A gelatin sponge microsphere product and preparation method thereof

Abstract

The present invention relates to the field of material technology, and in particular to a gelatin sponge microsphere product and a preparation method thereof. The present invention dissolves gelatin in deionized water to obtain a dispersed phase solution; mixes vegetable oil and a surfactant to obtain a continuous phase solution; mixes vegetable oil and a surfactant to obtain a receiving phase solution; injects the dispersed phase solution and the continuous phase solution into a microfluidic device respectively to obtain gelatin droplets; introduces the gelatin droplets into the receiving phase solution, cools, and solidifies to obtain a crude product; adds a first cross-linking solution to the crude product for preliminary cross-linking and fixation, removes the upper solution, and then adds a second cross-linking solution for further cross-linking and fixation to obtain a semi-finished microsphere product; removes the upper solution from the semi-finished microsphere product and washes it. The present invention uses a cross-linking solution containing alcohol to cross-link the microspheres, so that the cross-linking agent and the microspheres quickly contact and cross-link; it is conducive to the preparation of small-sized droplets, and can also maintain the droplet morphology, so that the surface of the cross-linked microspheres is smooth and wrinkle-free.

Description

Technical Field

[0001] The present invention relates to the field of material technology, in particular to a gelatin sponge microsphere product and a preparation method thereof. Background Art

[0002] Knee osteoarthritis (KOA) is a common chronic pain disorder with a very high incidence rate, making it the most common painful disease among middle-aged and elderly people. Currently, early treatment for KOA patients mainly includes oral medication, physical therapy, and intra-articular hyaluronic acid injections. However, the effects are often insignificant, and patients with severe or advanced KOA require surgical treatment, mainly including arthroscopic debridement and knee replacement. In 2015, Okuno et al. first reported the use of transcatheter genicular artery embolization (GAE) to relieve knee pain in patients with mild to moderate KOA, achieving good clinical results. Genicular artery embolization is a minimally invasive intra-arterial interventional method with the advantages of less trauma, high precision, and rapid onset of action, and can effectively relieve knee pain.

[0003] Hemoptysis refers to a clinical emergency in which the daily bleeding volume exceeds 500mL or more than 200mL each time. Due to the complex causes of hemoptysis, medical treatment is difficult to be effective in some patients. In 1974, French scholar Remy first reported the successful use of selective bronchial artery embolization (BAE) to treat massive hemoptysis. BAE is a minimally invasive interventional treatment method in which embolic material is selectively injected into a certain bronchial artery through a catheter to block the blood flow of the corresponding blood vessel, thereby controlling bleeding. This technology has the following significant advantages: precise control, using imaging guidance to accurately locate the lesion area and reduce damage to healthy tissue; minimally invasive treatment, only a rice-sized needle hole is needed to puncture the thigh root, and recovery is fast after surgery; treatment of various lung problems, in addition to stopping bleeding, it can also be used for recurrent bleeding of lung cancer and inhibiting tumor growth.

[0004] Lower gastrointestinal bleeding (LGIB) accounts for approximately 20% of acute gastrointestinal bleeding events, with an estimated incidence of 33–87 per 100,000 persons, and is defined as bleeding originating distal to the duodenojejunal junction (including the small intestine, colon, rectum, and anus). Although LGIB tends to resolve spontaneously and presents with milder clinical presentation than upper gastrointestinal bleeding (UGIB), mortality rates are closely related to comorbidities and range from 3% to 18%. Current guidelines recommend urgent CT angiography to localize the bleeding site, followed by endoscopic or interventional therapy, in hemodynamically unstable patients with suspected ongoing LGIB. Selective transarterial embolization (TAE) has emerged as an effective and well-tolerated treatment option for patients with LGIB, and current literature emphasizes its safety.

[0005] However, the embolic microspheres currently used for interventional treatment of these clinical symptoms are generally non-degradable, meaning they must remain permanently in the patient's body. Foreign matter residue can often lead to complications, and microspheres can also cause ectopic embolism during the embolization process. Gelatin, an animal protein composed of multiple amino acids, exhibits excellent water absorption and plasticity, good tissue compatibility, easy material collection, and in vivo biodegradability. Embolic microspheres made from gelatin are a novel peripheral vascular embolic agent developed in recent years.

[0006] Existing methods for preparing embolic microspheres often pre-freeze the microspheres using a freezing technique, then wash them with an oil solution, crosslink them with a crosslinking solution, and finally wash them to remove the crosslinker. This freezing, washing, crosslinking, and finally washing method has a major technical disadvantage: gelatin microspheres easily swell and enlarge in the crosslinking solution, resulting in larger microspheres, making it difficult to prepare small-diameter microspheres. Summary of the Invention

[0007] In view of this, the technical problem to be solved by the present invention is to provide a method for preparing gelatin sponge microspheres. The gelatin sponge microspheres prepared by the preparation method provided by the present invention have a smooth surface without wrinkles and are uniformly cross-linked.

[0008] The present invention provides a method for preparing a gelatin sponge microsphere product, comprising the following steps:

[0009] A) dissolving gelatin in deionized water to obtain a dispersed phase solution; mixing vegetable oil and a surfactant to obtain a continuous phase solution; and mixing the vegetable oil and the surfactant to obtain a receiving phase solution;

[0010] B) injecting the dispersed phase solution and the continuous phase solution into the microfluidic device separately to obtain gelatin droplets;

[0011] C) introducing the gelatin droplets into the receiving phase solution, cooling, and solidifying to obtain a crude gelatin sponge microsphere product;

[0012] D) adding the crude gelatin sponge microspheres to a first crosslinking solution for preliminary crosslinking and fixation, removing the upper solution, and then adding a second crosslinking solution for further crosslinking and fixation to obtain semi-finished gelatin sponge microspheres;

[0013] E) removing the upper layer solution from the semi-finished gelatin sponge microspheres and washing them to obtain a gelatin sponge microsphere product.

