CN119455066A - A kind of hemostatic nano hydrogel particle and its preparation method and application - Google Patents

Abstract

The invention relates to a hemostatic nano hydrogel particle, a preparation method and application thereof, wherein the hemostatic nano hydrogel particle is prepared from the following raw materials, by weight, 40-50 parts of mesoporous silica nano particles, 20-33 parts of thrombin, 25-50 parts of methacrylamide gelatin and 0.5-1.25 parts of photoinitiator. The invention also provides a hemostatic system based on the nano hydrogel particles and the initiation light source. The hemostatic hydrogel particles prepared by the invention can initiate rapid coagulation at a wound site under illumination and continuously adhere to the wound site, and have the effects of keeping thrombin activity, reducing the risk of bleeding after treatment and improving hemostatic capacity.

Description

Hemostatic nano hydrogel particle and preparation method and application thereof

Technical Field

The invention relates to the technical field of biomedical materials, in particular to a hemostatic nano hydrogel particle and a preparation method and application thereof.

Background

Thrombin, fibrin, etc. are often used as the main active ingredients in hemostatic materials, and can directly activate the coagulation cascade reaction pathway to promote the formation of blood clots during the coagulation process. Compared with organic hemostatic materials and inorganic hemostatic materials, the coagulation factors can better promote the generation of insoluble fibrin, and have hemostatic capability which cannot be achieved by other materials. Meanwhile, the bioactive substance has good degradability and the effect of accelerating wound repair, so the bioactive substance is widely developed and applied to hemostatic materials.

However, the existing thrombin hemostatic materials still have certain defects that 1, thrombin is directly exposed in the extreme environment of the digestive tract to weaken the coagulation activity, 2, most thrombin solution cannot stay at a bleeding part to play a role in hemostasis, and 3, the initially formed blood clot is not stable enough, and secondary bleeding is easy to occur when the initially formed blood clot is cracked or falls off. For example, after hemostasis in gastrointestinal surgery, re-bleeding may occur if substantial peristalsis occurs in the gastrointestinal tract. Therefore, the research and development of the hemostatic material with high blood coagulation speed, organized adhesion, capability of keeping the activity of thrombin under the acid-base condition, long duration of hemostatic effect and good biocompatibility has important significance.

Disclosure of Invention

Aiming at the problems that thrombin in the existing thrombin hemostatic material is directly exposed in the digestive tract environment to weaken the coagulation activity, most thrombin solution cannot stay at a bleeding part to play a role in hemostasis, and initially formed blood clots are unstable enough and easily bleed again when cracking or falling off, the invention provides a hemostatic nano hydrogel particle, a preparation method and application thereof.

Specifically, the invention provides the following technical scheme:

in a first aspect, the invention provides hemostatic nano hydrogel particles, which are characterized by being prepared from 40-50 parts of mesoporous silica nano particles, 20-33 parts of thrombin, 25-50 parts of methacrylamide gelatin and 0.5-1.25 parts of photoinitiator in parts by weight.

Preferably, the hemostatic nano hydrogel particles are characterized by being prepared from the following raw materials, by weight, 43-47 parts of mesoporous silica nanoparticles, 25-30 parts of thrombin, 30-40 parts of methacrylamide gelatin and 0.8-1 part of photoinitiator.

Preferably, the hemostatic nano hydrogel particles are characterized in that the mesoporous silica nanoparticles are selected from one of spherical mesoporous silica nanoparticles, dendritic mesoporous silica nanoparticles and hollow mesoporous silica nanoparticles;

and/or, the mesoporous silica nanoparticle is selected from one of MCM series, SBA series, MSU series and TDU series;

Preferably, the mesoporous silica nanoparticle is selected from aminated dendritic mesoporous silica nanoparticles.

Preferably, the hemostatic nano hydrogel particles are characterized in that the thrombin is one selected from the group consisting of porcine thrombin, bovine thrombin and human thrombin;

Preferably, the hemostatic nano hydrogel particles can maintain the thrombin activity for more than or equal to 45 days.

Preferably, the hemostatic nano hydrogel particles are characterized in that the amino substitution degree of the methacrylamide gelatin is 30-90%;

Preferably, the amino substitution degree of the methacrylamide gelatin is 60-90%.

Preferably, the hemostatic nano hydrogel particles are characterized in that the photoinitiator is selected from one of phenyl (2, 4, 6-trimethylbenzoyl) lithium phosphate (LAP), 2-hydroxy-40- (2-hydroxyethoxy) -2-methyl propenone (IC-2959), 2' -azo [ 2-methyl-N- (2-hydroxyethyl) propanamide (VA 086), triethanolamine (TEA), N-Vinyl Caprolactam (VC) and eosin-y (EY);

Preferably, the photoinitiator is selected from the group consisting of phenyl (2, 4, 6-trimethylbenzoyl) lithium phosphate (LAP).

Preferably, the hemostatic nano hydrogel particles are characterized in that the initiating light source of the hemostatic nano hydrogel particles is blue laser with the wavelength of 395-425nm, white light with the wavelength of 300-760 nm or ultraviolet light with the wavelength of 200-400 nm;

preferably, the hemostatic nano hydrogel particles have an initiation light source of blue laser with wavelength of 395-425 nm.

In a second aspect, the present invention provides a method for preparing the hemostatic nano hydrogel particles, the method comprising the steps of:

(1) Mixing mesoporous silica nano particles with thrombin, dissolving the mixture in pure water, carrying out ultrasonic treatment, and stirring to obtain thrombin-loaded mesoporous silica nano particle solution;

(2) Dissolving methacrylamide gelatin and a photoinitiator in a solvent to obtain GelMA premix;

(3) Mixing the thrombin-loaded mesoporous silica nanoparticle solution with GelMA premix, performing ultrasonic treatment, stirring, centrifuging, and taking out precipitate to obtain hemostatic nano hydrogel particles.

Preferably, in the preparation method of the hemostatic nano hydrogel particle, in the step (1), the concentration (g/ml) of the thrombin-loaded mesoporous silica nano particle solution is 12-16.6%;

preferably, the concentration (g/ml) of the thrombin-loaded mesoporous silica nanoparticle solution is 13.6-15.4%;

Further preferably, the mesoporous silica nanoparticle is mixed with thrombin in a mass ratio of 1 (2-2.44).

