WO2020045162A1

WO2020045162A1 - Drug delivery carrier

Info

Publication number: WO2020045162A1
Application number: PCT/JP2019/032411
Authority: WO
Inventors: 正人藤岡; 弘晃尾上; 佑太倉科; 英生宮原; 洋尚ジェイムス岡野; 知彦吉田; 優樹小川; 渉栗原; 央子宮内
Original assignee: 学校法人慶應義塾; 学校法人慈恵大学
Priority date: 2018-08-30
Filing date: 2019-08-20
Publication date: 2020-03-05
Also published as: JPWO2020045162A1

Abstract

The purpose of the present invention is to provide a drug delivery carrier which can be stably placed in a narrow tissue having a complex structure such as inner ear and enables the control of the sustained release of a drug, and a drug delivery system comprising the drug delivery carrier. The drug delivery carrier according to the present invention comprises a hydrogel which contains a polysaccharide having a crosslinkable functional group.

Description

Carrier for drug delivery

The present invention relates to a drug delivery carrier. More specifically, the present invention relates to a drug delivery carrier and a drug delivery system.

開発 In recent years, the development of new treatments for hearing loss has been active in Japan and overseas. In particular, with regard to inner ear deafness, there are many cases of presbycusis, sudden deafness, Meniere's disease, etc., and 30 to 40% of the Japanese population aged 65 or older is affected. Moreover, since the mammalian cochlea is poor in spontaneous regenerative ability, chronic sensorineural hearing loss is intractable. Because of this situation, a number of large and small companies abroad regard inner ear hearing loss as an unmet medical need, and have been competing in research into the development of gene therapy for the inner ear, especially for the curative treatment. For example, major pharmaceutical manufacturers are already conducting clinical trials for gene transfer with adenovirus. In gene transfer, in view of safety, a gene transfer medium using an adeno-associated virus (hereinafter, also referred to as AAV) is becoming mainstream.

The cochlea, which exists in the inner ear and converts sound into neural activity, is buried in the compact bone called the temporal bone, and contacts the middle ear cavity through a structure called a round window and an oval window. In particular, the former is a membrane-like structure located at the bottom of a recess called a round window fossa, and is expected as a route for drug administration to the inner ear.
For example, when AAV is used as a drug, AAV passes through the round window membrane and diffuses into the cochlea, while the middle ear surgery raises the eardrum and clearly approaches the round window fossa by visualization. However, there still remain problems of a drug delivery system (hereinafter, also referred to as DDS) such as a contact area and a residence time between the administered drug and the round window membrane and a sustained release. Research on DDS for topical administration to the inner ear has just begun in the world, and it is in the early stages of seeds exploration and development research.

徐 As for the DDS for the purpose of gene therapy, it has been reported that the collagen vector is slowly released to the wound by collagen (for example, see Non-Patent Document 1).

However, the technique of Non-Patent Document 1 is mainly applied to a body surface such as a wound, and is difficult to apply to a narrow and complicated tissue. Also, control at any timing of sustained release has not been realized.

The present invention has been made in view of the above circumstances, and provides a drug delivery carrier that can be stably placed in a tissue having a narrow and complicated structure such as the inner ear and that can control the sustained release of a drug. The purpose is to do.

