Gelatin sponge microsphere product and preparation method thereof

Abstract

The invention relates to the technical field of materials, in particular to a gelatin sponge microsphere product and a preparation method thereof. The preparation method comprises the following steps: dissolving gelatin into deionized water to obtain a dispersed phase solution; vegetable oil and a surfactant are mixed to prepare a continuous phase solution; vegetable oil and a surfactant are mixed, and a receiving phase solution is obtained; respectively injecting the dispersed phase solution and the continuous phase solution into a microfluidic device to obtain gelatin liquid drops; introducing the gelatin liquid drops into the receiving phase solution, cooling and solidifying to obtain a crude product; adding the crude product into a first cross-linking solution, carrying out primary cross-linking immobilization, removing an upper-layer solution, and continuously adding a second cross-linking solution for further cross-linking immobilization to obtain a semi-finished microsphere product; and removing the upper-layer solution from the microsphere semi-finished product, and cleaning to obtain the microsphere. According to the invention, the alcohol-containing cross-linking liquid is adopted to carry out microsphere cross-linking, so that the cross-linking agent is in rapid contact with the microspheres to carry out cross-linking; the preparation of small-size liquid drops is facilitated, and the morphology of the liquid drops can be maintained, so that the surfaces of the cross-linked microspheres are smooth and have no wrinkles.

Description

Technical Field

[0001] The present invention relates to the technical field of materials, and particularly relates to a gelatin sponge microsphere product and a preparation method thereof. Background Art

[0002] Knee osteoarthritis (KOA) is one of the common chronic pain diseases with a very high incidence rate, especially the most common painful disease among the middle-aged and elderly populations. At present, the early treatment for KOA patients mainly includes oral drug treatment, physical therapy, intra-articular injection of hyaluronic acid, etc. However, the effects are often not significant. Severe or late-stage KOA patients need to receive surgical treatment, mainly including arthroscopic debridement and knee joint replacement. In 2015, Okuno et al. first reported the use of transcatheter genicular artery embolization (GAE) to treat and relieve knee joint pain in mild and moderate KOA patients, achieving good clinical effects. Genicular artery embolization is a minimally invasive intra-arterial intervention method with the advantages of small trauma, high precision, and rapid onset, and can effectively relieve knee joint pain.

[0003] Hemoptysis refers to a clinical emergency of hemoptysis with a daily bleeding volume exceeding 500 mL or each episode exceeding 200 mL. Moreover, due to the complex causes of hemoptysis, medical treatment is difficult to be effective for some patients. In 1974, the French scholar Remy first reported the successful treatment of massive hemoptysis by selective bronchial artery embolization (BAE). BAE is a minimally invasive interventional treatment method. Through a catheter, embolization materials are selectively injected into a certain bronchial artery to block the blood flow of the corresponding blood vessel, thereby controlling bleeding. This technology has the following significant advantages: precise control, accurately positioning the lesion area through imaging guidance to reduce damage to healthy tissues; minimally invasive treatment, only need to puncture a pinhole the size of a rice grain at the root of the thigh, and the postoperative recovery is fast; treating various lung problems, in addition to hemostasis, it can also be used for repeated bleeding of lung cancer and inhibiting tumor growth.

[0004] Lower gastrointestinal bleeding (LGIB) accounts for about 20% of acute gastrointestinal bleeding events, with an estimated incidence rate of 33–87 / 100,000, and is defined as bleeding originating from the distal end of the duodenojejunal junction (including the small intestine, colon, rectum, and anus). Although LGIB tends to resolve spontaneously and the clinical manifestations are milder than those of upper gastrointestinal bleeding (UGIB), the mortality rate is closely related to complications, ranging from 3% to 18%. Current guidelines recommend emergency CT angiography for hemodynamically unstable patients suspected of persistent LGIB to locate the bleeding site, followed by endoscopic or interventional treatment. Selective transarterial embolization (TAE) has become an effective and well-tolerated treatment option for LGIB patients, and current literature emphasizes its safety.

[0005] However, at present, the embolization microspheres used to treat the above-mentioned clinical symptoms by interventional means are basically made of non-degradable materials, which means that these microspheres need to remain permanently in the patient's body. The residual foreign bodies always cause some complications. In addition, ectopic embolization may also occur during the embolization process of the microspheres. Gelatin is an animal protein composed of various amino acids, which has good water absorption and plasticity, good tissue compatibility, easy to obtain materials, and can be degraded in the body. The embolization microspheres prepared from gelatin are a new type of peripheral vascular embolizing agent developed in recent years.