Preferably, in the preparation method of the hemostatic nano hydrogel particles, in the step (1), the ultrasonic treatment time is 25-35min;

And/or the power of the ultrasound is 40-45khz;

Preferably, the stirring time is 25-35min.

Preferably, in the preparation method of the hemostatic nano hydrogel particle, in the step (2), the solvent is selected from one of pure water, physiological saline and phosphate buffer.

Preferably, in the preparation method of the hemostatic nano hydrogel particles, in the step (2), the concentration (g/ml) of the methacrylamide gelatin in the GelMA premix solution is 5-30%;

Preferably, the concentration (g/ml) of the methacrylamidoglycolate in the GelMA premix solution is 5-10%;

further preferably, the concentration (g/ml) of the methacrylamidoglycolate in the GelMA premix is 6-8%.

Preferably, in the preparation method of the hemostatic nano hydrogel particles, in the step (2), the concentration (g/ml) of the photoinitiator in the GelMA premix solution is 0.10% -0.40%;

Preferably, the concentration (g/ml) of the photoinitiator in the GelMA premix is 0.16-0.25%.

Preferably, in the preparation method of the hemostatic nano hydrogel particles, in the step (3), the thrombin-loaded mesoporous silica nano particle solution and the GelMA premix solution are mixed according to the volume ratio of 1:2-2:1;

preferably, the thrombin-loaded mesoporous silica nanoparticle solution is mixed with the GelMA premix solution according to the volume ratio of 1:1;

Further preferably, the time of the ultrasonic treatment is 25-35min;

And/or the power of the ultrasound is 40-45khz;

more preferably, the stirring time is 6 to 7 hours.

In a third aspect, the present invention provides a hemostatic hydrogel nanoparticle prepared by the method for preparing a hemostatic hydrogel nanoparticle.

In a fourth aspect, the present invention provides a hemostatic system, wherein the hemostatic system comprises the hemostatic hydrogel nanoparticle, a hemostatic hydrogel nanoparticle injection device, and an initiating light source generating device.

Preferably, the hemostatic system is characterized in that the hemostatic nano-hydrogel particle injection device comprises a syringe and a digestive endoscope tube;

preferably, the initiating light source generating device comprises a blue laser emitting lamp, a blue laser endoscope, a narrow-band imaging endoscope and an ultraviolet light emitting lamp.

In a fifth aspect, the present invention provides an application of the hemostatic nano hydrogel particle in preparing hemostatic materials or medicines.

In a sixth aspect, the present invention provides an application of the hemostatic nano hydrogel particle in preparing an organ hemostatic material or a medicament.

Preferably, the application is characterized in that the organ is a substantial organ or a hollow organ;

Preferably, the substantial organ is a heart, liver, spleen, lung, pancreas or kidney;

further preferably, the hollow organ is esophagus, stomach, duodenum, jejunum, ileum or colon.

The invention has the beneficial effects that:

The hemostatic nano hydrogel particles can rapidly stop bleeding through blue laser crosslinking, have strong tissue adhesion, can keep thrombin activity under the condition of acid and alkali, improve hemostatic effect and reduce risk of re-bleeding. Can be used for heart, liver, spleen, lung or kidney, and esophageal, gastric, duodenal, jejunum, ileum or colon cavity viscera, and can maintain thrombin activity for 45 days or more after lyophilization.

Drawings

FIG. 1 is a transmission electron microscope image of the hemostatic nano-hydrogel particles prepared in example 1-1, wherein A is an aminated dendritic mesoporous silica nanoparticle, B is a thrombin-loaded aminated dendritic mesoporous silica nanoparticle, C is a thrombin-loaded aminated dendritic mesoporous silica coated with GelMA, and D is a thrombin-loaded aminated dendritic mesoporous silica coated with GelMA after blue light excitation.

FIG. 2 shows the loading and encapsulation rates of mesoporous silica and thrombin prepared in example 1-1.

FIG. 3 is a Zeta potential diagram showing the hemostatic hydrogel nanoparticle prepared in example 1-1.

FIG. 4 is a graph showing the coagulation effect of the different materials of Experimental example 1 after they are blended with fresh blood. A is a blood coagulation effect graph after blood and normal saline are blended, B is a blood coagulation effect graph after blood and thrombin solution are blended, C is a blood coagulation effect graph after blood and aminated dendritic mesoporous silica are blended, D is a blood coagulation effect graph after blood and thrombin-loaded aminated dendritic mesoporous silica are blended, and E is a blood coagulation effect graph after blood and outer ring GelMA-coated aminated dendritic mesoporous silica loaded with thrombin are blended and crosslinked by ultraviolet light.

Fig. 5 is a scanning electron microscope image showing the formation of blood clots after blending different materials with fresh blood in experimental example 1. A is a scanning electron microscope image of blood and normal saline which are mixed to form blood clots, B is a scanning electron microscope image of blood and thrombin solution which are mixed to form blood clots, C is a scanning electron microscope image of blood and aminated dendritic mesoporous silica which are mixed to form blood clots, D is a scanning electron microscope image of blood and aminated dendritic mesoporous silica which is loaded with thrombin which are mixed to form blood clots, and E is a scanning electron microscope image of blood and aminated dendritic mesoporous silica which is loaded with thrombin and wrapped by outer ring GelMA which are mixed to form blood clots after ultraviolet crosslinking.

Fig. 6 shows a graph of the coagulation effect of the different materials for starting coagulation, and a statistical graph of the coagulation time of the different materials for recording the coagulation starting time after the different materials are blended with normal blood in experimental example 2.

Fig. 7 shows a graph of the coagulation effect of the different materials for starting coagulation, and a statistical graph of the coagulation time of the different materials for recording the coagulation starting time of the different materials after blending with the blood of the patient with cirrhosis in experimental example 2.

Fig. 8 shows the blood coagulation effect of the different materials treated by simulated gastric fluid and intestinal fluid in experimental example 3, wherein a is the blood coagulation effect of the different materials treated by simulated gastric fluid, and B is the blood coagulation effect of the different materials treated by simulated intestinal fluid.

FIG. 9 is a graph showing experimental results of the different materials used for hemostasis of rat liver in experimental example 4, wherein A is an untreated wound graph, B is a wound graph after thrombin solution is applied, C is a graph after hemostasis nano hydrogel particles are applied and subjected to blue laser crosslinking treatment, and D-F is a scanning electron microscope result graph of an in-vitro liver tissue wound specimen after hemostasis.