[0014] In some specific embodiments, the gelatin dissolving temperature in step A) is 35-70° C., the gelatin freezing strength is 100-300 bloom, the mass fraction of gelatin in the dispersed phase solution is 1%-50%, and the stirring time is 0.5-2 h.

[0015] In some specific embodiments, the vegetable oil in step A) includes one or more of soybean oil, peanut oil, and corn oil; the surfactant is one or more of Span80, Span85, and PGPR, and the mass fraction of the surfactant is 0.5% to 10%;

[0016] In the continuous phase solution, the mass ratio of vegetable oil to surfactant is 100:(1-10);

[0017] In the receiving phase solution, the mass ratio of vegetable oil to surfactant is 100:(0.1-2).

[0018] In some specific embodiments, in step B), the dispersed phase pressure is 0.1 kPa to 500 kPa, and the continuous phase pressure is 0.05 MPa to 1 MPa.

[0019] In some specific embodiments, in step D), the first cross-linking solution comprises water, a cross-linking agent, and an organic solvent, wherein the cross-linking agent is an aldehyde organic compound; the organic solvent is one of n-octanol, n-butanol, propylene glycol, isopropanol, or acetone solution; the volume fraction of the cross-linking agent in the first cross-linking solution is 0.1% to 1%, and the volume concentration of the organic solvent solution is 25% to 50%; and the time for the initial cross-linking fixation is 10 min to 60 min.

[0020] In some specific embodiments, in step D), the second cross-linking solution comprises water, a cross-linking agent, and an organic solvent, wherein the cross-linking agent is an aldehyde organic compound; the organic solvent is one of n-octanol, n-butanol, propylene glycol, isopropanol, or an acetone solution; the volume fraction of the cross-linking agent in the second cross-linking solution is 0.1% to 10%, and the volume concentration of the organic solvent solution is 25% to 50%; and the time for further cross-linking and fixation is 10 minutes to 2 hours.

[0021] In some specific embodiments, the cooling temperature in step C) is preferably <30°C; more preferably 0-8°C. In some specific embodiments, the cleaning in step E) is performed by cleaning with a detergent and then with deionized water; the detergent is one of n-octanol, n-butanol, propylene glycol, isopropanol or acetone solution; and the volume concentration of the detergent solution is not less than 75%.

[0022] In some specific embodiments, the microfluidic device in step B) includes a constant temperature environment box, a microfluidic constant pressure pump and a microfluidic chip, and the microfluidic chip adopts a multi-channel microfluidic chip with a flow focusing structure; the multi-channel microfluidic chip of the present invention is preferably a 72-channel microfluidic chip.

[0023] The multi-channel microfluidic chip consists of an upper cover plate containing a liquid inlet and a liquid outlet, an intermediate functional plate containing microchannels, and a lower cover plate containing a liquid outlet. The upper cover plate, the intermediate functional plate, and the lower cover plate are irreversibly bonded together by hot pressing.

[0024] The intermediate layer functional plate includes an upper continuous phase channel, an upper droplet shearing unit, and an upper droplet collection channel. The continuous phase enters through the continuous phase channel and the dispersed phase enters through the dispersed phase channel. The dispersed phase is sheared by the continuous phase in the droplet shearing unit to form droplets. The droplets are collected in the droplet collection channel and flow out to the receiving phase through the lower cover plate containing the liquid outlet; the lower cover plate includes a lower dispersed phase channel and a liquid outlet.

[0025] The upper droplet shearing unit is composed of a plurality of droplet micro-shearing units, each of which is composed of two continuous phase channels, a dispersed phase channel, a necking shear opening, and a droplet channel. The plurality of droplet micro-shearing units of the present invention is preferably 72 droplet micro-shearing units.

[0026] The present invention provides a gelatin sponge microsphere, which is prepared by the preparation method described in any one of the above technical solutions.

[0027] Compared with the prior art, the present invention provides a preparation method for a gelatin sponge microsphere product, comprising the following steps: A) dissolving gelatin in deionized water to obtain a dispersed phase solution; mixing vegetable oil and a surfactant to obtain a continuous phase solution; mixing the vegetable oil and the surfactant to obtain a receiving phase solution; B) injecting the dispersed phase solution and the continuous phase solution into a microfluidic device respectively to obtain gelatin droplets; C) introducing the gelatin droplets into a receiving phase solution, cooling, and solidifying to obtain a crude gelatin sponge microsphere product; D) adding the crude gelatin sponge microsphere product to a first cross-linking solution for preliminary cross-linking and fixation, removing the upper layer solution, and continuously adding a second cross-linking solution for further cross-linking and fixation to obtain a semi-finished gelatin sponge microsphere product; E) removing the upper layer solution from the semi-finished gelatin sponge microsphere product, and washing to obtain a gelatin sponge microsphere product. The present invention uses an alcohol-containing crosslinking solution to crosslink the microspheres. This, on the one hand, leverages the mutual solubility of oil solutions and alcohol to remove the oil solution from the microsphere surface, allowing the crosslinker to quickly contact the microspheres and initiate crosslinking. Furthermore, the osmotic pressure of the alcohol solution prevents microsphere swelling, facilitating the preparation of small droplets. Furthermore, using an alcohol-containing crosslinking solution to crosslink the microspheres maintains the droplet morphology, resulting in a smooth, wrinkle-free surface after crosslinking. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The size and morphology of microspheres obtained at different isopropanol concentrations;

[0029] Figure 2 Non-gradient cross-linking: cross-linking solution 2, glutaraldehyde volume fraction 0.1%;

[0030] Figure 3 Non-gradient cross-linking: cross-linking solution 2, glutaraldehyde volume fraction 10%;

[0031] Figure 4 Gradient cross-linking: the volume fraction of glutaraldehyde in cross-linking solution 1 and cross-linking solution 2 was 0.1%;

[0032] Figure 5 Gradient cross-linking: the volume fractions of glutaraldehyde in cross-linking solution 1 and cross-linking solution 2 were 0.1% and 10%, respectively;