[0006] In the existing preparation methods of embolization microspheres, most of them first use the freezing technology to pre-freeze the microspheres, then wash the oil solution, then crosslink the microspheres with the crosslinking solution, and finally wash the microspheres to remove the crosslinking agent. The main technical disadvantages of the method of freezing first, then washing, then crosslinking, and then washing again are as follows: the gelatin microspheres are easy to swell and become larger in the crosslinking solution, and the obtained microspheres are often larger in size, which is not conducive to the preparation of microspheres with small particle sizes. Summary of the Invention

[0007] In view of this, the technical problem to be solved by the present invention is to provide a preparation method of gelatin sponge microspheres, and the gelatin sponge microspheres prepared by the preparation method provided by the present invention have a smooth surface without wrinkles and uniform crosslinking.

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

[0009] A) Dissolve gelatin in deionized water to obtain a dispersed phase solution; mix vegetable oil and a surfactant to prepare a continuous phase solution; mix vegetable oil and a surfactant to obtain a receiving phase solution;

[0010] B) Inject the dispersed phase solution and the continuous phase solution into a microfluidic device respectively to obtain gelatin droplets;

[0011] C) Introduce the gelatin droplets into the receiving phase solution, cool and solidify to obtain crude gelatin sponge microspheres;

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

[0013] E) Remove the upper layer solution from the semi-finished gelatin sponge microspheres, wash to obtain a gelatin sponge microsphere product.

[0014] In some specific embodiments, the temperature of gelatin dissolution in step A) is 35-70 °C, the gel strength of gelatin 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 described 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 the vegetable oil to the surfactant is 100:(1 - 10);

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

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

[0019] In some specific embodiments, the first cross-linking solution in step D) 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 1%, and the volume concentration of the organic solvent solution is 25% to 50%; the time for preliminary cross-linking and fixation is 10 min to 60 min

[0020] In some specific embodiments, the second cross-linking solution in step D) 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 second cross-linking solution is 0.1% to 10%, and the volume concentration of the organic solvent solution is 25% to 50%; the time for further cross-linking and fixation is 10 min to 2 h.

[0021] In some specific embodiments, the temperature of cooling in step C) is preferably < 30°C; more preferably 0 to 8°C. In some specific embodiments, the cleaning in step E) is to clean with a cleaning agent and then with deionized water; 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%.

[0022] In some specific embodiments, the microfluidic device in step B) includes a constant temperature environmental chamber, a microfluidic constant pressure pump, and a microfluidic chip. The microfluidic chip is 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 is composed of an upper cover plate with a liquid inlet and a liquid outlet, an intermediate functional plate with microchannels, and a lower cover plate with a liquid outlet. The upper cover plate, the intermediate functional plate, and the lower cover plate are irreversibly bonded together by thermal pressing.

[0024] The intermediate functional plate includes an upper continuous phase channel, an upper droplet shearing unit, and an upper droplet collecting 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 collecting channel and flow out through the lower cover plate with a liquid outlet to the receiving phase. 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 droplet micro-shearing unit is composed of two continuous phase channels, one dispersed phase channel, one constriction shear opening, and one droplet channel. Preferably, the plurality of droplet micro-shearing units in the present invention are 72 droplet micro-shearing units.

[0026] The present invention provides a gelatin sponge microsphere 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, including the following steps: A) Dissolve gelatin in deionized water to obtain a dispersed phase solution; mix vegetable oil and a surfactant to prepare a continuous phase solution; mix vegetable oil and a surfactant to obtain a receiving phase solution; B) Inject the dispersed phase solution and the continuous phase solution into a microfluidic device respectively to obtain gelatin droplets; C) Introduce the gelatin droplets into the receiving phase solution, cool and solidify to obtain a crude gelatin sponge microsphere; D) Add the crude gelatin sponge microsphere to a first crosslinking solution for preliminary crosslinking and fixation. After removing the upper solution, continue to add a second crosslinking solution for further crosslinking and fixation to obtain a semi-finished gelatin sponge microsphere; E) Remove the upper solution from the semi-finished gelatin sponge microsphere, wash it to obtain a gelatin sponge microsphere product. The present invention uses a crosslinking solution containing alcohol for microsphere crosslinking. On the one hand, it can utilize the miscibility of the oil solution and alcohol to remove the oil solution on the surface of the microspheres, enabling the crosslinking agent to quickly contact the microspheres and crosslink. On the other hand, it can utilize the osmotic pressure effect of the alcohol solution to avoid the swelling of the microspheres and facilitate the preparation of small-sized droplets. At the same time, using a crosslinking solution containing alcohol for microsphere crosslinking can maintain the droplet morphology, making the surface of the crosslinked microspheres smooth and without wrinkles. Description of the Drawings

