WO2019181294A1

WO2019181294A1 - Hydrogel containing sulfonated cellulose nanofibers

Info

Publication number: WO2019181294A1
Application number: PCT/JP2019/005245
Authority: WO
Inventors: 昭広西口
Original assignee: 国立研究開発法人物質・材料研究機構
Priority date: 2018-03-23
Filing date: 2019-02-14
Publication date: 2019-09-26
Also published as: JPWO2019181294A1; JP6869518B2

Abstract

The purpose of the present invention is to provide an injectable hydrogel which can be easily delivered with a catheter, etc. and has excellent substance permeability. The hydrogel according to an embodiment of the present invention contains nanofibers of sulfonated cellulose having a sulfone group in an amount of 0.1-7 mmol/g and at least one biodegradable polymer containing primary amino groups which is selected from the group consisting of gelatin, chitosan, collagen, albumin, fibronectin, laminin, elastin, and derivatives thereof, the weight ratio of the nanofibers to the at least one biodegradable polymer being 1:99 to 70:30.

Description

Hydrogels containing sulfonated cellulose nanofibers

The present invention relates to a biodegradable hydrogel, and more particularly to an injectable hydrogel comprising a sulfonated cellulose nanofiber and a biodegradable polymer having a primary amino group.

Catheter treatment is widely used in medical settings such as cerebral aneurysm treatment as a minimally invasive treatment method. A cerebral aneurysm is a disease in which part of a blood vessel in the brain swells and causes subarachnoid hemorrhage when it ruptures. Currently, a treatment method for closing an aneurysm with an embolic material using a catheter is widespread, and it is important as a minimally invasive treatment method that does not require craniotomy.

Coils have been widely used as an embolic material so far, such as Matrix 2 coils coated with polyglycolic acid / lactic acid copolymer (Stryker, SA) and Hydrocoil embroidery system coated with non-degradable polymers (Microvention / Terumo, Japan) is used. However, there are problems such as the risk of rupturing the aneurysm during the operation, a low filling rate, insufficient endothelialization after filling, and non-degradability. The liquid embolic material can completely fill the aneurysm, while using a non-degradable polymer such as cyanoacrylate (NBCA) or ethylene vinyl alcohol (EVOH) copolymer (Onyx, Covidien). There is a risk of embolization in other organs due to high cytotoxicity and their leakage. Therefore, there is a demand for the development of an injectable embolic material that can be delivered by a catheter or the like.

As an injectable embolic material, a hydrogel in which cellulose nanocrystals or whiskers are added to the polymer to increase the elastic modulus has been proposed. For example, a hydrogel containing hyaluronic acid modified with adipic acid dihydrazide or the like and aldehyde-modified cellulose nanofiber (Non-patent Document 1), a hydrogel containing carboxymethyl cellulose, dextran and aldehyde-modified cellulose nanofiber (Non-Patent Document 2), A hydrogel containing gelatin and aldehyde-modified cellulose nanowhiskers (Non-patent Document 3) has been proposed.

The hydrogel containing cellulose nanocrystals or whiskers must be separately injected with nanocrystals or whiskers and hyaluronic acid using a double barrel syringe. Compared to annoying. This is because when nanocrystals or whiskers and hyaluronic acid are mixed, they immediately react with each other to form a cross-linked structure consisting of covalent bonds, resulting in gelation, and delivery becomes impossible or requires great force. This is because the gel structure may be damaged if it is injected into the gel. Moreover, the pore size in the gel is limited by the cross-linked structure, and the substance permeation may be inhibited.

Therefore, an object of the present invention is to provide an injectable hydrogel that can be more easily delivered with a catheter or the like and has excellent substance permeability.

That is, the present invention is as follows:
A sulfonated cellulose nanofiber having a sulfone group at 0.1 to 7 mmol / g, and a primary amino group selected from the group consisting of gelatin, chitosan, collagen, albumin, fibronectin, laminin, elastin and derivatives thereof A hydrogel comprising at least one biodegradable polymer having a weight ratio of 1:99 to 70:30.

The hydrogel of the present invention has thixotropic properties, and its viscosity decreases under shearing force. Therefore, it can be delivered without using a double barrel syringe. In addition, at least part of the cross-linked structure has a loose bond that reversibly returns to the sol due to stress, such as an electrostatic bond via a sulfone group, compared to a hydrogel that is densely cross-linked only by covalent bonds The pore size is large and the material permeability is excellent. Furthermore, since cellulose nanofibers are biodegradable, hydrogels are also biodegradable. In addition, the hydrogel has high cell adhesion and is excellent in biocompatibility and affinity.