The present invention includes the following aspects.
[1] A drug delivery carrier comprising a hydrogel containing a polysaccharide having a crosslinkable functional group.
[2] The carrier for drug delivery according to [1], wherein the polysaccharide having a crosslinkable functional group is alginic acid or a salt thereof.
[3] The carrier for drug delivery according to [1] or [2], wherein the hydrogel is in a sheet shape, a bead shape, or a fiber shape.
[4] The drug delivery carrier according to [3], wherein the hydrogel is in the form of beads.
[5] The drug delivery carrier according to [3], wherein the hydrogel is in the form of a fiber.
[6] The drug delivery carrier according to any one of [1] to [4], further comprising a fibrous extracellular matrix, wherein the extracellular matrix contains the hydrogel.
[7] A drug delivery system comprising the drug delivery carrier according to any one of [1] to [6] and a drug.
[8] The drug delivery system according to [7], further comprising a solubilizing agent for the hydrogel.
[9] The drug delivery system according to [7] or [8], which is a therapeutic agent for a disease.
[10] The drug delivery system according to [9], wherein the therapeutic agent for a disease is a therapeutic agent for hearing loss.
[11] The drug delivery system according to any one of [7] to [10], wherein the drug includes a nucleic acid.
[12] The drug delivery system according to [11], further comprising a viral vector, wherein the viral vector comprises the nucleic acid.
[13] The nucleic acid has a base sequence of any one of the following (A) to (D) and includes a gene encoding a protein capable of differentiating a supporting cell into a sensory hair cell: 11] or the drug delivery system according to [12]:
(A) the base sequence represented by SEQ ID NO: 1,
(B) a base sequence in which one or several bases are deleted, substituted, inserted, or added in the base sequence represented by SEQ ID NO: 1,
(C) a base sequence having an identity of 70% or more in the base sequence represented by SEQ ID NO: 1,
(D) a base sequence capable of hybridizing under stringent conditions to a gene consisting of a base sequence complementary to the gene consisting of the base sequence represented by SEQ ID NO: 1, and (E) the above-mentioned (A) to ( Degenerate isomer of the base sequence of D).

According to the present invention, it is possible to provide a drug delivery carrier that can be stably placed in a tissue having a narrow and complicated structure such as the inner ear and that can control the sustained release of a drug.

It is a fluorescence image of the microbead immediately after preparation. It is a fluorescence image of the microbeads after storing in a calcium chloride solution for 3 days. It is a stained image of a living cell in a test for judging the viability of a cell to which alginate lyase was dropped and cultured for 3 days. It is a stained image of a dead cell in a test for determining the viability of a cell to which alginate lyase was dropped and cultured for 3 days. It is a graph which shows the ratio of the GFP-expressing cell after 72 hours in the AAV-containing calcium alginate gel beads expressing GFP and the cells to which alginate lyase was dropped. FIG. 4 is a graph showing the results of a stability test of AAV-containing calcium alginate gel beads expressing GFP stored at 4 ° C. or −80 ° C. It is a graph which shows the result of the time-dependent change test of the infectivity of AAV containing calcium alginate gel beads. It is a graph which shows the test result of the virus infectivity after subjecting AAV containing collagen gel beads (Low Density and High Density) to alginate lyase treatment.

[Drug Delivery Carrier]
In one embodiment, the present invention provides a carrier for drug delivery comprising a hydrogel comprising a polysaccharide having a crosslinkable functional group.

(4) The polysaccharide having a crosslinkable functional group is crosslinked by a crosslinking agent to form a hydrogel. In the present invention, the hydrogel means a gel containing water or an aqueous solution. The method for crosslinking the polysaccharide is not limited as long as the hydrogel forms a three-dimensional network structure, and includes chemical crosslinking or physical crosslinking. Specifically, crosslinking by a covalent bond, a hydrogen bond, or an ionic bond can be mentioned, and crosslinking by an ionic bond is preferable. As the crosslinking by ionic bonding, crosslinking by divalent metal ions is preferable.

As the crosslinkable functional group, an acidic functional group is preferable, and examples thereof include a carboxyl group, an alcoholic hydroxyl group, and a phenolic hydroxyl group, and a carboxyl group is more preferable.
Examples of polysaccharides having a crosslinkable functional group include alginic acid, pectic acid, hyaluronic acid, cellulose, chitosan, chitin, starch, dextran, heparin, chondroitin, cationic guar, cationic starch, carboxymethyl cellulose, carboxymethyl chitosan, carboxymethyl dextran, Examples include carboxymethyl starch, xanthan, karaya gum, gum gutti, gellan, agar, heparin sulfate, chondroitin sulfate, or salts or derivatives thereof.
Among them, alginic acid, pectic acid, hyaluronic acid, or salts or derivatives thereof are preferred, and alginic acid or alginates are more preferred. Alginates include sodium alginate and potassium alginate.