[0028] Figure 1 Microsphere size and morphology obtained at different isopropanol concentrations;

[0029] Figure 2 Non-gradient crosslinking: The volume fraction of glutaraldehyde in the crosslinking solution 2 is 0.1%;

[0030] Figure 3 Non-gradient crosslinking: The volume fraction of glutaraldehyde in crosslinking solution 2 is 10%;

[0031] Figure 4 Gradient crosslinking: The volume fraction of glutaraldehyde in crosslinking solution 1 and crosslinking solution 2 is 0.1%;

[0032] Figure 5 Gradient crosslinking: The volume fractions of glutaraldehyde in crosslinking solution 1 and crosslinking solution 2 are 0.1% and 10% respectively;

[0033] Figure 6 Gradient crosslinking: The volume fractions of glutaraldehyde in crosslinking solution 1 and crosslinking solution 2 are 1% and 2% respectively;

[0034] Figure 7 Gradient crosslinking: The volume fractions of glutaraldehyde in crosslinking solution 1 and crosslinking solution 2 are 1% and 10% respectively;

[0035] Figure 8 Photographs of the morphology of microspheres crosslinked with crosslinking solutions of different organic solvents; (a) droplet, (b) isopropanol, (c) n-octanol, (d) n-butanol, (e) propylene glycol, (f) acetone;

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

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

[0038] Figure 11 Composition of the middle functional plate of the microfluidic chip (A) upper droplet collection channel; (B) upper continuous phase channel; (H) upper droplet shearing unit;

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

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

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

[0042] Figure 15 Microsphere morphologies obtained under different crosslinking solutions (a) Experimental group 1; (b) Experimental group 2; (c) Experimental group 3; (d) Experimental group 4;

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

[0044] Figure 17 Photographs of gelatin droplets (a) Necking structure; (b) Non-necking structure. Specific implementation manners

[0045] The present invention provides a gelatin sponge microsphere and a preparation method thereof. Those skilled in the art can draw on the content of this article and appropriately modify the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they all fall within the protection scope of the present invention. The method and application of the present invention have been described through preferred embodiments. Relevant personnel can obviously make changes or appropriate modifications and combinations to the methods and applications in this article 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 preparation method of a gelatin sponge microsphere product, comprising the following steps:

[0047] A) Dissolve gelatin in deionized water to obtain a dispersed phase solution; mix vegetable oil and a surfactant to prepare a continuous phase solution; mix vegetable oil and a surfactant to obtain a receiving phase solution;

[0048] B) Inject the dispersed phase solution and the continuous phase solution into a microfluidic device respectively to obtain gelatin droplets;

[0049] C) Introduce the gelatin droplets into the receiving phase solution, cool and solidify to obtain crude gelatin sponge microspheres;

[0050] D) Add the crude gelatin sponge microspheres to a first crosslinking solution for preliminary crosslinking and fixation. After removing the upper layer solution, continue to add a second crosslinking solution for further crosslinking and fixation to obtain semi-finished gelatin sponge microspheres;

[0051] E) Remove the upper layer solution from the semi-finished gelatin sponge microspheres, wash to obtain gelatin sponge microsphere products.

[0052] The preparation method of the gelatin sponge microsphere provided by the present invention first prepares solutions:

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

[0054] The gel strength of the gelatin in the present invention is 100 - 300 bloom, specifically 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, 300 bloom.

[0055] The mass fraction of gelatin in the disperse phase solution is 1% - 50%, specifically it can be 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 the range value between any two of the above.

[0056] The stirring time is 0.5 - 2 h; specifically it can be 0.5 h, 1 h or 2 h.

[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% - 10%; specifically it can be 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or the range value 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 the range values between any two of the above.

[0062] The above vegetable oil and surfactant have been clearly described and will not be elaborated here.

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

[0064] In some specific embodiments, the pressure of the dispersed phase is 0.1 kPa - 500 kPa, specifically, it can be: 1 kPa, 10 kPa, 50 kPa, 100 kPa, 150 kPa, 200 kPa, 250 kPa, 300 kPa, 350 kPa, 400 kPa, 450 kPa, 500 kPa; the pressure of the continuous phase is 0.05 MPa - 1 MPa. Specifically, it can be 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, 1 MPa.