FIG. 1 is an X-ray diffraction chart (left) and a graph (right) showing crystallinity of aldehyde cellulose. FIG. 2 is a graph showing the relationship between the amount of sodium pyrosulfite charged and the amount of sulfonate groups of the sulfonated cellulose nanofibers obtained. FIG. 3 is an electron micrograph of sulfonated cellulose nanofibers. FIG. 4 is a graph showing weight loss (%) of sulfonated cellulose nanofibers in a biodegradability test. FIG. 5 is a photograph of the hydrogel of the present invention obtained by mixing sulfonated cellulose nanofibers and a gelatin solution. FIG. 6 is an electron micrograph of a cross section of the hydrogel. FIG. 7 is a graph showing changes in the shear modulus of the hydrogel with temperature. FIG. 8 is a graph showing the relationship between the concentration of sulfonated cellulose nanofibers and gelatin and the shear modulus. FIG. 9 is a graph showing the relationship between the pH of the gelatin solution and the shear modulus of the hydrogel. FIG. 10 is a graph showing the thixotropy of the hydrogel. FIG. 11 is a graph showing the results of a viscoelasticity test of a PEG gel obtained by cross-linking gelatin with pentaerythritol-poly (ethylene glycol) ether tetrasuccinimidyl-glutarate instead of sulfonated cellulose nanofibers. . FIG. 12 is a graph showing a comparison of the permeability of hydrogel and PEG gel. FIG. 13 is an electron micrograph (upper) of cells cultured in a medium containing sulfonated cellulose nanofibers and a graph (lower) of the number of cells. FIG. 14 is a phase-contrast micrograph (left) of cells cultured in a medium containing sulfonated cellulose nanofibers, and a graph (right) showing the cell aspect ratio with respect to the concentration of sulfonated cellulose nanofibers. FIG. 15 is a phase contrast micrograph of cells (left: endothelial cells, right: fibroblasts) adhered to the hydrogel surface of the present invention. FIG. 16 is a phase contrast micrograph of fibroblasts encapsulated in the hydrogel of the present invention. FIG. 17 is a graph showing the relationship between the amount of sulfone groups per unit weight of sCNF contained in the hydrogel (mmol / g) and the adsorption amount of albumin. FIG. 18 is an image of hematoxylin and eosin (HE) staining of a tissue section of a tissue not implanted with hydrogel 14 days after implantation (control) and a tissue section of the tissue implanted with hydrogel. FIG. 19 is an HE-stained image several days after each hydrogel was implanted. FIG. 20 is a graph in which the distribution of cells infiltrating into the hydrogel is quantified and plotted. 0 represents the boundary between the tissue and the hydrogel, and 1 represents the central part of the gel. FIG. 21 is a graph in which the depth of cells infiltrating each hydrogel is quantified and plotted. FIG. 22 is an immunostained image using vimentin antibody and CD31 antibody 14 days after implantation. FIG. 23 is an immunostained image using vimentin antibody and CD31 antibody 14 days after implantation. FIG. 24 shows the result of quantifying the area of positive cells from a vimentin-stained image. FIG. 25 shows the result of quantifying the area of positive cells from the CD31-stained image. FIG. 26 shows the results of quantifying the number of luminal structures from CD31 stained images. FIG. 27 is an immunostained image using CD163 antibody and CD68 antibody 14 days after implantation. FIG. 28 shows the result of quantifying the area of positive cells from the CD163 stained image. FIG. 29 shows the result of quantifying the area of positive cells from a CD68-stained image. FIG. 30 shows the ratio of the area of positive cells obtained from the CD163-stained image and the area of positive cells obtained from the CD68-stained image.

<Sulfonated cellulose nanofiber>
The sulfonated cellulose is a cellulose having a sulfonated structure in which the ring position between the 3rd and 4th positions of the glucose unit represented by the following formula (1) is opened.

The above formula shows a state in which the sulfone group (—SO ₃ H) is dissociated in water or the like, and the counter cation is not limited to Na ⁺ and may be a proton, K ⁺ or the like.

The sulfonated cellulose can be synthesized by the route shown in the following formula described in, for example, Henrikki Liimatainen et al., Cellulose (2013) 20: 741-749.

First, raw material cellulose is dispersed in water. 50-200 mol% sodium periodate (NaIO ₄ ) was added to the dispersion when the amount of glucose units (k) in cellulose was 100 mol%, and the mixture was shielded from light at 40-60 ° C. Aldehydated cellulose is produced by stirring for 2-6 hours. An aqueous solution containing unreacted substances is removed by filtration under reduced pressure, and an operation of washing with ultrapure water is repeated several times, followed by freeze-drying to obtain a dry powder of aldehyde cellulose. In the above formula, when the initial value of k is 100 mol%, m is 1 to 80 mol%, preferably 10 to 50 mol%. If m is less than the lower limit, it is difficult to ensure biodegradability. On the other hand, when m exceeds the upper limit, hydrolysis tends to occur and it becomes difficult to prepare nanofibers. The amount of aldehyde groups can be determined by neutralization titration by conductivity measurement using an aqueous sodium hydroxide solution, and is 0.5 to 8 mmol / g, preferably 1 to 7 mmol / g, more preferably 1 to 5 Mmol / g.

As the raw material cellulose, for example, cellulose obtained from plants, animals, etc., such as softwood pulp, hardwood pulp and cotton pulp, paper using these, waste paper, and the like can be used.

Next, the aldehyde cellulose is dispersed in ultrapure water, and sodium pyrosulfite (Na ₂ S ₂ O ₅ ) is added at 20 to 200 mol%, preferably 50 to 150 mol%, with the amount of aldehyde groups being 100 mol%. The reaction is allowed to proceed with stirring at room temperature for 12-24 hours. The product is recovered by centrifugation, washed with ultrapure water, etc. to remove unreacted material and purified, and then homogenized with an ultrasonic homogenizer for about 10 to 30 minutes to obtain sulfonated cellulose nanofibers (“ may be abbreviated as “sCNF”). The yield is about 80-90%.

FIG. 3 is an electron micrograph of sCNF prepared in the example. The diameter of the fiber observed with an electron microscope is several nm to 100 nm, preferably 3 to 20 nm. The aspect ratio (fiber length / fiber diameter) is about 30 to 1000.