Fig. 10 is a graph showing the effect of different materials in experimental example 5 on hemostasis of a small fragrant pig under an endoscope with blue laser. A is an esophageal hemorrhage hemostasis effect diagram, B is a gastric hemorrhage hemostasis effect diagram, and C is a duodenal hemorrhage hemostasis effect diagram.

FIG. 11 is a schematic diagram showing the crosslinking reaction of a photo-excited light-emitting initiator.

FIG. 12 is a graph showing the effects of thrombin activity after freeze-drying and preservation of blood-stopping nano-hydrogel particles according to experimental example 6.

Detailed Description

The invention will be further illustrated with reference to the following specific examples, without limiting the invention to these specific embodiments.

The invention provides a hemostatic nano hydrogel particle which is prepared from the following raw materials in parts by weight: 40-50 parts of mesoporous silica nano particles, 20-33 parts of thrombin, 25-50 parts of methacrylamide gelatin and 0.5-1.25 parts of photoinitiator.

In some embodiments, the parts by weight of mesoporous silica nanoparticles in the hemostatic hydrogel particles may be any two values between 40 and 50, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

In some embodiments, the parts by weight of thrombin in the hemostatic hydrogel particles may be any two values between 20 and 33, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, or 33.

In some embodiments, the weight parts of methacrylamide gelatin in the hemostatic hydrogel nanoparticle can be any two values between 25 and 50, 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, or 50.

In some embodiments, the parts by weight of photoinitiator in the hemostatic hydrogel particles may be any two values between 0.5 and 1.25, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, or 1.25.

In some specific embodiments, the degree of substitution of amino groups in the methacrylamide gelatin (GelMA) is 30-90%, the molecular weight is 100-200kD, the degree of substitution of amino groups is the ratio of substitution of amino groups in the gelatin by methacrylate groups in the synthesis process of the methacrylamide gelatin (GelMA), and under the same conditions, the higher the degree of substitution, the stronger the strength of the solidified GelMA hydrogel.

In some specific embodiments, the photoinitiator is selected from one of phenyl (2, 4, 6-trimethylbenzoyl) lithium phosphate (LAP), 2-hydroxy-40- (2-hydroxyethoxy) -2-methyl propenone (IC-2959), 2' -azo [ 2-methyl-N- (2-hydroxyethyl) propanamide (VA 086), triethanolamine (TEA), N-Vinyl Caprolactam (VC) and eosin-y (EY), and the initiating light source is blue laser with a wavelength of 400-420 nm, white light with a wavelength of 300-500 nm or ultraviolet light with a wavelength of 200-400 nm.

The principle of the crosslinking reaction of the photo-induced luminescence initiator is that in the photo-crosslinking process, the photo-induced luminescence initiator absorbs the light energy with specific wavelength and is excited to an excited state, active substances such as free radicals or ions are generated, and the substances trigger the crosslinking reaction of acrylic acid groups in the methacrylamidoglycolate (GelMA), so that a three-dimensional network structure is formed, and the reaction is shown in figure 11.

The various reagents/instruments used in the examples and comparative examples of the present invention are conventional commercial products unless otherwise specified. The experimental materials and instrument information sources used in the present invention are shown in table 1 below:

TABLE 1

EXAMPLE 1 preparation of hemostatic hydrogel nanoparticles

Example 1-1

1. Preparation of aminated dendritic mesoporous silica nanoparticles

0.5G of cetyltrimethylammonium bromide (CTAB) was dissolved in an emulsion system consisting of 70mL H ₂O、0.8mL NH₄ OH, 15mL dehydrated ether and 5mL dehydrated alcohol. The emulsion is mixed vigorously by using a magnetic stirrer, and the stirring speed of 1000rpm is kept for 0.5h, so that the emulsion is mixed uniformly and stably. A mixture of 2.5mL Tetraethoxysilane (TEOS) and 0.1mL 3-aminopropyl triethoxysilane (APTES) was quickly dropped into the above emulsion, and after vigorously stirring at 1000rpm for 4 hours, 1mL concentrated hydrochloric acid was added to stop the base-catalyzed reaction. Centrifuging for 10min at 2500-10000rpm to obtain white precipitate, which is the amino dendritic mesoporous silica nanoparticle. The aminated dendritic mesoporous silica nano particles are respectively washed with absolute ethyl alcohol and deionized water for 3 times, and are dispersed in the ethyl alcohol for preservation. And removing CTAB in the amination dendritic mesoporous silica nano-particles by using an extraction method. The aminated dendritic mesoporous silica nanoparticles were sufficiently dispersed in a mixed solution consisting of 15mL of concentrated hydrochloric acid and 120mL of absolute ethanol, and stirred at 70 ℃ for 24 hours. Centrifuge at 8000rpm for 10min and wash 3 times with deionized water. The amination dendritic mesoporous silica nanoparticle is subjected to freeze drying to obtain about 700mg of amination dendritic mesoporous silica, and the amination dendritic mesoporous silica nanoparticle is stored for standby.

2. Preparation of thrombin-loaded aminated dendritic mesoporous silica nanoparticles

Firstly, exploring thrombin load capacity of the amination dendritic mesoporous silica nano-particles, weighing 0.1, 0.2, 0.5, 0.8, 1, 2, 3, 4 and 5mg of porcine thrombin powder and 1mL of pure water to prepare thrombin solutions with the concentration of 0.1, 0.2, 0.5, 0.8, 1, 2, 3, 4 and 5mg/mL, respectively mixing the thrombin solutions with 1mg of the amination dendritic mesoporous silica powder, measuring the change of the concentration of the porcine thrombin after the solution is loaded by using a BCA protein concentration measuring kit, calculating the content of the porcine thrombin according to the volume, substituting the content into a formula, and calculating the loading rate and the encapsulation rate (the loading rate=the mass of the loaded porcine thrombin/the mass of the silica nano-particles loaded with the porcine thrombin), wherein the encapsulation rate=the mass of the loaded porcine thrombin/the mass of the used porcine thrombin), and the optimal loading rate reaches 39.62% at the ratio of 1:2 as shown in a measuring result.