[0065] The microfluidic device of the present invention includes a constant temperature environmental chamber, a microfluidic constant pressure pump, and a microfluidic chip. The microfluidic chip is a multi-channel microfluidic chip with a flow focusing structure;

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

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

[0068] The intermediate 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 with a 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 each droplet micro-shearing unit is composed of two continuous-phase channels, a dispersed-phase channel, a necking shear opening, and a droplet channel.

[0070] The inventor of the present invention found that if the droplet micro-shearing unit does not have a necking shear opening design, wall sticking is likely to occur during the droplet preparation process, resulting in non-uniform droplet sizes (as Figure 7 shown). The design of the necking shear opening of the droplet micro-shearing unit can effectively solve this problem and ensure the stability of droplet shearing (as Figure 8 shown).

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

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

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

[0074] According to the present invention, the first cross-linking solution includes 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% isopropanol;

[0076] 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% - 1%, specifically, it can be 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%; or a range value between any two of the above.

[0077] The volume concentration of the organic solvent solution is 25% - 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 value between any two of the above.

[0078] The time for the preliminary cross-linking and fixation in the present invention is 30 min.

[0079] In some specific embodiments, the second crosslinking solution comprises water, a crosslinking agent and an organic solvent, the crosslinking agent being an aldehyde organic compound; the organic solvent being one of n-octanol, n-butanol, propylene glycol, isopropanol or acetone solution; the volume fraction of the crosslinking agent in the first crosslinking solution being 0.1% - 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 value between any two of the above.

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

[0081] In the present invention, the gradient crosslinking method is creatively adopted to avoid the rapid crosslinking of the surface of the gelatin droplet to form a dense shell layer when the gelatin droplet contacts a high-concentration crosslinking agent, which blocks the crosslinking agent from entering the interior of the droplet for crosslinking, resulting in inconsistent crosslinking degrees inside and outside the microsphere and forming a core-shell structure under stress pulling (as Figure 1 shown); while the gradient crosslinking method can first preliminarily crosslink and fix the inside and outside of the microsphere with a low-concentration crosslinking agent, retain the pores on the surface of the microsphere to facilitate the entry of the crosslinking agent, and then further crosslink and fix with a high-concentration crosslinking agent, so as to ensure consistent crosslinking degrees inside and outside the microsphere (as Figure 2 shown).

[0082] The present invention uses a crosslinking solution containing an organic solvent for crosslinking the droplets, which mainly has three advantages

[0083] 1) Vegetable oil will adhere to the surface of the microsphere, preventing the crosslinking agent from contacting the microsphere. However, the organic solvent can dissolve the vegetable oil on the surface of the microsphere, enabling the crosslinking agent to quickly contact and cure with the microsphere, shortening the crosslinking time of the microsphere

[0084] 2) Since the crosslinking agent and the oil solution are immiscible, the actual concentration of the crosslinking agent contacted by the microsphere is relatively high, and the surface of the microsphere is quickly crosslinked to form a dense structure, resulting in non-uniform crosslinking degrees inside and outside the microsphere. By using a crosslinking solution containing an organic solvent for crosslinking the droplets, the crosslinking agent can be uniformly mixed in the crosslinking solution to achieve a uniform crosslinking effect (as Figure 5 shown);

[0085] 3) A crosslinking solution containing an organic solvent at a certain concentration can maintain the osmotic pressure inside and outside the microsphere, prevent the microsphere from shrinking and swelling, so that the surface of the microsphere is smooth and without wrinkles, and is conducive to the preparation of small-sized microspheres (for example, when preparing 100-μm gelatin microspheres, if the microsphere expands, then the gelatin droplet needs to be <100 μm, and the process for smaller-sized microspheres will be more difficult) (as Figure 6 and Table 1).

[0086] Remove the upper solution from the gelatin sponge microsphere semi-finished product, and wash it to obtain gelatin sponge microspheres.

[0087] The washing in the present invention is to wash with a cleaning agent and then wash with deionized water; 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 solution from the gelatin sponge microsphere semi-finished product, add a cleaning agent and wash the gelatin sponge microspheres until there is no continuous-phase solvent residue; then wash the microspheres with deionized water to remove the residual cleaning agent to obtain gelatin sponge microspheres.

[0089] The present invention provides a gelatin sponge microsphere 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 and will not be elaborated herein.

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

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

[0093] In the present application, the term "and / or" describes the association relationship of associated objects and indicates that three relationships can exist. For example, A and / or B can represent: A exists alone, A and B exist simultaneously, and B exists alone. Where A and B can be singular or plural.

[0094] In the present application, "at least one" means one or more, and "a plurality" means two or more. "At least one of the following (items)" or its similar expressions refer to any combination of these items, including any combination of single items (items) or plural items (items).