SCNF is biodegradable. In the present invention, “biodegradability” was confirmed by reducing the weight of sCNF by 1% or more in one day in phosphate buffered saline (PBS) at pH 7.4 at 37 ° C. This biodegradability is defined as m + n, that is, the number of ring-opened units is at least 1 mol%, preferably 10 to 50 mol%, when the initial value of the amount (k) of glucose units in the raw cellulose is 100 mol%. It is thought to be due to some. Further, sCNF has a ring-opening unit, so that the crystallinity by X-ray diffraction measurement is 20 to 70%, which is lower than the raw cellulose having a crystallinity of about 90%.

Sulfonated cellulose nanofibers have sulfone groups at 0.1-7 mmol / g. The amount of the sulfone group can be determined by neutralization titration by conductivity measurement using an aqueous sodium hydroxide solution. In cellulose whose sulfone group is less than the lower limit, it is difficult to form nanofibers because the electrostatic repulsion between fibers is weak. The upper limit of the sulfone group is not particularly limited, but in practice, it is difficult to prepare one having more than the above value. Preferably, sCNF has a sulfone group content of 0.1 to 2.5 mmol / g, more preferably 0.1 to 2 mmol / g, in that nanofibers can be easily recovered by centrifugation. That is, sCNF represented by the following formula (2) is preferably used.

In the formula (2), k, m and n represent mol% of each repeating unit and are not particularly limited, but k is preferably 25 to 95 mol%, more preferably 40 to 90 mol%. m is preferably 4 to 70 mol%, more preferably 7 to 60 mol%. n is preferably 1 to 70 mol%, more preferably 10 to 30 mol%.

In addition, sCNF may have a repeating unit other than the above. Although it does not restrict | limit especially as repeating units other than the above, For example, the repeating unit hung up below is mentioned.

<Biodegradable polymer having primary amino group>
A biodegradable polymer having a primary amino group (hereinafter abbreviated as “biodegradable polymer”) is a polymer that can be decomposed by hydrolysis, enzymatic degradation, microbial degradation, etc. in the human body or body surface, It has a primary amino group. However, it goes without saying that in addition to the primary amino group, there may be a secondary amino group and a tertiary amino group capable of reacting with the sulfone group. Examples of such preferable biodegradable polymers include polysaccharides such as chitosan, polypeptides such as collagen, gelatin, albumin and laminin, glycoproteins such as fibronectin, fibers such as elastin, and derivatives thereof. These gene recombinants may be used. Of these, polypeptides are preferred, with gelatin or derivatives thereof being most preferred.

Gelatin may be derived from animals or fish. The gelatin may be acid-treated gelatin, alkali-treated gelatin, or genetically modified gelatin, preferably alkali-treated gelatin, more preferably low endotoxinized gelatin. The molecular weight range of the gelatin is preferably a weight average molecular weight (Mw) of 30,000 to 150,000, and more preferably 50,000 to 120,000. The molecular weight can be measured by gel permeation chromatography (GPC) according to a conventional method.

As the gelatin derivative, one containing a structure represented by the following formula (3) can be used.

GltnNH—CHR ¹ R ² (3)

In the above formula, “Gltn” is a gelatin residue, R ¹ is an alkyl group having 1 to 11 carbon atoms, and R ² is a hydrogen atom or an alkyl group having 1 to 11 carbon atoms. N is mainly derived from the ε-amino group of lysine (Lys) in gelatin. Preferably, R ² is a hydrogen atom. Since the derivative has an alkyl group, it has excellent adhesion to tissue and is also suitable as a scaffold material.

When R ² is an alkyl group having 1 to 5 carbon atoms, it may be the same as or different from R ¹ . The alkyl group may have a branch. Examples of the alkyl group include a methyl group, an ethyl group, a propyl group, and a butyl group. Preferably, R ¹ is a linear alkyl group having 1 to 3 carbon atoms, and R ² is a hydrogen atom.

The derivatization rate in the gelatin derivative is 20 to 80 mol%, preferably 30 to 70 mol% in terms of mol% of the imino group to which the hydrophobic group is bonded, based on the amount of amino group in the raw material gelatin. In other words, the imino group / amino group (molar ratio) in the obtained gelatin derivative is 20/80 to 80/20, preferably 30/70 to 70/30.

The gelatin derivative is prepared by adding an aldehyde or ketone, for example, dodecanal, tetradecanal, or decylethylketone, to a gelatin aqueous solution and stirring to form a Schiff base at 30 to 80 ° C. for 0.5 to 12 hours. It can be prepared by reduction using sodium borohydride (NaBH ₃ CN), sodium triacetoxyborohydride (NaBH (OAc) ₃ ), 2-picoline borane, pyridine borane and the like.

<Hydrogel>
Hydrogel means a gel containing water. The hydrogel of the present invention comprises sCNF and biodegradable polymer in a weight ratio of 1:99 to 70:30, preferably 10:90 to 40:60. Here, the weight ratio is a weight ratio in a dry state. The amount of water in the hydrogel is not particularly limited, but typically it is preferably prepared as appropriate at 50 to 99% by weight of the hydrogel. In particular, when the hydrogel is delivered by a catheter, the water content is preferably 90 to 99% by weight.

The hydrogel according to an embodiment of the present invention typically includes a liquid in which sCNF is dispersed in ultrapure water or a buffer having a pH of 6 to 8 at 0.5 to 2.5% by weight, and a biodegradable polymer. While heating a solution of 1 to 30% by weight in a buffer solution having a pH of 8 to 10 to about 30 to 40 ^° C., by a known stirring means such as a mixer, the amount ratio is within the above weight ratio range. It can be prepared by mixing. The viscosity increases immediately after mixing and the gelation reaction begins. However, as shown in Examples described later, it has been found that gelation reaches equilibrium and the elastic modulus reaches a maximum value, which may take time and depends on the pH of the solution. This tendency is remarkable when the biodegradable polymer is gelatin.
For example, when it is desired to form a hard embolus in a short time during surgery or the like, the pH of the gelatin aqueous solution is preferably 9-10.