As the cross-linking agent, those containing a divalent metal ion are preferable, and Pb ²⁺ , Cu ²⁺ , Cd ²⁺ , Ba ²⁺ , Sr ²⁺ , Ca ²⁺ , Co ²⁺ , Ni ²⁺ , Zn ²⁺ , Mn ²⁺ , Mg ²⁺ , those containing Ca ²⁺ and Ba ²⁺ are preferable, and those containing Ca ²⁺ are more preferable.

Further, since the crosslinking agent can be used in the form of an aqueous solution by dissolving in water, a salt containing the above divalent metal ion is preferable, and a halide, hydroxide, carbonate, nitrate containing the above cation, Acetates are more preferred, halogen salts are even more preferred, and calcium chloride is particularly preferred.

The hydrogel is obtained, for example, by extruding a solution containing a polysaccharide having a crosslinkable functional group into a solution containing a crosslinking agent.
The concentration of the crosslinking agent in the solution containing the crosslinking agent is preferably 1 mM to 10 M, more preferably 10 mM to 5 M, and particularly preferably 100 mM to 1000 mM.
The concentration of the polysaccharide in the solution containing the polysaccharide having a crosslinkable functional group is preferably 0.1% by weight to 50% by weight, more preferably 0.5% by weight to 20% by weight, and more preferably 1% by weight to 10% by weight. Is particularly preferred. The hydrogel can take any shape depending on the site to be delivered and the purpose. For example, a shape such as a bead, a sheet, and a fiber may be used, but is not limited thereto.

The hydrogel can be in the form of beads. Hydrogel beads can be produced, for example, by a microgel bead production method using centrifugal force (see Non-Patent Document 2).
The pore size (diameter) of the hydrogel beads is preferably 1 nm to 10 μm, more preferably 5 nm to 1 μm, and particularly preferably 10 nm to 200 nm.
The size (diameter) of the hydrogel beads is preferably 1 μm to 1000 μm, more preferably 10 μm to 500 μm, and particularly preferably 50 μm to 200 μm.

担体 The carrier for drug delivery of the present invention preferably contains a fibrous extracellular matrix containing a hydrogel, in which case the hydrogel is preferably in the form of beads. By making the drug delivery carrier of the present invention have a desired shape, the drug delivery carrier can be stably placed at a desired location in a living body. For example, by preparing the drug delivery carrier of the present invention in a fiber-like shape, the drug delivery carrier of the present invention can be appropriately placed in an auricular space having a specific shape such as a gap in a space where stapes are located. Can be detained.

The diameter of the fibrous extracellular matrix is preferably 1 μm to 1000 μm, more preferably 10 μm to 500 μm, and particularly preferably 50 μm to 200 μm.
The length of the fibrous extracellular matrix is preferably from 10 μm to 100 cm, more preferably from 100 μm to 10 cm, and particularly preferably from 1 mm to 1 cm.
A method for producing a fibrous extracellular matrix containing a hydrogel can be produced, for example, by pouring the extracellular matrix into a fiber-shaped mold.

As extracellular matrix, basal reconstituted from collagen (type I, II, III, V, XI, etc.), mouse EHS tumor extract (including type IV collagen, laminin, heparan sulfate proteoglycan, etc.) Examples include membrane components (trade name: Matrigel), glycosaminoglycans, hyaluronic acid, proteoglycans, gelatin, and the like.

Further, the hydrogel itself may be in the form of a fiber.
The diameter of the fibrous hydrogel is preferably 1 μm to 1000 μm, more preferably 10 μm to 500 μm, and particularly preferably 50 μm to 200 μm.
The length of the fibrous hydrogel is preferably from 10 μm to 100 cm, more preferably from 100 μm to 10 cm, and particularly preferably from 1 mm to 1 cm.