[0095] It should be understood that as long as the present invention is still operable, the order of steps or the order of performing certain actions is not important. In addition, two or more steps or actions can be performed simultaneously.

[0096] The use of any and all examples or exemplary language in this document, such as "for example" or "including", is merely intended 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 that any unclaimed element is essential for 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 values in the specific embodiments have been presented as precisely as possible herein. However, any value inherently and inevitably contains standard deviations due to individual testing methods. Therefore, unless otherwise clearly stated, it should be understood that all ranges, quantities, values, and percentages used in this disclosure are modified by "about". Here, "about" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a specific value or range.

[0098] It should be understood that in various embodiments of the present application, the magnitude of the serial numbers of the above processes does not mean the order of execution. Some or all of the steps may 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 to 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, in which the embodiments demonstrate certain implementation manners 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, a gelatin sponge microsphere and its preparation method provided by the present invention are described in detail below in conjunction with embodiments.

[0101] Example 1

[0102] A preparation method of gelatin sponge microspheres, belonging to the technical fields of biomedicine and polymer materials, comprises the following steps:

[0103] a. Prepare a dispersed phase solution: In a 60°C oil bath, dissolve gelatin with a bloom strength of 300 in deionized water to form a 25% by mass gelatin solution, and then place the gelatin solution in a 60°C oil bath environment and keep it warm for use;

[0104] b. Prepare a continuous phase solution: Take soybean oil, add PGPR, and stir at a speed of 300 rpm for 2 h to form a continuous phase solution with a PGPR mass fraction of 2%;

[0105] c. Prepare a receiving phase solution: Take soybean oil, add PGPR, and stir at a speed of 300 rpm for 2 h to form a receiving phase solution with a PGPR mass fraction of 0.5%, and place the receiving phase solution in an environment of 0 - 8°C;

[0106] d. Preparation of crosslinking solution 1: Take deionized water, add isopropanol and glutaraldehyde, and stir at a speed of 300 rpm for 2 h to prepare an organic solvent-containing crosslinking solution 1 with a glutaraldehyde volume fraction of 0.1%;

[0107] e. Preparation of crosslinking solution 2: Take deionized water, add isopropanol and glutaraldehyde, and stir at a speed of 300 rpm for 2 h to prepare an organic solvent-containing crosslinking solution 2 with a glutaraldehyde volume fraction of 1% and an isopropanol volume concentration consistent with that of crosslinking solution 1;

[0108] f. Inject the dispersed phase solution in step a and the continuous phase solution in step b into the microfluidic device respectively. The pressure of the dispersed phase is 0.06 MPa and the pressure of the continuous phase is 0.34 MPa to obtain gelatin droplets of the expected specifications. In this step, the droplet size reaches the expected target by controlling the dispersed phase pressure, the continuous phase pressure, and the size of the microfluidic device;

[0109] g. After the gelatin droplets obtained in step f are cooled and solidified by the receiving phase solution in step c, a crude product of gelatin sponge microspheres is obtained;

[0110] f. After removing the upper layer solution from the crude gelatin sponge microspheres in step g; perform gradient crosslinking on the crude gelatin sponge microspheres, that is, first add the crosslinking solution 1 in step d and crosslink for 30 min for preliminary crosslinking and fixation, then remove the upper layer solution again, add the crosslinking solution 2 in step e, and crosslink for 30 min for further crosslinking and fixation to obtain a semi-finished product of gelatin sponge microspheres;

[0111] g. After removing the upper layer solution from the semi-finished gelatin sponge microspheres in step f, add isopropanol solution to wash the crude gelatin sponge microspheres mentioned above until there is no continuous phase solvent residue 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] Then, the obtained gelatin sponge microspheres are detected under a microscope to check the shape characteristics of the microspheres. The results are as Figure 1 , Figure 1 Sizes and morphologies of microspheres obtained at different isopropanol concentrations; (a) droplets, (b) 25% isopropanol, (c) 30% isopropanol, (d) 50% isopropanol, (e) 90% isopropanol, (f) 100% isopropanol. It can be Figure 1 seen that under the same droplet conditions, with the increase of the isopropanol concentration, the size of the obtained microspheres gradually decreases, and the surface morphology and the uniformity of the microspheres gradually become worse. When the isopropanol concentration < 50%, the surface of the microspheres is relatively smooth; when the isopropanol concentration < 25%, the shrinkage of the microspheres is relatively small and there is a swelling situation.