Hydrogel is characterized by having thixotropic properties in addition to the biodegradability already described. This is not intended to limit the present invention, but the sCNF and the biodegradable polymer form a network structure based at least in part on weak physical interactions such as electrostatic interactions, hydrogen bonds, etc. It is thought to be due to The use of nanofibers with a higher aspect ratio than nanocrystals and whiskers is also considered to contribute. Since the viscosity of the hydrogel is lowered by applying a shearing force, it is suitable for injection with a catheter, a syringe, or the like.

Another feature of the hydrogel of the present invention is the internal structure of the gel. The hydrogel of the present invention comprising a colloid called nanofiber can be considered as a colloid gel, and its structure is greatly different from that of a molecular gel formed by crosslinking molecules generally used. In the case of a molecular gel, the pore size is about 5 nm, whereas the colloidal gel is considered to have a pore of several tens of nm. Therefore, the substance permeability is high, and it is expected that the nutrients and growth factors are further permeated.

<Additives>
Various drugs, proteins and the like to be delivered to the hydrogel of the present invention may be blended and used as a local delivery carrier or a sustained release delivery carrier. Examples of the drug include growth factors such as anti-inflammatory drugs such as steroids, antithrombotic drugs, antibiotics, fibroblast growth factor, vascular endothelial growth factor, and hepatocyte growth factor.

<Application>
The hydrogel of the present invention is suitably used as a medical injectable hydrogel. For example, it can be used for cerebral aneurysm treatment, coronary embolization therapy, and myocardial infarction treatment. Myocardial infarction is an irreversible pathological condition in which myocardial tissue is necrotized due to occlusion of blood vessels, and after infarction, heart failure may occur due to a decrease in cardiac function or arrhythmia. Injecting bioactive hydrogel into the infarct site as a scaffold keeps the ventricle wall thick, suppresses necrosis, inflammatory reaction, and myocardial remodeling, induces cellular infiltration from the surroundings, and forms new blood vessels It is expected that the regeneration of the organization can be promoted. Furthermore, by including cells and functional proteins for delivery, islet delivery therapy and growth factor delivery are expected. In addition, the hydrogel of the present invention may be used as an injectable artificial bone by mixing it with an artificial bone such as hydroxyapatite or calcium phosphate, as a wound dressing, or as an adhesive for living tissue.

The hydrogel of the present invention may be provided in the form of a hydrogel, but is provided in the form of a two-agent type comprising a first agent containing sulfonated cellulose nanofibers and a second agent containing a biodegradable polymer. It's okay. For example, the first agent is a dispersion of nanofibers of sulfonated cellulose, the second agent is an aqueous solution of a biodegradable polymer, these are mixed and injected at the time of treatment, or by a double barrel syringe You may inject.

EXAMPLES Hereinafter, although an Example demonstrates this invention, this invention is not limited to these.
<Preparation of sulfonated cellulose nanofiber (sCNF)>
With reference to Non-Patent Document 1, sulfonated cellulose 1-8 shown in Table 1 were synthesized to obtain sulfonated cellulose nanofibers (sCNF1-8).
As the first step, aldehyde-modified cellulose was prepared using cellulose as a raw material. 1 g of filter paper powder (ADVANTEC, mesh: 40-100, fiber length: 150-400 μm) was dispersed in 100 mL of ultrapure water while stirring with a stir bar. To the obtained dispersion, sodium periodate (NaIO ₄ ) was added at a ratio of 0.5 to 2 equivalents relative to the number of moles of glucose units in cellulose. Aldehydated cellulose was prepared by ring-opening between the 3rd and 4th positions in the glucose unit by stirring for 4 hours at 50 ^° C. in an oil bath while shielding from light. The reaction product was filtered under reduced pressure to remove the aqueous solution containing unreacted material, and washed with ultrapure water. This operation was repeated 3 times for washing, and finally freeze-dried to obtain a dry powder of aldehyde cellulose. The amount of aldehyde groups produced was quantified by neutralization titration.

Subsequently, sulfonated cellulose nanofibers were synthesized by sulfonation treatment. 1 g of the aldehyded cellulose obtained as described above was dispersed in 100 mL of ultrapure water, and sodium pyrosulfite (Na ₂ S ₂ O ₅ ) was added in an amount of 0.05 to 2.5 equivalents relative to the aldehyde group. Each was added in proportion. The mixture was allowed to react at room temperature for 12 to 24 hours with stirring to carry out sulfonation treatment. The obtained product was recovered by centrifugation at 4000 rpm for 30 minutes and washed with ultrapure water. By performing this operation three times, unreacted substances were removed and purified. Finally, sulfonated cellulose nanofibers were obtained by homogenizing with an ultrasonic homogenizer (150 W, 40 kHz, 60% power, BRANSON) for 10 to 30 minutes.

The percentage of ring opening of glucose units and the amount of aldehyde groups in each aldehyde cellulose were measured by a titration method. The results are shown in Table 1. As can be seen from the table, by changing the amount of sodium periodate to 0.5 to 2 equivalents per glucose unit, the amount of glucose units to be opened and the amount of aldehyde groups produced thereby can be controlled. .