[Drug delivery system]
In one embodiment, the present invention provides a drug delivery system including the above-described drug delivery carrier and a drug. As described later in Examples, the drug delivery system according to the present embodiment can deliver a drug to a desired organ without controlled cytotoxicity, with controlled sustained release.

薬物 The drug delivery system of the present embodiment can be used in a form containing a therapeutic agent for a disease, a contrast agent, and the like as a drug.

有効 Examples of the active ingredient of the therapeutic agent for a disease include low molecular weight compounds, nucleic acids, proteins, and peptides. As the low molecular weight compound, a compound whose size is adjusted by micelle formation or the like so as to be supported on the pores of the hydrogel beads is preferable. Examples of the nucleic acid include cDNA, mRNA, siRNA, shRNA, miRNA, antisense RNA and the like. Examples of proteins include enzymes and antibodies, and examples of peptides include ligands and vaccines.

用いる When the drug delivery system of the present embodiment is used for gene therapy, a nucleic acid is preferable as the disease therapeutic agent. When the expression of a wild-type gene is lost or suppressed in a patient due to a deletion, insertion or mutation of the gene sequence, it is preferable to use a cDNA of the wild-type gene as a therapeutic agent for a disease. When the therapeutic agent for a disease is a nucleic acid, it is preferable that the viral vector has a form containing the cDNA as the therapeutic agent for a disease, from the viewpoint of delivery and expression efficiency.

ウイルス The virus vector used here includes a retrovirus vector, an adenovirus vector, an adeno-associated virus vector (AAV), a herpes virus vector, and the like. From the viewpoint of low toxicity, an adeno-associated virus vector (AAV) is preferable.

用いる When the drug delivery system of the present embodiment is used for gene therapy for hearing loss, it is preferable to use an AAV containing a nucleic acid encoding an atonal-related factor. Atonal-related factors are transcription factors belonging to the basic helix-loop-helix family.

Examples of the gene encoding an atonal-related factor include a gene having a base sequence of any one of the following (A) to (D) and encoding a protein capable of differentiating a supporting cell into a sensory hair cell. Listed:
(A) the base sequence represented by SEQ ID NO: 1,
(B) a base sequence in which one or several bases are deleted, substituted, inserted, or added in the base sequence represented by SEQ ID NO: 1,
(C) a base sequence having an identity of 70% or more in the base sequence represented by SEQ ID NO: 1,
(D) a base sequence capable of hybridizing under stringent conditions to a gene consisting of a base sequence complementary to the gene consisting of the base sequence represented by SEQ ID NO: 1, and (E) the above (A) to ( A degenerate isomer of the base sequence of D).

遺伝子 In the above (A), the gene consisting of the nucleotide sequence represented by SEQ ID NO: 1 is cDNA of Hath1 (Human @ atonal @ homolog1).

In the above (B), the number of bases which may be deleted, substituted, inserted, or added is preferably 1 to 320, more preferably 1 to 160, still more preferably 1 to 80, and 1 to 80. The number is more preferably 40, particularly preferably 1 to 20, and most preferably 1 to 10.

において In the above (C), the identity is preferably at least 75%, more preferably at least 80%, further preferably at least 85%, particularly preferably at least 90%.

In the above (D), “under stringent conditions” means, for example, 5 × SSC (composition of 20 × SSC: 3 M sodium chloride, 0.3 M citric acid solution, pH 7.0), 0.1% by weight N Incubation at 55-70 ° C. for several hours to overnight in a hybridization buffer consisting of lauroyl sarcosine, 0.02% by weight SDS, 2% by weight nucleic acid hydridation blocking reagent and 50% formamide Thus, conditions for hybridization can be given. The washing buffer used for washing after incubation is preferably a 1 × SSC solution containing 0.1% by weight SDS, more preferably a 0.1 × SSC solution containing 0.1% by weight SDS.