And preparing the amination dendritic mesoporous silica nano particles loaded with the porcine thrombin, weighing 100mg of the amination dendritic mesoporous silica nano particles, mixing with 200mg of the porcine thrombin, dissolving the amination dendritic mesoporous silica nano particles and the porcine thrombin in 1ml of pure water according to the mass ratio of 1:2, carrying out ultrasonic treatment for 30min, carrying out stirring for 30min, loading the amination dendritic mesoporous silica nano particles with the thrombin through electrostatic adsorption, measuring the concentration of the porcine thrombin loaded with the solution through a BCA protein concentration measuring kit, and calculating the content of the porcine thrombin according to the volume. 100mg of the amination dendritic mesoporous silica nanoparticle is loaded with 66mg of porcine thrombin, and the concentration (g/ml) of the solution of the mesoporous silica nanoparticle loaded with the porcine thrombin is 16.6%.

3. Preparation of GelMA premix

0.1G of methacrylamidoglyco (GelMA) (the amino substitution degree of which is 60%) and 2.5mg of the photoinitiator phenyl (2, 4, 6-trimethylbenzoyl) lithium phosphate (LAP) were weighed and dissolved in 1mL of pure water to prepare a GelMA premix containing GelMA at a concentration (g/mL) of 10% and LAP at a concentration (g/mL) of 0.25%.

4. Preparation of hemostatic hydrogel nanoparticles

Mixing the amination dendritic mesoporous silica nanoparticle solution loaded with the pig thrombin with GelMA premix according to the volume ratio of 1:1, performing ultrasonic treatment for 30min, stirring for 6h, centrifuging to remove supernatant, and separating hemostatic nano hydrogel particles (the amination dendritic mesoporous silica sediment coated with the pig thrombin and loaded with the outer ring GelMA). And (3) after excitation by blue light of 400-420 nm, obtaining the blue light-excited GelMA-coated amination dendritic mesoporous silica loaded with the porcine thrombin. The prepared hemostatic hydrogel nanoparticle was analyzed by transmission electron microscope model HT7700, as shown in fig. 1. Wherein A is an amination dendritic mesoporous silica nanoparticle, B is a thrombin-loaded amination dendritic mesoporous silica nanoparticle, C is a thrombin-loaded amination dendritic mesoporous silica wrapped by GelMA, and D is a thrombin-loaded amination dendritic mesoporous silica wrapped by GelMA after blue light excitation. As can be seen from the graph D, the prepared hemostatic nano hydrogel particles are dendritic spherical particles, and GelMA is wrapped on the outer ring, and the diameter of the hemostatic nano hydrogel particles is about 100-200 nm.

And (3) measuring the prepared hemostatic nano hydrogel particles by a Zeta potential analyzer, wherein thrombin is detected to be negative, the aminated dendritic mesoporous silica is detected to be positive, the aminated dendritic mesoporous silica loaded with thrombin is detected to be negative, gelMA is detected to be negative, the aminated dendritic mesoporous silica loaded with thrombin and GelMA is detected to be more negative, the successful preparation of the material is proved, and a Zeta potential diagram is shown in figure 3.

Examples 1 to 2

1. Preparation of spherical mesoporous silica nanoparticles

0.5G of cetyltrimethylammonium bromide (CTAB) was dissolved in an emulsion system consisting of 70mL H ₂O、0.8mL NH₄ OH, 15mL dehydrated ether and 5mL dehydrated alcohol. The emulsion is mixed vigorously by using a magnetic stirrer, and the stirring speed of 1000rpm is kept for 0.5h, so that the emulsion is mixed uniformly and stably. 2.5mL of Tetraethoxysilane (TEOS) was quickly dropped into the above emulsion, and after vigorously stirring at 1000rpm for 4 hours, 1mL of concentrated hydrochloric acid was added to stop the base-catalyzed reaction. Centrifuging for 10min at 2500-10000rpm to obtain white precipitate 1g, which is spherical mesoporous silica nanoparticle.

2. Preparation of thrombin-loaded spherical mesoporous silica nanoparticles

Weighing 80mg of spherical mesoporous silica nano particles, mixing with 180mg of bovine thrombin solution, wherein the mass ratio of the spherical mesoporous silica nano particles to the bovine thrombin solution is 1:2.3, carrying out ultrasonic treatment for 25min, the ultrasonic power is 45khz, stirring for 25min, and loading the spherical mesoporous silica nano particles with bovine thrombin through electrostatic adsorption. The concentration of bovine thrombin after the loading of the solution was measured by BCA protein concentration measurement kit, and the bovine thrombin content was calculated from the volume. 80mg of spherical mesoporous silica nanoparticles are loaded with 40mg of bovine thrombin, and the concentration (g/ml) of the bovine thrombin loaded spherical mesoporous silica nanoparticle solution is 12%.

3. Preparation of GelMA premix

0.05G of methacrylamidoglyco (GelMA) (the amino substitution degree of which is 30%) and 1mg of the photoinitiator 2-hydroxy-40- (2-hydroxyethoxy) -2-methyl propenone (IC-2959) are weighed and dissolved in 1ml of phosphate buffer to prepare a GelMA premix which contains GelMA with a concentration (g/ml) of 5% and IC-2959 with a concentration (g/ml) of 0.1%.

4. Preparation of hemostatic hydrogel nanoparticles

Mixing spherical mesoporous silica nano particles loaded with bovine thrombin and GelMA premix according to a volume ratio of 1:1, performing ultrasonic treatment for 25min, performing ultrasonic treatment with power of 45khz, stirring for 7h, and centrifuging to remove supernatant, thereby separating hemostatic nano hydrogel particles (spherical mesoporous silica solution coated with GelMA and loaded with bovine thrombin). And (3) after white light with the wavelength of 300-500 nm is excited, obtaining the white light excited GelMA coated bovine thrombin-loaded spherical mesoporous silica nanoparticle.

Examples 1 to 3

1. Preparation of dendritic mesoporous silica nanoparticles

Tetraethyl orthosilicate (TETRAETHYL ORTHOSILICATE, TEOS) as a silicon source was dissolved in cyclohexane as an upper oil phase, CTAB as a template agent and triethanolamine (Triethanolamine, TEA) was dissolved in water as a lower aqueous phase, and an oil-water two-phase layered system was constructed. Into a 100ml single neck round bottom flask was added 36ml water, 24ml 25wt.% CTAB and 0.18g triethanolamine, and gentle stirring was performed at 60 ℃ oil bath temperature. After one hour, 20ml of a cyclohexane solution of tetraethyl orthosilicate in 20 v.% was carefully added dropwise to the upper layer of the aqueous phase. The temperature was kept at 60 ℃ and stirring was slow, the standard being that the lower aqueous phase was stirred uniformly and the upper oil phase was essentially stationary, and the reaction was carried out under these conditions for 12 hours. After 12 hours, the aqueous phase was centrifuged at 15000rpm for 30min, the product was collected and dispersed in ethanol solution, extracted 3 times at 60 ℃ for 12 hours each to remove surfactant CTAC. And finally, respectively washing the obtained precipitate with ethanol and deionized water for three times, centrifuging and vacuum drying at room temperature to obtain the dendritic mesoporous silica nanoparticle.