[0113] Table 1 Relationship between droplet and microsphere sizes

[0114]

[0115] Example 2

[0116] In this example, the dispersed phase solution, continuous phase solution, and receiving phase solution in Example 1 were used, and crosslinking solutions 1 and 2 were prepared with the isopropanol concentration of the second group. The method was as follows:

[0117] Preparation of crosslinking solution 1: Deionized water was taken, isopropanol and glutaraldehyde were added, and stirred at a speed of 300 rpm for 2 h to prepare a glutaraldehyde crosslinking solution 1 with an isopropanol volume concentration of 30%.

[0118] Preparation of crosslinking solution 2: Deionized water was taken, isopropanol and glutaraldehyde were added, and stirred at a speed of 300 rpm for 2 h to prepare a glutaraldehyde crosslinking solution 2 with an isopropanol volume concentration of 30%.

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

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

[0121] This example investigated the effects of non-gradient crosslinking and different glutaraldehyde concentration gradients on crosslinking, and finally on the morphology of the microspheres. The morphology of the microspheres is shown in the appendix Figures 2 - 7 . Figure 2 Non-gradient crosslinking: The volume fraction of glutaraldehyde in crosslinking solution 2 was 0.1%; Figure 3 Non-gradient crosslinking: The volume fraction of glutaraldehyde in crosslinking solution 2 was 10%; Figure 4 Gradient crosslinking: The volume fraction of glutaraldehyde in crosslinking solutions 1 and 2 was 0.1%; Figure 5 Gradient crosslinking: The volume fractions of glutaraldehyde in crosslinking solutions 1 and 2 were 0.1% and 10% respectively; Figure 6 Gradient crosslinking: The volume fractions of glutaraldehyde in crosslinking solutions 1 and 2 were 1% and 2% respectively; Figure 7 Gradient crosslinking: The volume fractions of glutaraldehyde in crosslinking solutions 1 and 2 were 1% and 10% respectively.

[0122] From Figures 2 - 7 it can be seen that when non-gradient crosslinking was used and the volume fraction of glutaraldehyde in crosslinking solution 2 was 10%, the microspheres showed a core-shell structure. The glutaraldehyde concentration on the surface of the microspheres was relatively high, forming a dense shell layer with a high degree of crosslinking, which restricted the diffusion of the crosslinking agent. The degree of crosslinking inside was relatively low, and shrinkage stress was generated during subsequent processing, resulting in the separation of the inside from the outer shell. And when the volume fraction of glutaraldehyde in crosslinking solution 2 was 10%, the overall density of the microspheres increased.

[0123] Table 2 Non-gradient crosslinking and gradient crosslinking parameter table

[0124]

[0125] Example 3

[0126] In this example, the dispersed phase solution, continuous phase solution, and receiving phase solution in Example 2 were used, and crosslinked solutions 1 and 2 containing organic solvents were prepared with the volume fractions of glutaraldehyde being 0.5% and 2% respectively. The method is as follows:

[0127] ① Preparation of crosslinked solution 1: Take deionized water, add n-octanol and glutaraldehyde, and stir at a speed of 300 rpm for 2 h to prepare crosslinked solution 1 with an n-octanol volume concentration of 30% and a glutaraldehyde volume fraction of 0.5%;

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

[0129] ② Preparation of crosslinked solution 1: Take deionized water, add n-butanol and glutaraldehyde, and stir at a speed of 300 rpm for 2 h to prepare crosslinked solution 1 with an n-butanol volume concentration of 30% and a glutaraldehyde volume fraction of 0.5%;

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

[0131] ③ Preparation of crosslinked solution 1: Take deionized water, add propylene glycol and glutaraldehyde, and stir at a speed of 300 rpm for 2 h to prepare crosslinked solution 1 with a propylene glycol volume concentration of 30% and a glutaraldehyde volume fraction of 0.5%;

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

[0133] ④ Preparation of crosslinked solution 1: Take deionized water, add acetone and glutaraldehyde, and stir at a speed of 300 rpm for 2 h to prepare crosslinked solution 1 with an acetone volume concentration of 30% and a glutaraldehyde volume fraction of 0.5%;

[0134] Preparation of crosslinked solution 2: Take deionized water, add acetone and glutaraldehyde, and stir at a speed of 300 rpm for 2 h to prepare crosslinked 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 microspheres were then detected under a microscope to observe the shape characteristics of the microspheres.

[0137] In this example, the effects of crosslinking solutions containing different organic solvents on the morphology of the microspheres were investigated. The morphology of the microspheres is shown in the appendix Figure 8 .