<X-ray diffraction measurement>
In order to evaluate how the crystallinity of cellulose changes due to the ring-opening reaction using sodium periodate, X-ray diffraction measurement (XRD) was performed (FIG. 1, left figure). From the results of XRD measurement, it was shown that the peak derived from cellulose (14.6, 16.5, 22.8 ^o ) decreased as the ring-opening ratio of glucose units decreased, and the crystallinity decreased. It was. When the crystallinity was calculated from the ratio of 18 ^o peak derived from the amorphous structure and 22.8 ^o , the crystallinity, which was originally 90%, gradually decreased with the ring-opening reaction, and 7% of glucose units. The sCNF1 with the ring opened has a crystallinity of 83%, the sCNF2 with the ring opened 30% has a crystallinity of 70%, and the sCNF3 with the ring opened 40% has a crystallinity of 68% (FIG. 1, right). Figure). With sCNF4 having a ring opening ratio of 62%, the degree of crystallinity is reduced to 23%, and it is predicted that the decomposability is improved. Thus, it was shown that the crystallinity and degradability of cellulose can be controlled by controlling the ring-opening reaction.

<Relationship between sodium pyrosulfite amount and sulfone group amount>
After each sulfonated cellulose nanofiber was treated with an ultrasonic homogenizer, the amount of sulfone group was measured by a titration method (Table 1). FIG. 2 shows the sulfone group amount of the sulfonated cellulose nanofiber when the amount of sodium pyrosulfite is 0.05 to 0.5 equivalent to the aldehyde group, respectively. As can be seen from the figure, the amount of sulfone groups in cellulose nanofibers could be controlled by changing the amount of sodium pyrosulfite even when the ring-opening ratio and the amount of aldehyde groups were the same.

<Electron microscope observation>
The sulfonated cellulose nanofiber containing 50 mol% of sodium pyrosulfite with an aldehyde group and 150 mol% of the sulfonated cellulose nanofiber is mechanically shredded by ultrasonic treatment to form sCNF as shown in the electron micrograph of FIG. Could be prepared.

<Biodegradability>
The biodegradability of sCNF was examined at 37 ° C. using sCNF1 with a glucose unit ring-opening ratio of 7% and sCNF4 with 62%. Each sCNF (100 mg) was dispersed in 5 mL of PBS (pH = 7.4), and the insoluble material was weighed every 24 hours to measure the weight loss. For comparison, raw material cellulose (filter paper powder, ADVANTEC) was used. The results are shown in FIG. sCNF1 decreased by about 5% in one day and sCNF4 decreased by about 15%. Thus, since biodegradability depends on the ring-opening ratio of glucose units, other sCNF is predicted to be a value between sCNF1 and 4. On the other hand, the weight of raw material cellulose hardly decreased in one day.

<Preparation of hydrogel>
A hydrogel (sCNF gel) composed of each sCNF and gelatin was synthesized. 2.5 wt% sCNF3 dispersion (ultra pure water, pH = 7) and 20 wt% pig skin alkali-treated gelatin (Nitta Gelatin) heated to 37 ^° C (0.1 M borate buffer) SCNF gel 3 was prepared by rapidly mixing an equal amount using a vortex mixer. The gelation reaction started from the moment of mixing and gelled within a few minutes as shown in the photograph of FIG. Similarly, sCNF gels were obtained for sCNF1, 2, 4, and 7. The obtained sCNF gel 3 was freeze-dried, immersed in liquid nitrogen and pulverized, and the cross section of the gel was observed with an electron microscope. FIG. 6 shows an electron micrograph. From the figure, it can be seen that the sCNF gel has pores with a diameter of about 10 to 80 μm. In FIG. 6, the high-magnification photograph (left) is an enlargement of the wall portion of the hole.

<Viscoelasticity evaluation>
In order to evaluate the mechanical properties of the obtained hydrogel, the viscoelasticity of the sCNF gel was evaluated using a viscoelasticity measuring device (Rheoplus, Anton Paar) (FIG. 7). A dispersion of 5% by weight of sCNF3 (ultra pure water, pH = 7) and 20% by weight of pig skin alkali-treated gelatin solution (0.1M borate buffer, pH = 8.5) heated to 37 ^° C. 100 μL of the solution obtained by quickly mixing 50 μL at a time is placed on the stage of a viscoelasticity measuring device, fixed with a disk-shaped jig having a diameter of 10 mm, measurement is started from 10 ^° C., and 1 ^° C. is increased per minute. Measurements were taken up to 50 ^° C. while warming. The experiment was conducted at a frequency of 1 Hz and a strain of 1%. Since gelatin has a phase transition temperature (gelation temperature) in the vicinity of 30 ^° C., it is gel at a low temperature, and dissolves and becomes liquid at 30 ^° C. or higher. This is because the hydrogen bond is cleaved by the temperature rise. On the other hand, in sCNF gel 3, phase transition does not occur even at 30 ^° C. or higher, and a high shear elastic modulus of 1 kPa is exhibited even at 37 ^° C., which is the temperature of living tissue, compared to the case of gelatin alone. Increased 1000 times. This is presumably because the network structure was formed by the interaction between sCNF3 and gelatin. From this, it was found that by combining sCNF and gelatin, gelation occurred, and a gel having a high elastic modulus could be produced even at 37 ^° C.