2. Preparation of thrombin-loaded dendritic mesoporous silica nanoparticles

86Mg of dendritic mesoporous silica nano-particles are weighed and mixed with 210mg of pig thrombin solution, the mass ratio of the dendritic mesoporous silica nano-particles to the pig thrombin solution is 1:2.4, the ultrasonic power is 40khz for 35min, the stirring is carried out for 35min, and the dendritic mesoporous silica nano-particles are loaded with the pig thrombin through electrostatic adsorption. The concentration of the pig thrombin after the solution loading is measured by the BCA protein concentration measuring kit, and the content of the pig thrombin is calculated according to the volume. 50mg of pig thrombin is loaded on 86mg of dendritic mesoporous silica nanoparticle, and the concentration (g/ml) of the dendritic mesoporous silica nanoparticle solution loaded with pig thrombin is 13.6%.

3. Preparation of GelMA premix

0.06G of methacrylamidoglyco (GelMA) (the degree of substitution of the amino group is 90%) and 1.6mg of the photoinitiator 2,2' -azo [ 2-methyl-n- (2-hydroxyethyl) propionamide (VA 086) were weighed and dissolved in 1ml of physiological saline to prepare a GelMA premix containing GelMA at a concentration (g/ml) of 6% and VA086 at a concentration (g/ml) of 0.16%.

4. Preparation of hemostatic hydrogel nanoparticles

Mixing dendritic mesoporous silica nano particles loaded with porcine thrombin with GelMA premix according to a volume ratio of 1:1, performing ultrasonic treatment for 35min, performing ultrasonic power of 40khz, stirring for 6h overnight, and centrifuging to remove supernatant, thereby separating hemostatic nano hydrogel particles (spherical mesoporous silica solution coated with GelMA and loaded with porcine thrombin). And after excitation, obtaining the dendritic mesoporous silica nanoparticle which is excited by ultraviolet light and is coated by GelMA and is loaded with porcine thrombin.

Examples 1 to 4

1. Preparation of hollow mesoporous silica nanoparticles

First, 1.6mL of ammonia was added to a mixture of 71.4mL of anhydrous ethanol and 1.6mL of water. 2mL of TEOS was added to the above mixture, and the reaction was carried out at 400rpm for 1 hour at room temperature. After 1h, the reaction solution was collected in a centrifuge tube, centrifuged at 10000rpm for 15min, washed with ethanol and ultrapure water 2 times, and the obtained precipitate was resuspended in 40mL of ultrapure water for use.

Next, 1.5g CTAB and 4.5mL of water were weighed into a 250mL flask, 53.4. Mu.L of triethanolamine and 60mL of deionized water were then added, respectively, and after stirring vigorously at 80℃for 5min, they were completely dissolved, 30mL of the above precipitate solution was added, stirring was continued for 20min, and after adding 450. Mu.L of TEOS, stirring was continued for 1h. After the reaction, the solution was cooled to 50℃and 5.67g of anhydrous sodium carbonate was dissolved in 9mL of water, the reaction was continued for 2 hours, and the solution was collected in a centrifuge tube, centrifuged at 10000rpm for 15 minutes, and then washed with anhydrous ethanol for 3 times.

Finally, 1.5g CTAB, 53.4. Mu.L TEA and 64.5mL water were thoroughly dissolved with vigorous stirring at 80 ℃. 30mL of the SiO ₂ suspension is added, stirring is continued for 20min, 450 mu LTEOS is added, incubation is carried out for 1h, cooling is carried out to 50 ℃, and then 9mL of saturated Na ₂CO₃ solution is added for reaction for 2h. After the reaction, the solution was collected and centrifuged (10000 rpm,15 min) to obtain hollow mesoporous silica nanoparticles.

2. Preparation of hollow mesoporous silica nanoparticle loaded with thrombin

94Mg of hollow mesoporous silica nano particles are weighed and mixed with 200mg of human thrombin solution, the mass ratio of the hollow mesoporous silica nano particles to the human thrombin solution is 1:2.1, the ultrasonic power is 40khz for 35min, the stirring is carried out for 35min, and the hollow mesoporous silica nano particles are loaded with human thrombin through electrostatic adsorption. The concentration of human thrombin after the loading of the solution was measured by BCA protein concentration measurement kit, and the content of human thrombin was calculated from the volume. 94mg of hollow mesoporous silica nanoparticle is loaded with 60mg of human thrombin, and the concentration (g/ml) of the hollow mesoporous silica nanoparticle solution loaded with human thrombin is 15.4%.

3. Preparation of GelMA premix

0.08G of methacrylamidoglyco (GelMA) (the amino substitution degree of which is 60%) and 2.0mg of Triethanolamine (TEA) serving as a photoinitiator were weighed and dissolved in 1ml of pure water to prepare a GelMA premix containing GelMA with a concentration (g/ml) of 8% and TEA with a concentration (g/ml) of 0.2%.

4. Preparation of hemostatic hydrogel nanoparticles

Mixing hollow mesoporous silica nanoparticles loaded with human thrombin with GelMA premix according to a volume ratio of 1:1, performing ultrasonic treatment for 35min with ultrasonic power of 40khz, stirring for 6h overnight, centrifuging to remove supernatant, and separating hemostatic nano hydrogel particles (hollow mesoporous silica nanoparticles coated with GelMA and loaded with human thrombin). And after excitation, obtaining the ultraviolet light-excited GelMA-coated hollow mesoporous silica nanoparticle loaded with human thrombin.

Examples 1 to 5

In comparison with example 1-1, N-Vinylcaprolactam (VC) was used as photoinitiator, the remainder being the same as in example 1-1.

Examples 1 to 6

In comparison with example 1-1, eosin-y (EY) was used as the photoinitiator, and the rest was the same as in example 1-1.