[0033] Figure 6 Gradient cross-linking: the volume fractions of glutaraldehyde in cross-linking solution 1 and cross-linking solution 2 were 1% and 2%, respectively;

[0034] Figure 7 Gradient cross-linking: the volume fractions of glutaraldehyde in cross-linking solution 1 and cross-linking solution 2 were 1% and 10%, respectively;

[0035] Figure 8 Photos of microsphere morphology after cross-linking in cross-linking solutions of different organic solvents; (a) droplet, (b) isopropanol, (c) n-octanol, (d) n-butanol, (e) propylene glycol, (f) acetone;

[0036] Figure 9 This is a model diagram of a multi-channel microfluidic chip;

[0037] Figure 10 The microfluidic chip consists of (a) upper cover plate; (b) middle functional board; (c) lower cover plate;

[0038] Figure 11 The functional plate in the middle layer of the microfluidic chip consists of (A) the upper droplet collection channel; (B) the upper continuous phase channel; (H) the upper droplet shear unit;

[0039] Figure 12 Design diagram of droplet micro-shear unit ( Figure 11 Partial view of H in (a) continuous phase channel; (b) dispersed phase channel; (c) necking shear; (d) droplet channel;

[0040] Figure 13 The lower cover plate of the microfluidic chip consists of (C) the lower dispersed phase channel; (D) the liquid outlet;

[0041] Figure 14 Droplets of different sizes: (a) 100 μm; (b) 200 μm; (c) 300 μm; (d) 400 μm; (e) 500 μm; (f) 600 μm; (g) 700 μm; (h) 800 μm; (i) 900 μm; (j) 1000 μm

[0042] Figure 15 Morphology of microspheres obtained under different cross-linking solutions (a) Experimental group 1; (b) Experimental group 2; (c) Experimental group 3; (d) Experimental group 4;

[0043] Figure 16 Design diagram of droplet micro-shear unit (non-necking structure);

[0044] Figure 17 Photos of gelatin droplets (a) necking structure; (b) non-necking structure. DETAILED DESCRIPTION

[0045] The present invention provides a gelatin sponge microsphere and a method for preparing the same. Those skilled in the art can refer to the contents of this article and appropriately improve the process parameters to achieve the same. It should be noted that all similar replacements and modifications are obvious to those skilled in the art and fall within the scope of protection of the present invention. The methods and applications of the present invention have been described through preferred embodiments. Relevant personnel can obviously modify or appropriately change and combine the methods and applications herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0046] The present invention provides a method for preparing a gelatin sponge microsphere product, comprising the following steps:

[0047] A) dissolving gelatin in deionized water to obtain a dispersed phase solution; mixing vegetable oil and a surfactant to obtain a continuous phase solution; and mixing the vegetable oil and the surfactant to obtain a receiving phase solution;

[0048] B) injecting the dispersed phase solution and the continuous phase solution into the microfluidic device separately to obtain gelatin droplets;

[0049] C) introducing the gelatin droplets into the receiving phase solution, cooling, and solidifying to obtain a crude gelatin sponge microsphere product;

[0050] D) adding the crude gelatin sponge microspheres to a first crosslinking solution for preliminary crosslinking and fixation, removing the upper solution, and then adding a second crosslinking solution for further crosslinking and fixation to obtain semi-finished gelatin sponge microspheres;

[0051] E) removing the upper layer solution from the semi-finished gelatin sponge microspheres and washing them to obtain a gelatin sponge microsphere product.

[0052] The preparation method of gelatin sponge microspheres provided by the present invention first prepares a solution:

[0053] Prepare the dispersed phase solution: dissolve gelatin in deionized water at 35-70°C, stir to prepare a gelatin solution, and then place the gelatin solution in an environment at 35-70°C and keep warm for use.

[0054] The freezing force of the gelatin of the present invention is 100-300 bloom, 100 bloom, 110 bloom, 120 bloom, 130 bloom, 140 bloom, 150 bloom, 160 bloom, 170 bloom, 180 bloom, 190 bloom, 200 bloom, 210 bloom, 220 bloom, 230 bloom, 240 bloom, 250 bloom, 260 bloom, 270 bloom, 280 bloom, 290 bloom, and 300 bloom.

[0055] The mass fraction of gelatin in the dispersed phase solution is 1% to 50%, specifically 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%; or ranges between any two of the above.

[0056] The stirring time is 0.5 to 2 hours; specifically, it can be 0.5 hours, 1 hour or 2 hours.

[0057] Prepare the continuous phase solution: at room temperature, take the continuous phase solvent, add the surfactant, and stir to prepare the continuous phase solution.

[0058] In the continuous phase solution, the mass ratio of vegetable oil to surfactant is 100:(1-10); specifically, it can be 100:1, 100:2, 100:3, 100:4, 100:5, 100:6, 100:7, 100:8, 100:9 or 100:10.

[0059] In some specific embodiments, the vegetable oil includes one or more of soybean oil, peanut oil, and corn oil; the surfactant is one or more of Span80, Span85, and PGPR, and the mass fraction of the surfactant is 0.5% to 10%; specifically, it can be 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or a range between any two of the above.

[0060] Prepare the receiving phase solution: at room temperature, take the continuous phase solvent, add the surfactant, stir to prepare the receiving phase solution, and place the receiving phase solution in an environment of 0-8°C.

[0061] In the receiving phase solution of the present invention, the mass ratio of vegetable oil to surfactant is 100:(0.1-2); specifically, it can be 100:0.1, 100:0.2, 100:0.3, 100:0.4, 100:0.5, 100:0.6, 100:0.7, 100:0.8, 100:0.9, 100:1.0, 100:1.1, 100:1.2, 100:1.3, 100:1.4, 100:1.5, 100:1.6, 100:1.7, 100:1.8, 100:1.9, 100:2.0; or a range value between any two of the above.

[0062] The above-mentioned vegetable oils and surfactants have been clearly described and will not be repeated here.

[0063] The dispersed phase solution and the continuous phase solution were injected into the microfluidic device respectively to obtain gelatin droplets.