<Influence of sCNF and gelatin concentration>
In order to clarify the relationship between the preparation conditions of sCNF gel and the mechanical properties, the effects of the concentrations of sCNF and gelatin were examined. An apparatus for measuring viscoelasticity 100 μL of a solution obtained by rapidly mixing 50 μL each of a 0.5 to 2.5 wt% sCNF3 solution (ultra pure water, pH = 7) and 1 to 30 wt% gelatin each solution And was fixed with a disk-shaped jig having a diameter of 10 mm, and viscoelastic properties at 37 ^° C. were evaluated (FIG. 8). The experiment was conducted at a frequency of 1 Hz and a strain of 1%. As shown in FIG. 8, the shear modulus increased with increasing concentrations of sCNF and gelatin. By adjusting the concentrations of sCNF and gelatin, the mechanical properties could be varied widely from several Pa to 1000 Pa.

<Effect of pH of gelatin solution>
The effect on the gelation rate was examined by changing the pH of a 2.5 wt% solution of sCNF3 (ultra pure water, pH = 7) and the 20 wt% gelatin solution to 8-10 (FIG. 9). Although it gelled in 5 minutes at pH = 8, it took 6 hours for the hydroelastic shear modulus to reach equilibrium. On the other hand, when gelatin solutions with pH = 9 and pH = 10 were used, gelation occurred immediately and the shear modulus reached 1 kPa within 10 minutes.

<Viscoelasticity test of hydrogel>
A solution of 2.5% by weight of sCNF3 (ultra-pure water, pH = 7) and a 20% by weight gelatin solution were mixed quickly by 50 μL, and 100 μL of the solution was placed on the stage of the viscoelasticity measuring device, and the diameter was 10 mm. The thixotropy was evaluated by changing the strain from 1% to 1000% every 5 minutes at 37 ^° C. The gel was once destroyed by the 1000% strain and the elastic modulus was lowered, but when the strain was returned to 1%, the elastic modulus was restored again (FIG. 10). This indicates that the binding mode of the hydrogel of the present invention is reversible. On the other hand, as a comparative object, gelatin with 4-branched polyethylene glycol (pentaerythritol-poly (ethylene glycol) ether tetrasuccinimidyl glutarate, Mw = 20,000) having a carboxyl group modified with terminal N-hydroxysuccinimide A similar experiment was conducted using a gel crosslinked with (hereinafter referred to as “PEG-G” or “PEG gel”) (FIG. 11). As a result, after 3 cycles, the storage elastic modulus was reduced to about 20%. On the other hand, the hydrogel (sCNF-G) of the present invention recovered to its original value after 3 cycles, indicating that it has thixotropic properties.

<Permeability test>
Gel permeability was tested using dextran (Mw = 2,000,000 Da) fluorescently labeled with fluorescein (FITC) having a high molecular weight (FIG. 12). A dispersion of 2.5% by weight of sCNF3 (ultra pure water, pH = 7) and 20% by weight of pig skin alkali-treated gelatin solution (0.1M borate buffer, pH = 8.5) heated to 37 ^° C. 100 μL of the solution obtained by quickly mixing 50 μL each in a 24-well transwell insert (Corning), gelled for 1 day, and then added with 2 mL of ultrapure water to swell for 1 day. . For comparison, the above PEG-G was treated in the same manner. Thereafter, 200 μL of 1 mg / mL FITC-dextran (Mw = 2,000,000 Da, hydrodynamic radius˜50 nm) was added, and 1 mL of PBS was added to the plate side. Every predetermined time, 50 μL of the solution was taken out from the lower side of the insert and supplemented with 50 μL of PBS. The fluorescence intensity of the obtained solution was measured using a microplate reader, and the transmittance was evaluated from a calibration curve. As a result, it became clear that the sCNF gel 3 permeates a huge molecule of 50 nm more than the molecular gel (PEG-G). The rate constant is 6.4 × 10 ⁻⁵ h ^{−1 for} sCNF gel and 1.6 × 10 ⁻⁵ h ⁻¹ for PEG-G, indicating that the permeability is about 4 times better. It was.

<Effect of sCNF on cell function>
The effect on cell function was evaluated by in vitro test by dispersing sCNF in a cell culture medium and giving it to cultured cells. L929 mouse fibroblasts (RIKEN) were cultured in a 37 ^° C., 5% CO ₂ incubator using RPMI 1640 medium (10% fetal bovine serum, 1% penicillin streptomycin). 5 × 10 ³ L929 cells were seeded in a 96-well plate and cultured for 24 hours. Thereafter, a medium in which sCNF3 was suspended at a concentration of 12.5 μg / mL was added to L929 cells, and the culture was continued. The day when sCNF was added was defined as day 0. After 1, 2, and 3 days, the number of cells was quantified using a cell count counting kit (WST-8, DOJINDO), and the sample after 3 days was fixed and dehydrated. After performing, SEM observation was performed (the upper part of FIG. 13). sCNF accumulated on the substrate to form a film-like structure, suggesting that it is involved in adhesion as a cell scaffold.
Regarding the number of cells, the cells to which sCNF had been added proliferated in the same manner as the samples to which sCNF had not been added, and no significant difference was observed (lower row in FIG. 13).

On the other hand, when the cell morphology was observed with a phase-contrast microscope, sCNF concentration-dependent cell extension was observed (FIG. 14, left, x40). From this image observation, the ratio (aspect ratio) of the major axis to the minor axis of the cell was calculated and compared, and at a concentration of sCNF of 50 μg / mL or more, cell extension was promoted predominately compared with no addition. (FIG. 14, right, x40). This is thought to be because the sulfone group on sCNF adsorbs proteins (such as fibronectin) in the medium, and a matrix as a cell scaffold was constructed in the culture. Thus, the hydrogel of the present invention is expected to be applied to regenerative medicine.