Examples 1 to 7

In contrast to example 1-1, the mesoporous silica nanoparticles were selected from the MCM family, the remainder being the same as in example 1-1.

Examples 1 to 8

In contrast to example 1-1, the mesoporous silica nanoparticles were selected from the SBA series, the remainder being the same as in example 1-1.

Examples 1 to 9

In contrast to example 1-1, the mesoporous silica nanoparticles were selected from the MSU series, the remainder being the same as in example 1-1.

Examples 1 to 10

In contrast to example 1-1, the mesoporous silica nanoparticles were selected from the TDU series, the remainder being the same as in example 1-1.

Experimental example 1 hemostatic Effect verification of different hemostatic materials

The hemostatic capacity of the hemostatic hydrogel nanoparticle prepared in example 1-1 was verified. Preparing fresh rat blood containing sodium citrate anticoagulant, respectively taking 100 mu L of blood sample, placing the blood sample into a 4mL centrifuge tube, adding different materials for testing, (1) adding 50 mu L of physiological saline, (2) adding 50 mu L of thrombin solution loaded by the hemostatic nano hydrogel particles in the same way as in example 1-1, standing for 30s, (3) adding 50 mu L of 100mg/mL of amino dendritic mesoporous silica solution, standing for 30s, (4) adding 50 mu L of 100mg/mL of thrombin-loaded amino dendritic mesoporous silica solution, standing for 30s, and (5) simultaneously adding 50 mu L of 100mg/mL of GelMA-coated amino dendritic mesoporous silica solution loaded with thrombin for 30s, and irradiating by a 365nm ultraviolet lamp. The centrifuge tube was then placed upside down for observation. The results are shown in fig. 4, wherein A is a blood coagulation effect graph obtained by blending blood and normal saline, B is a blood coagulation effect graph obtained by blending blood and thrombin solution, C is a blood coagulation effect graph obtained by blending blood and aminated dendritic mesoporous silica, D is a blood coagulation effect graph obtained by blending blood and thrombin-loaded aminated dendritic mesoporous silica, and E is a blood coagulation effect graph obtained by blending blood and thrombin-loaded aminated dendritic mesoporous silica coated with GelMA and blue laser crosslinking. In the graph E, after the GelMA-coated thrombin-loaded amination dendritic mesoporous silica solution is added and ultraviolet light is crosslinked, blood forms a stable blood clot, and liquid cannot flow downwards after a centrifuge tube is inverted, so that the hemostatic nano hydrogel particles prepared in the embodiment 1-1 can promote blood coagulation rapidly.

To investigate the mechanism of the hydrogel prepared in example 1-1 to promote in vitro coagulation, observation was carried out using a scanning electron microscope of Hitachi S-3400N. The method comprises the following steps:

And (3) respectively adding glutaraldehyde solution with the volume ratio of 2.5% into each group of blood clots to fix for 2-3h, and sequentially dehydrating with ethanol solution with the volume ratio of 30%, 50%, 70%, 90% and 100% diluted by pure water for three times, wherein each concentration is dehydrated for 10 minutes. The water and ethanol were then displaced with a t-butanol solution 3 times for 10 minutes each. And finally, performing metal spraying on the treated sample, and observing by a scanning electron microscope.

The results are shown in fig. 5, wherein A is a scanning electron microscope image of blood and normal saline after being mixed to form blood clots, B is a scanning electron microscope image of blood and thrombin solution after being mixed to form blood clots, C is a scanning electron microscope image of blood and amino dendritic mesoporous silica after being mixed to form blood clots, D is a scanning electron microscope image of blood and thrombin-loaded amino dendritic mesoporous silica after being mixed to form blood clots, and E is a scanning electron microscope image of blood and thrombin-loaded amino dendritic mesoporous silica wrapped by outer ring GelMA after being mixed and crosslinked by ultraviolet light. From figure E, the hemostatic nano-hydrogel particles self-assemble into a fibrous network structure in blood and enwind blood cells, which proves that the hemostatic nano-hydrogel can promote blood coagulation. Among them, thrombin promotes the formation of a large amount of fibrin network, and GelMA cross-links the network to further strengthen hemostasis.

Experimental example 2 verification of hemostatic time of different hemostatic materials for normal blood and blood of patient

The hemostatic nano-hydrogel particles prepared in example 1-1 were validated for hemostatic time in normal blood and blood of patients with cirrhosis.

Normal human blood in a blood collection tube is added into an EP tube, then 0.1M calcium chloride solution is added and mixed uniformly, wherein after mixing for 10 seconds, 50 mu L of the blood mixture is added into a 96-well plate, 20 mu L of each group of different materials (a control group, an amino dendritic mesoporous silica group, a thrombin group, an amino dendritic mesoporous silica group loaded with thrombin and GelMA, the control group is normal saline, the amino dendritic mesoporous silica groups loaded with thrombin and GelMA need to be irradiated by a blue laser irradiation lamp), and when 5s, 10s, 30s, 60s, 180s, 300s and 420s are mixed, the plate is washed by normal saline to stop coagulation, the liquid is rapidly pumped and repeatedly washed until the solution becomes clear, and all soluble blood components are removed. If a uniform clot formation is observed, clotting time is recorded and a statistical plot is drawn with origin, the significance level is set to P <0.05 for significant differences, P <0.01 for very significant differences, and ns for no significant differences. The results are shown in FIG. 6, wherein A is a graph of the coagulation effect of the different materials for initiating coagulation, and B is a statistical graph of the coagulation time of the different materials.

Fresh liver cirrhosis patient blood was prepared, liver cirrhosis patient blood in blood collection tube was added to EP tube, then 0.1M calcium chloride solution was added and mixed well, wherein the ratio of blood to calcium chloride solution was 10:1, after mixing 10s, 50 μl of blood mixture was added to 96 well plate, respectively, 20 μl of each of the different material sample solutions (control group, aminated dendritic mesoporous silica group, thrombin-loaded aminated dendritic mesoporous silica group and thrombin-and GelMA-loaded aminated dendritic mesoporous silica group, control group was normal saline, thrombin-and GelMA-loaded aminated dendritic mesoporous silica group was irradiated with blue laser light), the well plate was rinsed with physiological saline at time intervals of 5s, 10s, 30s, 60s, 180s, 300s, 420s and 480s, respectively, the liquid was rapidly aspirated and repeatedly rinsed until the solution became clear, indicating that all soluble blood components were removed. If a uniform clot formation is observed, clotting time is recorded and a statistical plot is drawn with origin, the significance level is set to P <0.05 for significant differences, P <0.01 for very significant differences, and ns for no significant differences. The results are shown in FIG. 7, wherein A is a graph of the coagulation effect of the different materials for initiating coagulation, and B is a statistical graph of the coagulation time of the different materials. As can be seen from fig. 6 and fig. 7, the start coagulation time of the hemostatic nano hydrogel particle group is significantly earlier than that of the thrombin group for the hemostatic time of the normal blood and the blood of the liver cirrhosis patient, which indicates that the hemostatic nano hydrogel particles have rapid and efficient hemostatic capability for the normal blood and the blood of the liver cirrhosis patient.