[0064] In some specific embodiments, the dispersed phase pressure is 0.1 kPa to 500 kPa, specifically 1 kPa, 10 kPa, 50 kPa, 100 kPa, 150 kPa, 200 kPa, 250 kPa, 300 kPa, 350 kPa, 400 kPa, 450 kPa, and 500 kPa. The continuous phase pressure is 0.05 MPa to 1 MPa, specifically 0.1 MPa, 0.2 MPa, 0.3 MPa, 0.4 MPa, 0.5 MPa, 0.6 MPa, 0.7 MPa, 0.8 MPa, 0.9 MPa, and 1 MPa.

[0065] The microfluidic device of the present invention comprises a constant temperature environment box, a microfluidic constant pressure pump and a microfluidic chip, wherein the microfluidic chip adopts a multi-channel microfluidic chip with a flow focusing structure;

[0066] The present invention adopts a multi-channel microfluidic chip, which can not only obtain gelatin microspheres of uniform size, but also increase the yield of microspheres and reduce production costs.

[0067] The multi-channel microfluidic chip consists of an upper cover plate containing a liquid inlet and a liquid outlet, an intermediate functional plate containing microchannels, and a lower cover plate containing a liquid outlet. The upper cover plate, the intermediate functional plate, and the lower cover plate are irreversibly bonded together by hot pressing.

[0068] The intermediate layer functional plate includes an upper continuous phase channel, an upper droplet shearing unit, and an upper droplet collection channel. The continuous phase enters through the continuous phase channel and the dispersed phase enters through the dispersed phase channel. The dispersed phase is sheared by the continuous phase in the droplet shearing unit to form droplets. The droplets are collected in the droplet collection channel and flow out to the receiving phase through the lower cover plate containing the liquid outlet; the lower cover plate includes a lower dispersed phase channel and a liquid outlet.

[0069] The upper layer droplet shearing unit is composed of a plurality of droplet micro-shearing units, and the droplet micro-shearing unit is composed of two continuous phase channels, a dispersed phase channel, a necking shearing port and a droplet channel.

[0070] The inventors have found that if the droplet micro-shearing unit has no necking shearing opening design, the wall adhesion phenomenon is likely to occur during the droplet preparation process, resulting in uneven droplet size (such as Figure 17 (b)). The design of the necking shearing port of the droplet micro-shearing unit can effectively solve this problem and ensure the stability of the droplet shearing (as shown in Figure 17 (as shown in (a)).

[0071] The gelatin droplets are introduced into the receiving phase solution, cooled, and solidified to obtain a crude gelatin sponge microsphere product.

[0072] In some specific embodiments, the cooling temperature in step C) is <30°C; more preferably 0-8°C.

[0073] The crude gelatin sponge microspheres are added to the first cross-linking solution for preliminary cross-linking and fixation, and the upper solution is removed and then the second cross-linking solution is added for further cross-linking and fixation to obtain semi-finished gelatin sponge microspheres.

[0074] According to the present invention, the first cross-linking solution comprises water, a cross-linking agent and an organic solvent.

[0075] According to the present invention, the cross-linking agent is an aldehyde organic compound; specifically, it can be formaldehyde, glutaraldehyde, or a glutaraldehyde solution containing 30% isopropyl alcohol;

[0076] The organic solvent is one of n-octanol, n-butanol, propylene glycol, isopropanol or acetone solution; the volume fraction of the crosslinking agent in the first crosslinking solution is 0.1% to 1%, specifically 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%; or a range between any two of the above.

[0077] The volume concentration of the organic solvent solution is 25% to 50%; specifically, it can be 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%; or a range between any two of the above.

[0078] The time for the initial cross-linking and fixing of the present invention is 30 minutes.

[0079] In some specific embodiments, the second cross-linking solution includes water, a cross-linking agent and an organic solvent, the cross-linking agent is an aldehyde organic compound; the organic solvent is one of n-octanol, n-butanol, propylene glycol, isopropanol or acetone solution; the volume fraction of the cross-linking agent in the first cross-linking solution is 0.1% to 10%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%; or a range between any two of the above.

[0080] The volume concentration of the organic solvent solution is 25% to 50%; and the time for further cross-linking and fixing is 30 minutes.

[0081] The invention creatively adopts a gradient cross-linking method to avoid the gelatin droplets from rapidly cross-linking to form a dense shell when they come into contact with a high concentration of cross-linking agent, which blocks the cross-linking agent from entering the droplets and cross-linking, resulting in inconsistent cross-linking levels inside and outside the microspheres, and forming a core-shell structure under stress pulling (such as Figure 3 As shown in ); Gradient cross-linking can first make the microspheres preliminarily cross-linked and fixed inside and outside at a low concentration of cross-linking agent, retaining the pores on the surface of the microspheres to facilitate the entry of the cross-linking agent, and then further cross-linked and fixed at a high concentration of cross-linking agent, thereby ensuring that the degree of cross-linking inside and outside the microspheres is consistent (as shown in Figure 7 shown).

[0082] The present invention uses a cross-linking solution containing an organic solvent to cross-link the droplets, which has three main advantages:

[0083] 1) Vegetable oil will adhere to the surface of the microspheres, preventing the crosslinker from contacting the microspheres, while organic solvents can dissolve the vegetable oil on the surface of the microspheres, allowing the crosslinker to quickly contact and solidify the microspheres, shortening the crosslinking time of the microspheres;

[0084] 2) Since the cross-linking agent and the oil solution are not miscible, the concentration of the cross-linking agent that the microspheres actually contact is relatively high, and the surface of the microspheres is rapidly cross-linked to form a dense structure, resulting in uneven cross-linking inside and outside the microspheres. However, using a cross-linking solution containing an organic solvent to cross-link the droplets can make the cross-linking agent evenly mixed in the cross-linking solution, achieving a uniform cross-linking effect (such as Figure 15 );

[0085] 3) A certain concentration of cross-linking solution containing organic solvent can maintain the osmotic pressure inside and outside the microspheres, prevent the microspheres from shrinking and expanding, thereby making the surface of the microspheres smooth and wrinkle-free, and is conducive to the preparation of small-sized microspheres (for example, to prepare 100 μm gelatin microspheres, if the microspheres expand, then the gelatin droplets need to be less than 100 μm, and the process for smaller-sized microspheres will be more difficult) (such as Figure 1 and Table 1).