<Cell adhesion test to hydrogel>
A dispersion of 2.5% by weight of sCNF3 (ultra pure water, pH = 7) and 20% by weight of pig skin alkali-treated gelatin solution (0.1M borate buffer, pH = 8.5) heated to 37 ^° C. 200 μL of a solution obtained by quickly mixing 100 μL each) was added to a 48-well plate and gelled for 1 day, and then 2 mL of ultrapure water was added to swell for 1 day. The resulting gel was sterilized by UV irradiation for 1 hour. 1 × 10 ⁴ human umbilical vein-derived vascular endothelial cells (HUVEC, Lonza) or 2 × 10 ⁴ human skin-derived fibroblasts (NHDF, Lonza) were seeded on a gel. Were cultured in FGM medium (Lonza) for 24 hours in an incubator of 37 ^° C. and 5% CO ₂ , respectively. Then, it observed with the phase-contrast microscope (FIG. 15, * 40). Both cells were confirmed to have good adhesion and extension on the gel surface. It was shown that the hydrogel of the present invention has high cell adhesion and affinity.

<Cell inclusion experiment in hydrogel>
Dispersion of 1 × 10 ⁵ NHDFs mixed with 20% by weight pig skin alkali-treated gelatin solution (0.1M borate buffer, pH = 8) heated to 37 ^° C. and 2.5% by weight of sCNF3 200 μL of a solution obtained by quickly mixing 100 μL of the liquid (ultra pure water, pH = 7) was added to a 48-well plate and gelled. After standing for 3 minutes, 1 mL of FGM medium was added and cultured in an incubator of 37 ^° C., 5% CO ₂ . Then, it observed with the phase-contrast microscope (FIG. 16, * 40). It was confirmed that the NHDF encapsulated in the gel was well bonded and stretched inside the gel. It was shown that the hydrogel of the present invention can be applied to cell delivery because it can encapsulate cells.

<Protein adsorption experiment to hydrogel>
A sCNF dispersion (ultra pure water, pH = 7) having a sulfone group amount of 0.5 mmol / g and 37 ^° C. was prepared by the same method as described as “6” in Table 1. A vortex mixer is used to quickly mix an equal amount of a 20% by weight pig skin alkali-treated gelatin (Nitta Gelatin) solution (0.1M borate buffer, pH = 8.5) heated to A sCNF hydrogel was prepared by placing in a 1 mm thick silicon mold and incubating at 4 ° C. for 24 hours. The hydrogel was washed in PBS for 24 hours, punched out with a 10 mm punch, immersed in a bovine serum albumin solution labeled with 1 mg / mL fluorescein isocynate, and incubated at 37 ° C. for 24 hours. Next, after washing three times with PBS, the gel was dissolved with a 100 μg / mL collagenase solution, transferred to a 96-well plate, and the fluorescence intensity was measured with a microplate reader. The amount of albumin adsorbed on the hydrogel was calculated from a calibration curve.

Also, instead of the above sCNF, it was prepared by the same method as described in Table 1 as “7”, and the amount of sulfone group was 1.2 mmol / g, and in Table 1, “8” was described. The same test was performed using sCNF produced by the same method and having a sulfone group amount of 1.7 mmol / g. The results are shown in FIG.

From the results shown in FIG. 17, the amount of adsorbed albumin increased as the amount of sulfone groups per unit weight (mmol / g) of sCNF contained in the hydrogel increased.
This is considered to be due to the electrostatic interaction between the negatively charged sulfone group and the positively charged portion of albumin. From this, the hydrogel according to the embodiment of the present invention is expected to exhibit functions in the body by adsorbing cytokines and growth factors in the body.

More specifically, when the amount of sulfonic group of sCNF contained in the hydrogel is 0.4 mmol / g or more, the hydrogel has an excellent albumin adsorption performance, and the amount of sulfone group exceeds 0.5 mmol / g. When the hydrogel has a better protein adsorption performance and the sulfone group amount is 1.0 mmol / g or more, the hydrogel has a better protein adsorption performance and the sulfone group amount is 1.0 mmol / g. If it exceeds g, the hydrogel has particularly excellent protein adsorption performance, and if the amount of the sulfone group is 1.7 mmol / g or more, the hydrogel has the most excellent protein adsorption performance.

<Hydrogel implantation test in rats>
This experiment was conducted based on an experimental design approved by the Animal Experiment Safety Committee of the National Institute for Materials Science. Mice (C57BL / 6J, female, 6-8 weeks old) under inhalation anesthesia with 2.5% isoflurane, shaved back hair and sterilized with 70% ethanol, then subcutaneously in the shape of a disc with a diameter of 5 mm and a thickness of 1 mm The hydrogel was embedded.

The hydrogel is prepared by mixing an equivalent amount of 2% by weight of sCNF and 20% gelatin (similar to the protein adsorption experiment). In addition, sCNF was produced by the same method as described in Table 1 as “6”, “7”, and “8”, and the sulfone group amounts were 0.5 mmol / g, 1.2 mmol / g, What was 1.7 mmol / g was used.
Table 2 summarizes the “sample name” of the hydrogel used, the method for producing sCNF contained in the hydrogel, and the amount of sulfone groups.

