The present disclosure relates to an injectable hydrogel rod using a crosslinked hydrogel, a manufacturing method thereof, and a biomedical use thereof.
Hydrogel, a jelly-like substance through physicochemical crosslinking of polymers dissolved in water, is used as a support for tissue engineering or drug delivery because it is able to easily absorb water due to its excellent hydrophilicity while its strength and shape are easily adjustable. In addition, due to outstanding biocompatibility, biodegradability, and minimally invasive properties to regulate drug release and achieve phase transition after in vivo injection, hydrogels are being actively researched as an alternative to methods showing low efficiency in the in vivo drug delivery. As a drug delivery carrier, its main purpose is to eliminate the need for frequent injections, which may cause side effects and discomfort such as pain through sustained release of drugs above the effective concentration for a long period of time at a site of the disease. Typically, in the case of wet age-related macular degeneration among ophthalmic diseases, an anti-VEGF agent, which is a protein drug, should be injected intraocularly for treatment, but the intraocular half-life is less than two (2) weeks, and the duration of the drug efficacy is short, lasting 1-2 months. Therefore, in order to derive the maximum effectiveness of the anti-VEGF agent, intraocular injection is required almost every month, which not only causes a great deal of discomfort and side effects to patients, but also poses a substantial economic burden, such that injectable hydrogels are being developed as a promising intraocular protein drug delivery carrier.
However, injectable hydrogels show limitations in controlling a rate of drug release in diseases where long-term maintenance of drug effectiveness is required, since an excess amount of drug is released at an initial stage before phase transition occurs after injection. In order to control the rate of drug release, it is necessary to inject hydrogel that had undergone crosslinking and phase transition, but the crosslinked hydrogel is solidified and in an irregular form which cannot be injected into the body. Accordingly, the present inventors have newly developed a long-term protein drug release-controlled injectable implant (hydrogel rod) using a crosslinked hydrogel.
The present disclosure relates to an injectable hydrogel rod for control of release of a protein drug using a crosslinked hydrogel, a manufacturing method thereof, and a use thereof, and an object of the present disclosure is to provide an injectable hydrogel rod which exhibits sustained release properties by controlling the contents of a polymer, an enzyme, and hydrogen peroxide.
In order to achieve the above object, the present disclosure provides an injectable hydrogel rod including a rod-shaped hydrogel matrix which includes a polymer substituted with phenol derivatives and in which the phenol derivatives introduced into side chains of the polymer are bonded to each other and crosslinked; and a drug loaded in the hydrogel matrix.
In addition, the present disclosure provides an injectable implant material including the injectable hydrogel rod described above.
In addition, the present disclosure provides a drug delivery carrier including the injectable hydrogel rod as described above.
In addition, the present disclosure provides a method of manufacturing an injectable hydrogel rod, including preparing a mold having a rod-shaped hole; preparing a lysate in which a polymer substituted with a phenol derivative, a drug, and an enzyme are dissolved; injecting the lysate and hydrogen peroxide sequentially into the hole provided in the mold and then crosslinking hydrogel; separating the crosslinked hydrogel from the mold; and freeze-drying the separated hydrogel to manufacture a hydrogel rod,
wherein a mixing ratio of the polymer substituted with the phenol derivative, the enzyme, and the hydrogen peroxide is 10:0.1˜0.5:0.1˜0.5 in a volume ratio.
According to the present disclosure, by manufacturing an injectable hydrogel rod by controlling the contents of a polymer, an enzyme, and hydrogen peroxide in a certain ratio, it is possible to control an initial burst release amount of drug released from the hydrogel rod to ensure sustained release above a concentration of drug efficacy for a long period of time compared to existing in situ forming hydrogels, and remarkably decrease elimination to the surrounding tissues, thereby significantly reducing side effects.
Hereinafter, in order to explain the present disclosure in more detail, preferred example embodiments according to the present disclosure will be described in more detail with reference to the attached drawings. However, the present disclosure is not limited to the example embodiments described herein but may be implemented in other forms.
The present inventors have completed the present disclosure through the design and effectiveness evaluation on a manufacturing method of a long-term protein drug release-controlled injectable hydrogel rod using a crosslinked hydrogel.
The present disclosure provides an injectable hydrogel rod which includes a rod-shaped hydrogel matrix which includes a polymer substituted with phenol derivatives and in which the phenol derivatives introduced into side chains of the polymer are bonded to each other and crosslinked; and a drug loaded in the hydrogel matrix.
The hydrogel matrix may have a degree of crosslinking of 30% to 80%, 40% to 70%, or 45% to 60%. With the degree of crosslinking as described above, the hydrogel rod may exhibit sustained release properties for more than 3 or 4 months by controlling an initial release rate to 10% or less, 7% or less, or 5% or less.