[0086] The upper solution of the semi-finished gelatin sponge microspheres is removed and the gelatin sponge microspheres are obtained after washing.

[0087] The cleaning method of the present invention is to use a cleaning agent and then use deionized water for cleaning; the cleaning agent is one of n-octanol, n-butanol, propylene glycol, isopropanol or acetone solution; the volume concentration of the cleaning agent solution is not less than 75%.

[0088] In some embodiments, after removing the upper layer solution from the semi-finished gelatin sponge microspheres, a cleaning agent is added to clean the gelatin sponge microspheres until no continuous phase solvent remains; the microspheres are then washed with deionized water to remove the residual cleaning agent to obtain gelatin sponge microspheres.

[0089] The present invention provides a gelatin sponge microsphere, which is prepared by the preparation method described in any one of the above technical solutions.

[0090] The present invention has clearly described the above preparation method, which will not be repeated here.

[0091] It should be understood that the expression "one or more of" includes individually each of the items recited after the expression and various combinations of two or more of the recited items, unless otherwise apparent from the context and usage. The expression "and / or" in conjunction with three or more recited items should be understood to have the same meaning, unless otherwise apparent from the context.

[0092] The terms "comprising", "having" or "containing", including their grammatical synonyms, should generally be understood as open and non-restrictive, e.g., not excluding other unrecited elements or steps, unless otherwise specifically stated or understood from the context.

[0093] In this application, the term "and / or" describes the association relationship between associated objects, indicating that three relationships may exist. For example, A and / or B can mean: A exists alone, A and B exist at the same time, and B exists alone. A and B can be singular or plural.

[0094] In this application, "at least one" means one or more, and "more than one" means two or more. "At least one of the following" or similar expressions refers to any combination of these items, including any combination of single or plural items.

[0095] It should be understood that the order of steps or the order in which certain actions are performed are not important as long as the present invention remains operable. Additionally, two or more steps or actions may be performed simultaneously.

[0096] The use of any and all examples or exemplary language such as "for example" or "including" herein is intended only to better illustrate the present invention and does not limit the scope of the present invention. No language in this specification should be construed as indicating any non-claimed element is essential to the practice of the present invention.

[0097] In addition, the numerical ranges and parameters used to define the present invention are approximate values. The relevant numerical values ​​in the specific examples have been presented as accurately as possible. However, any numerical value inherently inevitably contains standard deviations due to individual testing methods. Therefore, unless otherwise expressly stated, all ranges, amounts, values, and percentages used in this disclosure should be understood to be modified by the word "about." As used herein, "about" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a specified value or range.

[0098] It should be understood that in the various embodiments of the present application, the size of the serial numbers of the above-mentioned processes does not mean the order of execution. Some or all of the steps can be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0099] Some cases are described in the embodiments and comparative examples of the present invention, wherein the embodiments illustrate certain implementations of the present invention. However, this does not mean that the effects of the present invention can only be achieved in these cases.

[0100] To further illustrate the present invention, the gelatin sponge microspheres and the preparation method thereof provided by the present invention are described in detail below with reference to the examples.

[0101] Example 1

[0102] A method for preparing gelatin sponge microspheres belongs to the technical field of biomedicine and polymer materials, and comprises the following steps:

[0103] a. Preparation of dispersed phase solution: Dissolve 300 bloom gelatin in deionized water at 60°C in an oil bath to obtain a gelatin solution having a mass fraction of 25%. The gelatin solution was then placed in a 60°C oil bath and kept warm for later use.

[0104] b. Preparation of the continuous phase solution: soybean oil was added to PGPR and stirred at 300 rpm for 2 h to obtain a continuous phase solution having a mass fraction of 2% PGPR;

[0105] c. Preparation of receiving phase solution: soybean oil was added to PGPR, stirred at 300 rpm for 2 h to obtain a receiving phase solution having a mass fraction of 0.5% PGPR, and the receiving phase solution was placed in an environment of 0 to 8 ° C;

[0106] d. Preparation of cross-linking solution 1: deionized water, isopropanol and glutaraldehyde were added, and the mixture was stirred at 300 rpm for 2 h to prepare a cross-linking solution 1 containing an organic solvent with a volume fraction of 0.1% glutaraldehyde;

[0107] e. Preparation of cross-linking solution 2: Deionized water was added with isopropanol and glutaraldehyde, and stirred at 300 rpm for 2 h to prepare a cross-linking solution 2 containing an organic solvent having a glutaraldehyde volume fraction of 1% and an isopropanol volume concentration consistent with that of the cross-linking solution 1;

[0108] f. The dispersed phase solution in step a and the continuous phase solution in step b were injected into the microfluidic device, the dispersed phase pressure was 0.06MPa, the continuous phase pressure was 0.34MPa, and gelatin droplets of the desired specifications were obtained. In this step, the droplet size was achieved by controlling the dispersed phase pressure, the continuous phase pressure, and the size of the microfluidic device to achieve the desired target;

[0109] g. The gelatin droplets obtained in step f are cooled and solidified by the receiving phase solution in step c to obtain crude gelatin sponge microspheres;

[0110] f. After removing the upper layer of the crude gelatin sponge microspheres in step g; the crude gelatin sponge microspheres were subjected to gradient cross-linking, i.e., the cross-linking solution 1 of step d was first added, cross-linked for 30 min, and a preliminary cross-linking fixation was performed, and then the upper layer of the solution was removed, and the cross-linking solution 2 of step e was added, cross-linked for 30 min, and further cross-linked and fixed to obtain semi-finished gelatin sponge microspheres;

[0111] g. After removing the upper layer of the semi-finished gelatin sponge microspheres in step f, add isopropanol solution to wash the crude gelatin sponge microspheres until no continuous phase solvent remains on the surface of the microspheres; then wash the microspheres with deionized water 2-5 times to remove the isopropanol solution and other residues to obtain gelatin sponge microspheres that meet the requirements.

