell that loses water and shrinks. The appropriate proportion of PDMS makes the oil shell have sufficient toughness.

[0075] Test example 5: embolic test.

[0076] The double emulsion droplet drug-loaded microsphere embolic agent prepared in Example 1 was injected into a PDMS microfluidic chip simulating tumor blood vessels for embolization (the schematic diagram of the experimental device is shown in FIG. Figure 9 The embolic properties of drug-loaded microspheres as embolic agents were evaluated based on the pressure changes in the microchannels before and after embolization. At the same time, the pressure monitoring chip was able to prevent the embolic chip from being damaged by excessive pressure from continuously flowing into it after it was blocked.

[0077] like Figure 10 As shown, after the drug-loaded microsphere embolic agent is injected into the embolized chip, the microspheres are mechanically squeezed and deformed in the narrow channel with a height of 50μm, releasing the internal aqueous phase, and at the same time blocking the microchannel to achieve embolization and drug delivery. The chip channel is blocked. The phenomenon shows that the pressure rises due to the blockage of the channel, resulting in the failure of the blue dye solution to pass through the channel, proving the embolic property of the drug-loaded microspheres as embolic agents.

[0078] The foregoing are merely preferred embodiments of the present invention. These embodiments are all different implementations based on the overall concept of the present invention. The scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by a person skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be based on the scope of protection of the claims.

Claims

1. A method for preparing temperature-stable double-emulsion drug-loaded microspheres, characterized in that: The method: Step 1: Add the drug to deionized water to obtain an inner phase solution, add PDMS and its curing agent to white oil to obtain an intermediate phase solution; dissolve Tween 80 in deionized water to obtain an outer phase solution; the concentration of the inner phase solution is 0.6-1 mg / mL, the mass ratio of PDMS, curing agent and white oil in the intermediate phase solution is (3-5):1:(5-7), and the concentration of Tween 80 in the outer phase solution is 0.5-1.5wt%; Step 2: Fill each channel of the microfluidic glass capillary microfluidic chip with deionized water, and then inject the inner phase solution, the middle phase solution, and the outer phase solution into the corresponding inlets of the glass capillary microfluidic chip to generate double emulsion droplets; Step 3: The double emulsion droplets are reintroduced into the external phase solution described in step 1 and solidified at room temperature. The solidification temperature is 20-37° C. and the solidification time is 24-48 h to obtain temperature-stable double emulsion droplet drug-loaded microspheres.

2. The method according to claim 1, characterized in that In step 2, the injection flow rate of the inner phase solution is 500-900 μL / h, the injection flow rate of the intermediate phase solution is 500-700 μL / h, and the injection flow rate of the outer phase solution is 25-30 mL / h.

3. The temperature-stable double-emulsion drug-loaded microspheres prepared by the method according to any one of claims 1 to 2, characterized in that: The size of the microspheres is 150-200 μm.

4. Use of the drug-loaded microspheres according to claim 3 as embolic agents, characterized in that: The drug-loaded microspheres are mixed with a storage solution and used as an embolic agent. The storage solution consists of propylene glycol, gum arabic and deionized water.

5. The use according to claim 4, characterized in that The concentration of propylene glycol in the storage solution is 1-3 wt %, and the concentration of gum arabic is 0.5-1 wt %.

6. A liver vascular embolic agent having both fluidity and embolic properties, characterized in that: The embolic agent comprises the drug-loaded microspheres according to claim 3 and the storage solution according to claim 4, wherein the active drug in the drug-loaded microspheres is a microalgae suspension at a concentration of 3-5 mg / mL.

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