[0138] Figure 8 Photographs of the morphology of microspheres crosslinked with crosslinking solutions of different organic solvents; (a) droplet, (b) isopropanol, (c) n-octanol, (d) n-butanol, (e) propylene glycol, (f) acetone.

[0139] From Figure 8 it can be seen that there is no significant difference in the morphology and shrinkage rate of the microspheres after crosslinking the droplets with crosslinking solutions prepared with different organic solvents.

[0140] Table 3 Statistical table of the effects of crosslinking solutions containing different organic solvents on the morphology of microspheres

[0141]

[0142] Example 4

[0143] In this example, the dispersed phase solution, continuous phase solution, and receiving phase solution in Example 2 were used, and gelatin droplets of different sizes were prepared through different microfluidic chips. The design diagrams of the microfluidic chips are shown in Figures 9 - 13 .

[0144] Each of the different microfluidic chips consists of an upper cover plate with an inlet and an outlet, an intermediate functional plate with microchannels, and a lower cover plate with an outlet. The upper cover plate, intermediate functional plate, and lower cover plate are irreversibly bonded together by thermal pressing.

[0145] The intermediate functional plates of the different microfluidic chips all include 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, and the droplets are collected in the droplet collection channel and flow out to the receiving phase through the lower cover plate with an outlet. The lower cover plate includes a lower dispersed phase channel and an outlet.

[0146] The upper droplet shearing units of the different microfluidic chips are all composed of multiple droplet micro-shearing units. Each droplet micro-shearing unit consists of two continuous phase channels, one dispersed phase channel, one necking shear opening, and one droplet channel.

[0147] The preparation steps of the gelatin droplets in this example are the same as those in Example 1.

[0148] The obtained gelatin droplets were then observed and measured under a microscope, and the size distribution of the gelatin droplets was statistically analyzed.

[0149] In this example, the preparation of gelatin droplets with different sizes was investigated by controlling the dispersed phase pressure, continuous phase pressure, and the size of the microfluidic device. The morphology of the gelatin droplets is shown in the appendix Figure 14 .

[0150] From Figure 14 it can be seen that droplets of different sizes can be prepared by using different chip structure designs, different dispersed phase pressures, and continuous phase pressures

[0151] Table 4 Statistical table of gelatin droplets prepared with different dispersed phase pressures, continuous phase pressures, and microfluidic chip sizes

[0152]

[0153] Example 5

[0154] In this example, the effects of crosslinking with a crosslinking solution containing an organic solvent and a crosslinking solution without an organic solvent on the morphology of the microspheres were investigated. The morphology of the microspheres is shown in the appendix Figure 15 .

[0155] In this example, for the experimental group 1 crosslinked with a crosslinking solution containing an organic solvent, the dispersed phase solution, continuous phase solution, and receiving phase solution in Example 1 were used, and crosslinking was carried out with crosslinking solution 1 with a glutaraldehyde volume fraction of 1% and an isopropanol volume concentration of 30% for 30 min. The remaining steps were the same as the preparation method in Example 1

[0156] In this example, for the experimental group 2 crosslinked with a crosslinking solution without an organic solvent, the dispersed phase solution and continuous phase solution in Example 1 were used, and the receiving phase solution was a soybean oil solution with a glutaraldehyde volume fraction of 1% to crosslink the droplets for 30 min. The remaining steps were the same as the preparation method in Example 1

[0157] In this example, for the experimental group 3 crosslinked with a crosslinking solution without an organic solvent, the dispersed phase solution, continuous phase solution, and receiving phase solution in Example 1 were used to crosslink with a crosslinking solution with a glutaraldehyde volume fraction of 1% for 30 min. The remaining steps were the same as the preparation method in Example 1

[0158] In this example, for the experimental group 4 crosslinked with a crosslinking solution without an organic solvent, the dispersed phase solution, continuous phase solution, and receiving phase solution in Example 1 were used. After the gelatin droplets were cooled and solidified by the receiving phase solution, they were washed with isopropanol and then crosslinked with a crosslinking solution with a glutaraldehyde volume fraction of 1%. The method was as follows

[0159] (1) Gelatin droplets were obtained by using the same preparation method as in Example 1. After the obtained gelatin droplets were cooled and solidified by the receiving phase solution, crude gelatin sponge microspheres were obtained

[0160] (2) After removing the upper layer solution from the crude gelatin sponge microspheres in step (1), isopropyl alcohol solution was added to wash the crude gelatin sponge microspheres until there was no residual continuous phase solvent on the surface of the microspheres; then crosslinking was carried out with a crosslinking solution with a glutaraldehyde volume fraction of 1% for 30 min to obtain semi-finished gelatin sponge microspheres.