The hydrogel was sterilized by UV (ultraviolet) irradiation. Next, the day when the hydrogel was implanted was defined as

day

0, and 3, 7, and 14 days later, somnopentyl was overdosed and the mice were sacrificed. Thereafter, the surrounding tissue was taken out and fixed with 10% formalin to prepare a tissue section (center part of the material) and stained. The results are shown in FIGS.
FIG. 18 is an image of hematoxylin and eosin (HE) staining of a tissue section of a tissue not implanted with hydrogel 14 days after implantation (control) and a tissue section of the tissue implanted with hydrogel. In addition, the range enclosed with the black broken line in FIG. 18 has shown the embedded hydrogel. Note that the scale bar in FIG. 18 corresponds to 1 mm.
FIG. 19 is an HE-stained image several days after each hydrogel was implanted. In FIG. 19, the white broken line indicates the boundary between the host structure and the hydrogel, and the range between the broken lines indicates the hydrogel. Moreover, in the range sandwiched by the broken lines, the portion that appears black is hydrogel, and the portion that appears gray is infiltrated cells and extracellular matrix components produced by the cells. Note that the scale bar in FIG. 19 corresponds to 300 μm.
FIG. 20 quantifies the distribution of cells infiltrated into the hydrogel. 0 represents the boundary between the tissue and the hydrogel, and 1 represents the central part of the gel.
FIG. 21 quantifies the depth of cells infiltrating each hydrogel.

As shown in FIGS. 18 to 21, no cell infiltration was observed in any sample 3 days after implantation, but invasion occurred 7 days later, in particular 1.2-sNC and 1. In 7-sNC, after 14 days, the host cells completely invaded the cells.

Next, in order to examine which cells were infiltrated, the samples after 14 days were subjected to immunostaining with vimentin and CD31. The results are shown in FIGS.
FIG. 22 and FIG. 23 are immunostained images using vimentin antibody and CD31 antibody 14 days after implantation. In the figure, the part that appears black in vimentin staining is fibroblast, and the part that appears black in CD31 staining is vascular endothelial cell. Note that the scale bar in FIGS. 22 and 23 corresponds to 100 μm.
FIG. 24 shows the result of quantifying the area of positive cells from a vimentin-stained image.
FIG. 25 shows the result of quantifying the area of positive cells from the CD31-stained image.
FIG. 26 shows the results of quantifying the number of luminal structures from CD31 stained images.

From the results of vimentin staining described above, it was revealed that many fibroblasts were infiltrated into the gel. In 1.2-sNC and 1.7-sNC, the number of blood vessels was larger than in other samples. From these, it is suggested that when the amount of sCNF sulphonic group contained in the hydrogel is 0.7 mmol / g or more, cell infiltration and angiogenesis are further promoted to induce tissue remodeling more easily. It was done. From the above viewpoint, the amount of the sulfonic group of sCNF is preferably 1.0 mmol / g or more, and more preferably 1.2 mmol / g or more.

In order to understand the mechanism in more detail, macrophage staining was performed. The results are shown in FIGS.
FIG. 27 is an immunostained image using CD163 antibody and CD68 antibody 14 days after implantation. Note that the scale bar in FIG. 27 corresponds to 100 μm.
FIG. 28 shows the result of quantifying the area of positive cells from the CD163 stained image.
FIG. 29 shows the result of quantifying the area of positive cells from a CD68-stained image.
FIG. 30 shows the ratio of the area of positive cells obtained from the CD163-stained image and the area of positive cells obtained from the CD68-stained image.

There are two types of macrophages, M1 type involved in inflammation and M2 type related to tissue regeneration. By examining the number of M2 type macrophages in the gel, we thought that the relationship with tissue regeneration could be understood. CD163 is a marker for M2 type macrophages. From the results of FIG. 27 to FIG. 30, when the amount of the sulfonic group of sCNF contained in the hydrogel is in the above numerical range, it is apparent that the number of CD163 positive cells is clearly increased and more M2-type macrophages are present. I understood. It is considered that tissue regeneration was further induced by producing humoral factors that promote tissue regeneration.

The hydrogel of the present invention is a biodegradable thixotropic material and is very useful for medical use as an injectable hydrogel.

Claims

A sulfonated cellulose nanofiber having a sulfone group at 0.1 to 7 mmol / g and a primary amino group selected from the group consisting of gelatin, chitosan, collagen, albumin, fibronectin, laminin, elastin and derivatives thereof A hydrogel containing at least one biodegradable polymer in a weight ratio of 1:99 to 70:30.
The hydrogel according to claim 1, wherein the biodegradable polymer having a primary amino group is gelatin or a derivative thereof.
The hydrogel according to claim 1 or 2, wherein the sulfonated cellulose nanofiber has a sulfone group at 0.1 to 2 mmol / g.
The hydrogel according to claim 2 or 3, wherein the gelatin or a derivative thereof is derived from an alkali-treated animal or fish.
The hydrogel according to any one of claims 2 to 4, wherein the gelatin or a derivative thereof is a low endotoxinized gelatin.
The gelatin derivative has a structure represented by the following formula (3):

GltnNH—CHR 1 R 2 (3)

(In the above formula, Gltn is a gelatin residue, R 1 is an alkyl group having 1 to 11 carbon atoms, and R 2 is a hydrogen atom or an alkyl group having 1 to 11 carbon atoms)
The hydrogel according to any one of claims 2 to 5, which is a gelatin derivative containing
7. The method according to claim 1, wherein the first agent comprising the sulfonated cellulose nanofibers and the second agent comprising the second agent comprising the biodegradable polymer having a primary amino group are used. The hydrogel according to item 1.
The hydrogel according to claim 7, wherein the first agent is a dispersion of nanofibers of the sulfonated cellulose, and the second agent is an aqueous solution of a biodegradable polymer having a primary amino group.
A medical injectable hydrogel comprising the hydrogel according to any one of claims 1 to 8.
The medical injectable hydrogel according to claim 9, which is a vascular embolization material.