Experimental example 3 PH tolerance and thrombin Activity protection ability of hemostatic hydrogel nanoparticle

100Mg of each of the different materials (thrombin group, thrombin-loaded aminated dendritic mesoporous silica group and thrombin-and GelMA-loaded aminated dendritic mesoporous silica group, which were irradiated with blue laser irradiation lamp) were mixed with 1ml of simulated gastric fluid and 1ml of simulated intestinal fluid for 5s, 10s, 30s, 60s, 3min, 5min, 10min, 30min, 60min, respectively, and then the mixed solution was added to a test tube containing 100. Mu.L of fresh blood containing sodium citrate anticoagulant, left standing for 30s, and the test tube was placed upside down for observation. The results are shown in FIG. 8, wherein A is a graph of the coagulation effect of the different materials after the simulated gastric fluid treatment, and B is a graph of the coagulation effect of the different materials after the simulated intestinal fluid treatment. The hemostatic nano hydrogel particle group still keeps a certain blood coagulation effect in simulated gastric fluid for 60min, the blood coagulation effect of the thrombin group is completely ineffective in 5min, and the blood coagulation effect of the hemostatic nano hydrogel particle group and the thrombin group in simulated intestinal fluid is not obviously reduced. The hemostatic nano hydrogel particles can effectively resist the degradation of artificial gastric juice and intestinal juice, and have the potential of being used for hemostasis of digestive tract mucous membranes in vivo.

Experimental example 4 hemostatic nanohydrogel particles for liver hemostasis in mice

The experimental unit is the second affiliated hospital gastroenterology laboratory of army medical university, and the experimental batch number is AMUWEC20235153.

Normal-bred 20-25g mice were anesthetized with 1% pentobarbital solution prepared from physiological saline, then the liver was exposed, a part of liver lobules was cut off with tissue scissors to construct a liver hemorrhage model, then 100 μl of thrombin solution and 100 μl of hemostatic nano hydrogel particles of example 1-1 were simultaneously added dropwise with a pipette, and irradiated with 405nm blue laser for 30s. The control was left untreated and wound bleeding was observed and recorded by photographing.

The results are shown in FIG. 9, wherein A is an untreated wound map, B is a wound map after thrombin solution is applied, C is a map after hemostatic nano hydrogel particles are applied and subjected to ultraviolet crosslinking treatment, and D-F is a scanning electron microscope result map of an isolated liver tissue wound site specimen after hemostasis. The best hemostatic effect of the hemostatic nano-hydrogel group can be seen in the figures A-C. D-F shows that no fibrin net is generated in the control group, the thrombin group only contains fibrin net, and the fibrin net structure and the methacrylamide gelatin in the blood clot of the hemostatic nano hydrogel particle group are crosslinked into a net structure to be mutually wrapped to form a complex.

Experimental example 5 hemostatic nanoparticle for hemostatic of piglets under blue laser endoscope

Experimental Unit, army medical university, second affiliated Hospital gastroenterology laboratory, experimental lot number AMUWEC20235153.

An endoscope model FUJIFILM (EG-L590 ZW) was used as a device for injecting the hydrogel hemostatic agent of example 1-1, and a blue laser light source provided in the endoscope itself was used as an irradiation light source. Firstly, respectively constructing a small fragrant pig acute non-varicose upper gastrointestinal tract bleeding model through mucous membrane layers of the jaw parts of esophagus, stomach and duodenum, respectively injecting 1.5mL 100mg/ml hemostatic nano hydrogel particle solution into a wound bleeding part by using a single-pass endoscope catheter, and irradiating by using a blue laser mode.

The results are shown in FIG. 10, wherein A is an esophageal hemorrhage hemostasis effect graph, B is a gastric hemorrhage hemostasis effect graph, and C is a duodenal hemorrhage hemostasis effect graph. Under the endoscope, the hemostatic nano hydrogel particles can be observed to be respectively attached to the wounds of esophagus, stomach and duodenum, and the hemostatic nano hydrogel particles can be hemostatic to form a stable blood clot after being irradiated by blue laser for less than 30 seconds, so that the bleeding part is effectively blocked, and the bleeding amount is obviously reduced. After 5min observation, the wound has no active bleeding, and the nano hydrogel particles and blood clots formed by blood are firmly attached to the bleeding part after flushing, so that the hydrogel can realize visual hemostasis under a blue laser endoscope and rapidly and effectively plug the gastrointestinal mucosa bleeding.

Experimental example 6 preservation time verification of hemostatic hydrogel nanoparticles

Freeze-drying hemostatic nano hydrogel particles, storing in a refrigerator at 4 ℃ for 45 days, preparing 100mg/mL solution by pure water, collecting 100 mu L of fresh blood of a rat into a 4mL centrifuge tube, simultaneously dripping 50 mu L of hemostatic nano hydrogel particle solution, irradiating for 30s by a 365nm ultraviolet lamp, and placing the centrifuge tube upside down for observation. As shown in FIG. 12, the blood is coagulated and does not flow down along the wall of the tube, and it is verified that the hemostatic nano-hydrogel particles can maintain the thrombin activity for 45 days or longer.

While the foregoing describes the embodiments of the present invention, it is not intended to limit the scope of the present invention, and on the basis of the technical solutions of the present invention, various modifications or variations may be made by those skilled in the art without the need for inventive labor.

Claims (20)

1. The hemostatic nano hydrogel particle is characterized by being prepared from 40-50 parts of mesoporous silica nano particles, 20-33 parts of thrombin, 25-50 parts of methacrylamide gelatin and 0.5-1.25 parts of photoinitiator by weight.