[0161] Then, the obtained gelatin sponge microspheres were detected under a microscope to check the shape characteristics of the microspheres.

[0162] Figure 15 The morphologies of the microspheres obtained under different crosslinking solutions (a) Experimental group 1; (b) Experimental group 2; (c) Experimental group 3; (d) Experimental group 4; From Figure 15 It can be seen that the surface morphology of the microspheres crosslinked with the crosslinking solution containing organic solvent is relatively smooth and the crosslinking degree is uniform; for the microspheres crosslinked with the crosslinking solution without organic solvent in Experimental group 2 and Experimental group 3, the crosslinking degree is non-uniform, and the crosslinking degree inside the microspheres is inconsistent with that on the surface of the microspheres; most of the microspheres crosslinked with the crosslinking solution without organic solvent in Experimental group 4 show shrinkage, which is due to the shrinkage of the microspheres after being washed with isopropyl alcohol and the microspheres failing to fully recover during the crosslinking process.

[0163] Example 6

[0164] In this example, the droplet uniformity (see Figure 12 ) prepared by the droplet micro-shearing unit with a necking structure design (see Figure 16 ) and without a necking structure design (see Figure 17 ) was investigated.

[0165] The remaining steps are the same as the preparation method in Example 1.

[0166] The obtained gelatin droplets were observed under a microscope.

[0167] 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, because the shearing force of the shearing unit of the non-necking structure chip is weaker and the shearing stability is lower than that of the necking structure chip.

[0168] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A preparation method of a gelatin sponge microsphere product, characterized in that, It includes the following steps: A) Dissolve gelatin in deionized water to obtain a dispersed phase solution; mix vegetable oil and surfactant to prepare a continuous phase solution; mix vegetable oil and surfactant to obtain a receiving phase solution; B) Inject the dispersed phase solution and the continuous phase solution into a microfluidic device respectively to obtain gelatin droplets; C) Introduce the gelatin droplets into the receiving phase solution, cool and solidify to obtain crude gelatin sponge microspheres; D) Add the crude gelatin sponge microspheres to a first cross-linking solution for preliminary cross-linking and fixation. After removing the upper layer solution, continue to add a second cross-linking solution for further cross-linking and fixation to obtain semi-finished gelatin sponge microspheres; the first cross-linking solution includes water, a cross-linking agent and an organic solvent, and the organic solvent is one of n-octanol, n-butanol, propylene glycol, isopropanol or acetone solution; E) Remove the upper layer solution from the semi-finished gelatin sponge microspheres, wash them to obtain gelatin sponge microsphere products.

2. The preparation method according to claim 1, wherein In step A), the temperature for dissolving the gelatin is 35~70°C, the gel strength of the gelatin 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.

3. The preparation method according to claim 1, characterized in that, In step A), 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%~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~500 kPa, and the pressure of the continuous phase solution is 0.05 MPa~1 MPa.

5. The preparation method according to claim 1, characterized in that, In step D), the cross-linking agent is an aldehyde organic compound; the volume fraction of the cross-linking agent in the first cross-linking solution is 0.1%~1%, and the volume concentration of the organic solvent solution is 25%~50%; the time for preliminary cross-linking and fixation is 10 min~60 min.

6. The preparation method according to claim 1, characterized in that, In step D), 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 second cross-linking solution is 0.1%~10%, and the volume concentration of the organic solvent solution is 25%~50%; the time for further cross-linking and fixation is 10 min~2 h.

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

8. The preparation method according to claim 1, wherein, In step E), the washing is to wash with a cleaning agent and then with deionized water; 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%.

9. The preparation method according to claim 1, characterized in that, In step B), the microfluidic device includes a constant temperature environmental chamber, a microfluidic constant pressure pump and a microfluidic chip, and 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 with a liquid inlet and a liquid outlet, an intermediate functional plate with micro-channels, and a lower cover plate with a liquid outlet. The upper cover plate, the intermediate functional plate, and the lower cover plate are irreversibly bonded together by thermal compression bonding; 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, and the droplets are collected in the droplet collection channel and flow out to the receiving phase through the lower cover plate with a liquid outlet; The upper droplet shearing unit is composed of a plurality of droplet micro-shearing units, and each droplet micro-shearing unit is composed of two continuous phase channels, one dispersed phase channel, one necking shear opening, and one droplet channel.

10. A gelatin sponge microsphere product, characterized in that, Prepared by the preparation method according to any one of claims 1 to 9.

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