[0112] The obtained gelatin sponge microspheres were then examined under a microscope to check the shape characteristics of the microspheres. Figure 1 , Figure 1 The size and morphology of microspheres obtained at different isopropanol concentrations; (a) droplet, (b) 25% isopropanol, (c) 30% isopropanol, (d) 50% isopropanol, (e) 90% isopropanol, (f) 100% isopropanol. Figure 1 It can be seen that under the same droplet conditions, as the isopropanol concentration increases, the size of the resulting microspheres gradually decreases, and the surface morphology and uniformity of the microspheres gradually deteriorate. When the isopropanol concentration is less than 50%, the microsphere surface is relatively smooth; while when the isopropanol concentration is less than 25%, the microspheres shrink less and show some swelling.

[0113] Table 1 Relationship between droplet and microsphere size

[0114]

[0115] Example 2

[0116] In this example, the dispersed phase solution, continuous phase solution, and receiving phase solution in Example 1 were used to prepare crosslinking solution 1 and crosslinking solution 2 at the second isopropanol concentration, and the method was as follows:

[0117] Prepare crosslinking solution 1: Add isopropanol and glutaraldehyde to deionized water and stir at 300 rpm for 2 h to prepare glutaraldehyde crosslinking solution 1 with an isopropanol volume concentration of 30%;

[0118] Preparation of cross-linking solution 2: Deionized water was added with isopropyl alcohol and glutaraldehyde, and the mixture was stirred at 300 rpm for 2 h to prepare a glutaraldehyde cross-linking solution 2 with an isopropyl alcohol volume concentration of 30%.

[0119] The remaining steps are the same as the preparation method in Example 1.

[0120] The obtained gelatin sponge microspheres were then examined under a scanning electron microscope to check the shape characteristics of the microspheres.

[0121] This example investigates the effects of non-gradient cross-linking and gradient cross-linking at different glutaraldehyde concentrations on the microsphere morphology. Figure 2-7 . Figure 2 Non-gradient cross-linking: cross-linking solution 2, glutaraldehyde volume fraction 0.1%; Figure 3 Non-gradient cross-linking: cross-linking solution 2, glutaraldehyde volume fraction 10%; Figure 4 Gradient cross-linking: the volume fraction of glutaraldehyde in cross-linking solution 1 and cross-linking solution 2 was 0.1%; Figure 5 Gradient cross-linking: the volume fractions of glutaraldehyde in cross-linking solution 1 and cross-linking solution 2 were 0.1% and 10%, respectively; Figure 6 Gradient cross-linking: the volume fractions of glutaraldehyde in cross-linking solution 1 and cross-linking solution 2 were 1% and 2%, respectively; Figure 7 Gradient cross-linking: The volume fractions of glutaraldehyde in cross-linking solution 1 and cross-linking solution 2 were 1% and 10%, respectively.

[0122] from Figure 2-7 It can be seen that when using non-gradient crosslinking, when the volume fraction of glutaraldehyde in the crosslinking solution 2 is 10%, the microspheres exhibit a core-shell structure. The high concentration of glutaraldehyde on the microsphere surface forms a dense shell with a high degree of crosslinking, which restricts the diffusion of the crosslinker. However, the low degree of crosslinking inside the microspheres generates shrinkage stress during subsequent processing, leading to separation of the inner and outer shells. Furthermore, when the volume fraction of glutaraldehyde in the crosslinking solution 2 is 10%, the overall density of the microspheres increases.

[0123] Table 2 Non-gradient cross-linking and gradient cross-linking parameters

[0124]

[0125] Example 3

[0126] In this example, the dispersed phase solution, continuous phase solution, and receiving phase solution of Example 2 were used to prepare organic solvent-containing crosslinking solution 1 and crosslinking solution 2 with glutaraldehyde volume fractions of 0.5% and 2%, respectively, by the following method:

[0127] ① Preparation of cross-linking solution 1: Add n-octanol and glutaraldehyde to deionized water and stir at 300 rpm for 2 h to prepare a cross-linking solution 1 with a volume concentration of 30% n-octanol and a volume fraction of 0.5% glutaraldehyde;

[0128] Preparation of cross-linking solution 2: Take deionized water, add n-octanol and glutaraldehyde, and stir at 300 rpm for 2 hours to prepare a cross-linking solution 2 with a volume concentration of n-octanol of 30% and a volume fraction of glutaraldehyde of 2%.

[0129] ② Prepare crosslinking solution 1: Add n-butanol and glutaraldehyde to deionized water and stir at 300 rpm for 2 h to prepare a crosslinking solution 1 with a volume concentration of 30% n-butanol and a volume fraction of 0.5% glutaraldehyde;

[0130] Preparation of cross-linking solution 2: Take deionized water, add n-butanol and glutaraldehyde, and stir at 300 rpm for 2 hours to prepare a cross-linking solution 2 with a volume concentration of n-butanol of 30% and a volume fraction of glutaraldehyde of 2%.

[0131] ③ Prepare crosslinking solution 1: Add propylene glycol and glutaraldehyde to deionized water and stir at 300 rpm for 2 h to prepare a crosslinking solution 1 with a propylene glycol volume concentration of 30% and a glutaraldehyde volume fraction of 0.5%;

[0132] Preparation of cross-linking solution 2: Take deionized water, add propylene glycol and glutaraldehyde, and stir at 300 rpm for 2 hours to prepare a cross-linking solution 2 with a propylene glycol volume concentration of 30% and a glutaraldehyde volume fraction of 2%.