アミノ酸 For amino acids other than methionine and tryptophan, multiple codons correspond to one amino acid. This is called the degeneracy of the genetic code. In the above (E), the degenerate isomer of the base sequence means another base sequence corresponding to an amino acid encoded by a certain base sequence.

From the viewpoint of controlling the sustained release, the drug delivery system of the present embodiment preferably further includes a solubilizing agent for the hydrogel.
Examples of the solubilizing agent for the hydrogel include a chelating agent for removing divalent metal ions when crosslinked by ionic bonding. In addition, an enzyme that decomposes a polysaccharide having a crosslinkable functional group itself is also included.

Examples of the chelating agent include citric acid, ethylenediamine, ethylenediaminetetraacetic acid, nitrilotriacetic acid, diethylenetriaminepentaacetic acid, and glycol ether diaminetetraacetic acid.
Examples of the enzyme include alginate lyase.

The drug delivery system of the present embodiment can be reliably placed inside a complex tissue by adjusting the size or shape of the hydrogel, or by enclosing the hydrogel in a polymer structure such as a gel fiber. can do.
Furthermore, by subjecting the hydrogel beads carrying the AAV to degradation by an enzyme such as alginate lyase, or irradiation of ultrasonic waves or the like, the AAV can be inactivated at desired timing without deactivation of AAV or damage to surrounding tissues. Can be sustained released. The sustained-release AAV can introduce a desired gene into a target tissue.

Further, when using a contrast agent as a drug in the drug delivery system of the present embodiment, as the drug, barium sulfate, bismuth subcarbonate, bismuth oxide, zirconium oxide, ytterbium fluoride, iodoform, barium apatite, barium titanate, lanthanum glass Contrast agents for computed tomography (CT) such as iodine contrast agents; contrast agents for MRI such as gadolinium preparations, superparamagnetic iron oxide preparations (Super Paramagnetic Iron Oxide, SPIO); technetium ^{99m (99m} Tc), molybdenum ^{99 (99 Mo) Single photon emission} computed tomography (SPECT) for radioactive isotopes such like good It can be cited as Shii drug.

[Method of treatment]
In one embodiment, the present invention provides a method of treating a disease caused by a defect or loss of sensory hair cells, comprising administering a drug delivery system as described above to a patient in need of treatment.

Diseases caused by sensory hair cell defect or loss include hearing loss or impaired balance, with hearing loss being preferred. Since the inner ear hair cells are destroyed when exposed to loud sounds for a long period of time and do not regenerate spontaneously, the hydrogel carrying AAV containing the nucleic acid encoding the atonal-related factor described above was used as a drug delivery system. Preferably, gene therapy is performed.
The carrier for drug delivery preferably contains a fibrous hydrogel or a fibrous extracellular matrix containing a beaded hydrogel. By having such a shape, the drug delivery carrier of the present invention can be stably placed in a space in the inner ear, such as a gap in the space where the stapes are located, while confirming the endoscope.

した After placement, alginate lyase may be administered to the inner ear several times periodically. By such administration, the sustained release of AAV containing the nucleic acid encoding the atonal-related factor carried on the hydrogel can be controlled.

The dose of AAV supported on the hydrogel is 10 ⁵ gc / mL to 10 ¹² gc / mL, and the lower limit is 10 ⁵ gc / mL, 10 ⁶ gc / mL, 10 ⁷ gc / mL and 10 ⁷ gc / mL. ⁸ gc / mL, 10 ⁹ gc / mL, 10 ¹⁰ gc / mL, 10 ¹¹ gc / mL, and the upper limit is 10 ¹² gc / mL, 10 ¹¹ gc / mL, 10 ¹⁰ gc / mL, 10 ⁹ gc / mL, 10 ⁸ gc / mL, 10 ⁷ gc / mL, and 10 ⁶ gc / mL.

The dose of alginate lyase is 1 μg to 10 mg per administration, the lower limit is 1 μg, 10 μg, 100 μg, 1 mg, and the upper limit is 10 mg, 1 mg, 100 μg, 10 μg. No.