2. The hemostatic hydrogel nanoparticle according to claim 1, wherein the hemostatic hydrogel nanoparticle is prepared from 43-47 parts by weight of mesoporous silica nanoparticles, 25-30 parts by weight of thrombin, 30-40 parts by weight of methacrylamide gelatin and 0.8-1 part by weight of a photoinitiator.

3. The hemostatic hydrogel particle of claim 1 or 2, wherein the mesoporous silica nanoparticle is selected from one of a spherical mesoporous silica nanoparticle, a dendritic mesoporous silica nanoparticle, and a hollow mesoporous silica nanoparticle;

and/or, the mesoporous silica nanoparticle is selected from one of MCM series, SBA series, MSU series and TDU series;

Preferably, the mesoporous silica nanoparticle is selected from aminated dendritic mesoporous silica nanoparticles.

4. The hemostatic nano hydrogel particle according to any one of claims 1-3 wherein the thrombin is selected from one of porcine thrombin, bovine thrombin and human thrombin;

Preferably, the hemostatic nano hydrogel particles can maintain the thrombin activity for more than or equal to 45 days.

5. The hemostatic hydrogel nanoparticle of any one of claims 1-4, wherein the methacrylamide gelatin has an amino substitution degree of 30-90%;

Preferably, the amino substitution degree of the methacrylamide gelatin is 60-90%.

6. The hemostatic hydrogel particle of any one of claims 1-5, wherein the photoinitiator is selected from one of phenyl (2, 4, 6-trimethylbenzoyl) lithium phosphate (LAP), 2-hydroxy-40- (2-hydroxyethoxy) -2-methyl propenone (IC-2959), 2' -azo [ 2-methyl-N- (2-hydroxyethyl) propanamide (VA 086), triethanolamine (TEA), N-Vinylcaprolactam (VC), and eos-y (EY);

Preferably, the photoinitiator is selected from the group consisting of phenyl (2, 4, 6-trimethylbenzoyl) lithium phosphate (LAP).

7. The hemostatic hydrogel nanoparticle according to any one of claims 1-6, wherein the hemostatic hydrogel nanoparticle has an initiating light source of blue laser light having a wavelength of 395-425nm, white light having a wavelength of 300-760 nm, or ultraviolet light having a wavelength of 200-400 nm;

preferably, the hemostatic nano hydrogel particles have an initiation light source of blue laser with wavelength of 395-425 nm.

8. A method of preparing the hemostatic nano hydrogel particles according to any one of claims 1-7, the method comprising the steps of:

(1) Mixing mesoporous silica nano particles with thrombin, dissolving the mixture in pure water, carrying out ultrasonic treatment, and stirring to obtain thrombin-loaded mesoporous silica nano particle solution;

(2) Dissolving methacrylamide gelatin and a photoinitiator in a solvent to obtain GelMA premix;

(3) Mixing the thrombin-loaded mesoporous silica nanoparticle solution with GelMA premix, performing ultrasonic treatment, stirring, centrifuging, and taking out precipitate to obtain hemostatic nano hydrogel particles.

9. The method for preparing hemostatic hydrogel nanoparticle according to claim 8, wherein in step (1), the concentration (g/ml) of the thrombin-loaded mesoporous silica nanoparticle solution is 12-16.6%;

preferably, the concentration (g/ml) of the thrombin-loaded mesoporous silica nanoparticle solution is 13.6-15.4%;

Further preferably, the mesoporous silica nanoparticle is mixed with thrombin in a mass ratio of 1 (2-2.44).

10. The method for preparing hemostatic nano hydrogel particles according to claim 8 or 9, wherein in step (1), the time of ultrasonic treatment is 25-35min;

And/or the power of the ultrasound is 40-45khz;

Preferably, the stirring time is 25-35min.

11. The method for preparing hemostatic hydrogel particles according to any one of claims 8-10, wherein in step (2), the solvent is selected from one of pure water, physiological saline and phosphate buffer.

12. The method for preparing hemostatic nano hydrogel particles according to any one of claims 8-11, wherein in step (2), the concentration (g/ml) of methacrylamidoglycolate in the GelMA premix is 5-30%;

Preferably, the concentration (g/ml) of the methacrylamidoglycolate in the GelMA premix solution is 5-10%;

further preferably, the concentration (g/ml) of the methacrylamidoglycolate in the GelMA premix is 6-8%.

13. The method for preparing hemostatic nano hydrogel particles according to any one of claims 8-12, wherein in step (2), the concentration (g/ml) of the photoinitiator in the GelMA premix is 0.10% -0.40%;

Preferably, the concentration (g/ml) of the photoinitiator in the GelMA premix is 0.16-0.25%.

14. The method for preparing hemostatic nano hydrogel particles according to any one of claims 8-13, wherein in the step (3), the thrombin-loaded mesoporous silica nanoparticle solution and the GelMA premix are mixed in a volume ratio of 1:2-2:1;

preferably, the thrombin-loaded mesoporous silica nanoparticle solution is mixed with the GelMA premix solution according to the volume ratio of 1:1;

Further preferably, the time of the ultrasonic treatment is 25-35min;

And/or the power of the ultrasound is 40-45khz;

more preferably, the stirring time is 6 to 7 hours.

15. A hemostatic nano hydrogel particle prepared according to the method of preparing the hemostatic nano hydrogel particle according to any one of claims 8-14.

16. A hemostatic system comprising hemostatic hydrogel nanoparticle according to any one of claims 1-7 or 15, a hemostatic hydrogel nanoparticle injection device, and an initiating light source generating device.

17. The hemostatic system of claim 16, wherein the hemostatic hydrogel nanoparticle injection device comprises a syringe and a digestive endoscope tube;

preferably, the initiating light source generating device comprises a blue laser emitting lamp, a blue laser endoscope, a narrow-band imaging endoscope and an ultraviolet light emitting lamp.

18. Use of the hemostatic nano hydrogel particle according to any one of claims 1-7 or 15 in the preparation of a hemostatic material or medicament.

19. Use of the hemostatic nano hydrogel particles according to any one of claims 1-7 or 15 in the preparation of an organ hemostatic material or medicament.

20. The use according to claim 19, wherein the organ is a parenchymal organ or a hollow organ;

Preferably, the substantial organ is a heart, liver, spleen, lung, pancreas or kidney;

further preferably, the hollow organ is esophagus, stomach, duodenum, jejunum, ileum or colon.