[0133] ④ Prepare crosslinking solution 1: Add acetone and glutaraldehyde to deionized water and stir at 300 rpm for 2 h to prepare a crosslinking solution 1 with a volume concentration of 30% acetone and 0.5% glutaraldehyde;

[0134] Preparation of cross-linking solution 2: Take deionized water, add acetone and glutaraldehyde, and stir at 300 rpm for 2 hours to prepare a cross-linking solution 2 with an acetone volume concentration of 30% and a glutaraldehyde volume fraction of 2%.

[0135] The remaining steps are the same as the preparation method in Example 1.

[0136] The obtained gelatin sponge microspheres are then tested under a microscope to check the shape characteristics of the microspheres.

[0137] This example investigates the effect of cross-linking solutions containing different organic solvents on the morphology of microspheres. Figure 8 .

[0138] Figure 8 Photos of the microsphere morphology after cross-linking in cross-linking solutions of different organic solvents; (a) droplets, (b) isopropanol, (c) n-octanol, (d) n-butanol, (e) propylene glycol, and (f) acetone.

[0139] from Figure 8 It can be seen that there is no obvious difference in the morphology and shrinkage of the microspheres after cross-linking the droplets using cross-linking solutions prepared with different organic solvents.

[0140] Table 3 Statistical table of the effects of cross-linking solutions containing different organic solvents on microsphere morphology

[0141]

[0142]

[0143] Example 4

[0144] In this embodiment, the dispersed phase solution, continuous phase solution, and receiving phase solution in Example 2 were used to prepare gelatin droplets of different sizes using different microfluidic chips. The microfluidic chip design diagram is shown in FIG. Figures 9-13 .

[0145] Different microfluidic chips are composed of an upper cover plate containing a liquid inlet and outlet, an intermediate functional plate containing a microchannel, and a lower cover plate containing a liquid outlet. The upper cover plate, the intermediate functional plate, and the lower cover plate are irreversibly bonded together by hot pressing.

[0146] The middle functional plate of each microfluidic chip includes an upper continuous phase channel, an upper droplet shearing unit, and an upper droplet collection channel. The continuous phase enters through the continuous phase channel, and the dispersed phase enters through the dispersed phase channel. The dispersed phase is sheared by the continuous phase in the droplet shearing unit to form droplets. The droplets collect in the droplet collection channel and flow out to the receiving phase through the lower cover plate containing the liquid outlet. The lower cover plate includes the lower dispersed phase channel and the liquid outlet.

[0147] The upper droplet shearing units of different microfluidic chips are composed of multiple droplet micro-shearing units, and the droplet micro-shearing unit is composed of two continuous phase channels, a dispersed phase channel, a necking shear port and a droplet channel.

[0148] The steps for preparing the gelatin droplets in this example are the same as those in Example 1.

[0149] The obtained gelatin droplets are then observed and measured under a microscope to calculate the size distribution of the gelatin droplets.

[0150] This example investigates the preparation of gelatin droplets of different sizes by controlling the dispersed phase pressure, continuous phase pressure, and microfluidic device size. The morphology of the gelatin droplets is shown in the attached figure. Figure 14 .

[0151] from Figure 14 It can be seen that droplets of different sizes can be prepared by using different chip structure designs and different dispersed phase pressures and continuous phase pressures.

[0152] Table 4 Statistics of gelatin droplets prepared at different dispersed phase pressures, continuous phase pressures, and microfluidic chip sizes

[0153]

[0154] Example 5

[0155] This example investigates the effects of cross-linking with a cross-linking solution containing an organic solvent and a cross-linking solution without an organic solvent on the morphology of the microspheres. Figure 15 .

[0156] In this embodiment, a cross-linking liquid containing an organic solvent is used for cross-linking experimental group 1, which uses the dispersed phase solution, continuous phase solution, and receiving phase solution in Example 1 and a cross-linking solution 1 with a glutaraldehyde volume fraction of 1% and an isopropanol volume concentration of 30% for cross-linking for 30 minutes. The remaining steps are the same as the preparation method in Example 1.

[0157] In this embodiment, a cross-linking liquid without an organic solvent was used to cross-link the droplets in experimental group 2 using the dispersed phase solution and continuous phase solution in Example 1, and a soybean oil solution with a glutaraldehyde volume fraction of 1% was used as the receiving phase solution. The cross-linking was performed for 30 minutes, and the remaining steps were the same as the preparation method in Example 1.

[0158] In this embodiment, cross-linking experimental group 3 was cross-linked using a cross-linking solution without organic solvent. The dispersed phase solution, continuous phase solution, and receiving phase solution in Example 1 were cross-linked with a cross-linking solution having a volume fraction of glutaraldehyde of 1% for 30 minutes. The remaining steps were the same as the preparation method in Example 1.

[0159] In this embodiment, a cross-linking solution without an organic solvent was used in experimental group 4. The dispersed phase solution, continuous phase solution, and receiving phase solution in Example 1 were used. After the gelatin droplets were cooled and solidified in the receiving phase solution, they were washed with isopropyl alcohol and then cross-linked with a cross-linking solution having a volume fraction of 1% glutaraldehyde. The method was as follows:

[0160] (1) using the same preparation method as in Example 1 to obtain gelatin droplets, cooling and solidifying the obtained gelatin droplets through a receiving phase solution to obtain a crude gelatin sponge microsphere;

[0161] (2) After removing the upper layer of the crude gelatin sponge microspheres in step (1), an isopropyl alcohol solution is added to clean the crude gelatin sponge microspheres until no continuous phase solvent remains on the surface of the microspheres; and then cross-linking is performed with a cross-linking solution having a volume fraction of 1% glutaraldehyde for 30 minutes to obtain a semi-finished gelatin sponge microsphere.

[0162] The obtained gelatin sponge microspheres are then tested under a microscope to check the shape characteristics of the microspheres.