代わり Instead of administration of alginate lyase, ultrasound may be applied to a predetermined part of the ear to physically dissolve the hydrogel placed in the inner ear.

[Other Embodiments]

In one embodiment, the present invention provides a drug delivery system for the treatment of a disease caused by a defect or loss of sensory hair cells. The same drug delivery system as described above can be used.

In one embodiment, the present invention provides a pharmaceutical composition for the treatment of a disease caused by defect or loss of sensory hair cells, comprising a drug delivery carrier and a drug. . As the drug delivery carrier and drug, those similar to those described above can be used.

In one embodiment, the present invention provides a drug delivery system for use as a treatment for a disease caused by a defect or loss of sensory hair cells. The same drug delivery system as described above can be used.

Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to the following examples.

[Experimental example 1]
(Stability test of microbeads containing silica beads)
As a test for producing calcium alginate gel beads having a diameter of about 100 μm carrying AAV, fluorescent silica beads having a diameter similar to that of AAV and having a diameter of 20 nm were regarded as AAV, and encapsulated in calcium alginate gel microbeads.
A solution was prepared so that the final concentration was 2% by weight of sodium alginate and 10% by weight of fluorescent silica, and microgel beads were prepared using centrifugal force (K Maeda, et al., Controlled Synthesis of 3D Multi-Compartmental Particles with Centrifuge-Based Microdroplet Formation from a Multi-Barrelled Capillary, Adv. Mater., 24, 1340-1346 (2012)).
The stability of the microbeads after 3 days in a calcium chloride solution was evaluated.

FIG. 1A is a fluorescence image of microbeads immediately after preparation, and FIG. 1B is a fluorescence image of microbeads after storage in a calcium chloride solution for 3 days. As shown in FIG. 1B, it was confirmed that the silica beads were stably encapsulated in the microgel beads.

[Experimental example 2]
(Sustained release test of microbeads)
Next, a sustained release test of the encapsulated silica beads was performed. An aqueous solution of 3% by weight of sodium alginate was injected into a 100 mM calcium chloride solution in the same manner as in Experimental Example 1 to produce calcium alginate gel microbeads (about 80 μm in diameter).
After the microbeads were dropped on the dish in which the NIH / 3T3 cells were cultured, alginate lyase was dropped on the dish such that the final concentration became 50 μg / mL, and the state until the beads were dissolved was observed. Successfully decomposed. Thereafter, the cells to which the alginate lyase had been dropped were cultured for 3 days, and then evaluated by a viability test. Calcein-AM was used to stain live cells (see FIG. 2A), and ethidium homodimer was used to stain dead cells (see FIG. 2B). As can be seen from the results of FIGS. 2A and 2B, almost all of the cultured cells were alive, and it was confirmed that the cells were not damaged by the sustained release process.