[0163] Figure 15 The morphology of microspheres obtained under different cross-linking solutions (a) Experimental group 1; (b) Experimental group 2; (c) Experimental group 3; (d) Experimental group 4; Figure 15 It can be seen that the surface morphology of the microspheres obtained by cross-linking with a cross-linking solution containing an organic solvent is relatively smooth, and the degree of cross-linking is uniform; the degree of cross-linking of the microspheres obtained in experimental groups 2 and 3 by cross-linking with a cross-linking solution without an organic solvent is uneven, and the degree of cross-linking inside the microspheres is inconsistent with that on the surface of the microspheres; most of the microspheres obtained in experimental group 4 by cross-linking with a cross-linking solution without an organic solvent are wrinkled. This is because the microspheres shrink after being washed with isopropyl alcohol, and the microspheres fail to fully recover during the cross-linking process.

[0164] Example 6

[0165] This example investigates the design of a droplet micro-shear unit containing a necking structure (see Figure 12 ) and designs without necking (see Figure 16 ) uniformity of droplets prepared (see Figure 17 ).

[0166] The remaining steps are the same as the preparation method in Example 1.

[0167] The obtained gelatin droplets were observed under a microscope.

[0168] from Figure 17 It can be seen that the uniformity of the gelatin droplets prepared by the non-necking structure is lower than that of the gelatin droplets prepared by the necking structure. This is because the shear force of the shear unit of the non-necking structure chip is weaker and the shear stability is lower than that of the necking structure chip.

[0169] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for preparing a gelatin sponge microsphere product, characterized in that: The steps include: A) dissolving gelatin in deionized water to obtain a dispersed phase solution; mixing vegetable oil and a surfactant to obtain a continuous phase solution; and mixing the vegetable oil and the surfactant to obtain a receiving phase solution; B) injecting the dispersed phase solution and the continuous phase solution into the microfluidic device separately to obtain gelatin droplets; C) introducing the gelatin droplets into the receiving phase solution, cooling, and solidifying to obtain a crude gelatin sponge microsphere product; D) adding the crude gelatin sponge microspheres to a first crosslinking solution for preliminary crosslinking and fixation, removing the upper solution, and then adding a second crosslinking solution for further crosslinking and fixation to obtain a semi-finished gelatin sponge microsphere; the first crosslinking solution comprises water, a crosslinking agent, and an organic solvent, the organic solvent being one of n-octanol, n-butanol, propylene glycol, isopropanol, or acetone solution; the crosslinking agent is an aldehyde organic compound; the volume fraction of the crosslinking agent in the first crosslinking solution is 0.1% to 1%, and the second crosslinking solution comprises water, a crosslinking agent, and an organic solvent, the crosslinking agent is an aldehyde organic compound; the volume fraction of the crosslinking agent in the second crosslinking solution is 0.1% to 10%; the volume fraction of the crosslinking agent in the first crosslinking solution is lower than the volume fraction of the crosslinking agent in the second crosslinking solution; E) removing the upper layer solution from the semi-finished gelatin sponge microspheres and washing them to obtain a gelatin sponge microsphere product.

2. The preparation method according to claim 1, characterized in that Step A) The gelatin is dissolved at a temperature of 35-70° C., the gelatin has a freezing strength of 100-300 blooms, the mass fraction of gelatin in the dispersed phase solution is 1%-50%, and the stirring time is 0.5-2 hours.

3. The preparation method according to claim 1, characterized in that Step A) The vegetable oil comprises one or more of soybean oil, peanut oil, and corn oil; the surfactant comprises one or more of Span80, Span85, and PGPR, and the mass fraction of the surfactant is 0.5% to 10%; In the continuous phase solution, the mass ratio of vegetable oil to surfactant is 100:(1:~10); In the receiving phase solution, the mass ratio of vegetable oil to surfactant is 100:(0.1-2).

4. The preparation method according to claim 1, characterized in that In step B), the pressure of the dispersed phase solution is 0.1 kPa to 500 kPa, and the pressure of the continuous phase solution is 0.05 MPa to 1 MPa.

5. The preparation method according to claim 1, characterized in that Step D) the volume concentration of the organic solvent solution is 25% to 50%; the time for the preliminary cross-linking and fixing is 10 minutes to 60 minutes.

6. The preparation method according to claim 1, characterized in that In step D), the organic solvent is one of n-octanol, n-butanol, propylene glycol, isopropanol or acetone solution; the volume concentration of the organic solvent solution is 25% to 50%; and the further cross-linking and fixing time is 10 minutes to 2 hours.

7. The preparation method according to claim 1, characterized in that The cooling temperature in step C) is 0-8°C.

8. The preparation method according to claim 1, characterized in that In step E), the cleaning is performed by using a cleaning agent and then deionized water; the cleaning agent is one of n-octanol, n-butanol, propylene glycol, isopropanol or acetone solution; and the volume concentration of the cleaning agent solution is not less than 75%.

9. The preparation method according to claim 1, characterized in that Step B) the microfluidic device comprises a constant temperature environment box, a microfluidic constant pressure pump and a microfluidic chip, wherein the microfluidic chip is a multi-channel microfluidic chip with a flow focusing structure; The multi-channel microfluidic chip is composed of an upper cover plate containing a liquid inlet and a liquid outlet, an intermediate functional plate containing microchannels, and a lower cover plate containing a liquid outlet. The upper cover plate, the intermediate functional plate, and the lower cover plate are irreversibly bonded together by hot pressing. The intermediate functional plate includes an upper continuous phase channel, an upper droplet shearing unit, an upper droplet collection channel, and a lower dispersed phase channel. The continuous phase enters through the continuous phase channel, and the dispersed phase enters through the dispersed phase channel. The dispersed phase is sheared by the continuous phase in the droplet shearing unit to form droplets. The droplets are collected in the droplet collection channel and flow out to the receiving phase through the lower cover plate having a liquid outlet. The upper layer droplet shearing unit is composed of a plurality of droplet micro-shearing units, and the droplet micro-shearing unit is composed of two continuous phase channels, a dispersed phase channel, a necking shearing port and a droplet channel.

10. A gelatin sponge microsphere product, characterized in that: The compound is prepared by the preparation method according to any one of claims 1 to 9.

Images