[Experimental example 3]
(Sustained release test of AAV-containing calcium alginate gel beads)
A verification test of DDS using AAV instead of silica beads was performed. Specifically, AAV prepared to express GFP in cells by infection was encapsulated in micro-beads of calcium alginate gel, and the same sustained release test as in Example 2 was performed. Example 3 demonstrates whether a DDS carrier can maintain the infectious function of AAV.
In the same manner as in Experimental Examples 1 and 2, an aqueous solution of 3% by weight of sodium alginate containing AAV expressing 1.7 × 10 ¹⁰ gc / mL of GFP was placed in a 100 mM, 500 mM, or 1000 mM calcium chloride solution. In the same manner as in Example 1, injection was performed to produce calcium alginate gel microbeads (about 80 μm in diameter).
The prepared AAV-containing calcium alginate gel beads expressing GFP and alginate lyase at final concentrations of 10 μg / mL and 30 μg / mL were dropped on HeLa cells, and the proportion of GFP-expressing cells 72 hours later was measured. The results are shown in FIG. In FIG. 3, from the left lane, Control indicates AAV-containing calcium alginate gel beads expressing GFP and HeLa cells without addition of alginate lyase, and AAV + Lyase_0 indicates HeLa cells with only AAV expressing GFP added. AAV + Lyase_10 indicates AAV expressing GFP and HeLa cells to which alginate lyase at a final concentration of 10 μg / mL was added, and AAV + Lyase_30 indicates AAV expressing GFP and an alginate lyase at a final concentration of 30 μg / mL. AAV-Beads + Lyase_0 indicates HeLa cells to which only AAV-containing calcium alginate gel beads expressing GFP prepared in a 100 mM calcium chloride solution were added, AAV-Beads + Lyase_30 indicates AAV-containing calcium alginate gel beads expressing GFP prepared in 100 mM calcium chloride solution, and HeLa cells to which alginate lyase at a final concentration of 30 μg / mL was added. AAV-containing calcium alginate gel beads expressing GFP prepared in a calcium solution, and HeLa cells to which alginate lyase was added at a final concentration of 30 μg / mL are shown. AAV-Beads + Lyase_30 + CaCl2_1000 shows GFP prepared in a 1000 mM calcium chloride solution. Expressed AAV-containing calcium alginate gel beads and alginate lyase at a final concentration of 30 μg / mL Show the added HeLa cells.

示す As shown in FIG. 3, in HeLa cells to which only AAV-containing calcium alginate gel beads expressing GFP were added, AAV infection was suppressed, and it was confirmed that the calcium alginate gel could carry AAV. Furthermore, by adding alginate lyase, the sustained release of AAV expressing GFP was controlled, and the expression of the GFP gene in cultured cells was confirmed.

[Experimental example 4]
(Stability test of AAV-containing calcium alginate gel beads expressing GFP stored at 4 ° C. or −80 ° C.)
In the same manner as in Experimental Example 3, an aqueous solution of 3% by weight of sodium alginate containing AAV expressing 1.7 × 10 ¹⁰ gc / mL of GFP was injected into a 100 mM calcium chloride solution, and a calcium alginate gel was obtained. Microbeads (about 80 μm in diameter) were prepared.
A bead suspension obtained by suspending the prepared GFP-expressing AAV-containing calcium alginate gel beads in PBS was stored at 4 ° C. or −80 ° C. for 72 hours, and the same infection experiment as in Experimental Example 3 was performed. FIG. 4 shows the results. From the left lane in FIG. 4, Fresh B (-) L (-) indicates HeLa cells to which only AAV expressing fresh GFP without preservation was added, and Fresh B (-) L (+) indicates AAV expressing fresh GFP without preservation and HeLa cells supplemented with alginate lyase at a final concentration of 30 μg / mL. Fresh B (+) L (+) shows fresh AAV-containing calcium alginate gel beads containing GFP expressing and HeLa cells supplemented with alginate lyase at a final concentration of 30 μg / mL are shown. 4B (+) L (+) shows AAV expressing GFP stored at 4 ° C. for 72 hours. Containing calcium alginate gel beads and HeLa microspheres added with a final concentration of 30 μg / mL alginate lyase. Are shown, -80B (+) L (+) shows AAV-containing calcium alginate gel beads expressing GFP stored at -80 ° C. 72 h, and HeLa cells was added alginate lyase a final concentration of 30 [mu] g / mL.
As shown in FIG. 4, the AAV-containing calcium alginate gel beads can be stored at 4 ° C. or −80 ° C. since there is no difference in the infection efficiency even after storage at 4 ° C. or −80 ° C. as compared with the control. Was confirmed.

[Example 5]
(Evaluation test of infectivity of virus after elapse of time after AAV encapsulation in hydrogel microbeads)
AAV prepared to express GFP in cells by infection was encapsulated in calcium alginate gel microbeads, (i) a sample retained for up to 72 hours after encapsulation, and (ii) alginate lyase immediately after encapsulation in microbeads. Samples that were degraded and retained for up to 72 hours after encapsulation, and (iii) samples that were retained for 24 hours after encapsulation in microbeads and then degraded by alginate lyase to retain for up to 72 hours after encapsulation were prepared. The lyase concentration was 33 μg / mL. The samples (i) to (iii) were used at a MOI of 1 × 10 ⁶ for 5 × 10 ³ cells per well. The cell infection rate was determined for samples (i) to data (iii) and controls (positive and negative). FIG. 5 shows the results.

感染 As shown in FIG. 5, infectivity was observed in all of the samples (i) to (iii). Infectivity in sample (i) was significantly lower than infectivity in samples (ii) and (iii). This result indicates that AAV infection was suppressed by the beads.

[Example 6]
(Evaluation of the infectivity of samples that have been subjected to encapsulation of AAV-containing beads in a collagen gel and subsequent degradation treatment)
Regarding beads containing AAV prepared to express GFP in cells by infection (Low Density, High Density), (i) a sample in which Low Density beads were encapsulated in a collagen gel and held for 72 hours, (ii) Low Density Immediately after encapsulating the beads in the collagen gel, the sample was degraded by alginate lyase and retained for up to 72 hours after encapsulation, (iii) High Density beads were encapsulated in the collagen gel and retained for 72 hours, Immediately after the Density beads were encapsulated in the collagen gel, the density beads were digested with alginate lyase to prepare a sample that was retained for up to 72 hours after the encapsulation. High Density beads have 1/10 the number of beads and 10 times the virus concentration of Low density.
The lyase concentration was 33 μg / mL. The samples (i) to (iv) were used at a MOI of 1 × 10 ⁶ for 5 × 10 ³ cells per well. The infection rate of the cells was determined for the samples (i) to (iv) and the controls (positive and negative). FIG. 6 shows the results.

感染 As shown in FIG. 6, infectivity was observed in sample (i), sample (ii), and sample (iv) except sample (iii).

Claims

(4) A drug delivery carrier comprising a hydrogel containing a polysaccharide having a crosslinkable functional group.
The drug delivery carrier according to claim 1, wherein the polysaccharide having a crosslinkable functional group is alginic acid or a salt thereof.
薬物 The drug delivery carrier according to claim 1 or 2, wherein the hydrogel is in the form of a sheet, a bead, or a fiber.
薬物 The drug delivery carrier according to claim 3, wherein the hydrogel is in the form of beads.
担体 The drug delivery carrier according to claim 3, wherein the hydrogel is in the form of a fiber.
The drug delivery carrier according to any one of claims 1 to 4, further comprising a fibrous extracellular matrix, wherein the extracellular matrix comprises the hydrogel.
(8) A drug delivery system comprising the drug delivery carrier according to any one of (1) to (6) and a drug.
The drug delivery system according to claim 7, further comprising a solubilizing agent for the hydrogel.
The drug delivery system according to claim 7 or 8, which is an agent for treating a disease.
The drug delivery system according to claim 9, wherein the therapeutic agent for a disease is a therapeutic agent for hearing loss.
薬物 The drug delivery system according to any one of claims 7 to 10, wherein the drug includes a nucleic acid.
12. The drug delivery system of claim 11, further comprising a viral vector, wherein the viral vector comprises the nucleic acid.
12. The nucleic acid according to claim 11, wherein the nucleic acid has a base sequence of any one of the following (A) to (D) and includes a gene encoding a protein capable of differentiating a supporting cell into a sensory hair cell. A drug delivery system according to item 12,
(A) the base sequence represented by SEQ ID NO: 1,
(B) a base sequence in which one or several bases are deleted, substituted, inserted, or added in the base sequence represented by SEQ ID NO: 1,
(C) a base sequence having an identity of 70% or more in the base sequence represented by SEQ ID NO: 1,
(D) a base sequence capable of hybridizing under stringent conditions to a gene consisting of a base sequence complementary to the gene consisting of the base sequence represented by SEQ ID NO: 1, and (E) the above (A) to ( A degenerate isomer of the base sequence of D).