The rod-shaped hydrogel matrix may be 0.5 to 0.7 mm, 0.7 to 0.9 mm, or 0.9 to 1.2 mm in diameter.
In addition, the rod-shaped hydrogel matrix may be 4 to 6 mm, 6 to 8 mm, or 8 to 10 mm in length.
The polymer may be one or more polymers selected from the group consisting of gelatin, chitosan, heparin, cellulose, dextran, dextran sulfate, chondroitin sulfate, keratan sulfate, dermatan sulfate, alginate, collagen, albumin, fibronectin, laminin, elastin, vitronectin, hyaluronic acid, fibrinogen, and polybranched-polymers.
The phenol derivative may be one or more selected from the group consisting of hydroxyphenyl propionic acid, 4-hydroxyphenyl acetic acid, tyrosine, tyramine, tetronic tyramine, and PEG-tyramine.
The drug may be one or more drugs selected from intraocularly injectable protein therapeutic agents, chemotherapeutic drugs, low molecular weight therapeutic agents, genes, cells, or cell derivatives. Specifically, protein therapeutic agents may consist of anti-vascular endothelial growth factors used in macular degeneration and diabetic retinopathy, in other words, anti-VEGF ranibizumab, aflibercept, bevacizumab, brolucizumab, and other protein drugs used in diseases such as rituximab, adalimumab, or infliximab; the chemotherapeutic drug may consist of dexamethasone, triamcinolone, ganciclovir, methotrexate, or vancomycin; the low molecular weight therapeutic agent may consist of sugars, lipids, amino acids, fatty acids, phenolic compounds, or alkaloids; the gene may consist of siRNAs (anti-VEGF); and the cell may consist of retinal and intraocular cells such as RPE and photoreceptors, and stem cells. The following example embodiments using bevacizumab as a drug are described, but the present disclosure is not limited to these types of drugs and may include protein therapeutic agents, chemotherapeutic drugs, low molecular weight therapeutic agents, genes or cells of any ophthalmic and various diseases.
The hydrogel may form a rod-shaped hydrogel matrix by controlling concentration of the polymer, an enzyme, and hydrogen peroxide.
Specifically, the polymer substituted with the phenol derivatives, the enzyme, and the hydrogen peroxide may be mixed in a volume ratio of 10:0.1˜0.5:0.1˜0.5 to form a rod-shaped hydrogel matrix.
In addition, the present disclosure provides an injectable implant material including the injectable hydrogel rod as described above. Specifically, the injectable implant material may be an intraocularly injectable implant material.
The implant material may be applied to any one selected from the group consisting of intraocular and extraocular spaces in the body, subcutaneous tissues, and other interiors of organs.
In addition, the present disclosure provides a drug delivery carrier including the injectable hydrogel rod as described above.
In addition, the present disclosure provides a method of manufacturing an injectable hydrogel rod, including preparing a mold having a rod-shaped hole; preparing a lysate in which a polymer substituted with a phenol derivative, a drug, and an enzyme are dissolved; injecting the lysate and hydrogen peroxide sequentially into the hole provided in the mold and then crosslinking hydrogel; separating the crosslinked hydrogel from the mold; and freeze-drying the separated hydrogel to manufacture a hydrogel rod,
wherein a mixing ratio of the polymer substituted with the phenol derivative, the enzyme, and the hydrogen peroxide is 10:0.1˜0.5:0.1˜0.5 in a volume ratio.
In the preparing of the mold, the mold may be prepared to have a rod-shaped hole inside one type of mold selected from the group consisting of gelatin, metal, and Teflon.
The rod-shaped hole may have a diameter of 0.5 to 1.2 mm and a length of 4 to 10 mm.
Specifically, the preparing of the mold may include preparing a gelatin mold by pouring a gelatin solution on the outside of a needle and then hardening at a temperature of 1 to 10° C.
The needle may have a diameter of 0.5 to 1.2 mm and a length of 4 to 10 mm.
The gelatin solution may include 5 to 20 wt % or 5 to 15 wt % of gelatin.
In the crosslinking of the hydrogel, one or more phenol groups introduced into a side chain of the polymer may be bonded to each other and crosslinked through an enzymatic crosslinking reaction of the enzyme and the hydrogen peroxide. Specifically, it is possible to form a rod-shaped hydrogel through the crosslinking of hydrogel.
The polymer may be any one or more polymers selected from the group consisting of gelatin, chitosan, heparin, cellulose, dextran, dextran sulfate, chondroitin sulfate, keratan sulfate, dermatan sulfate, alginate, collagen, albumin, fibronectin, laminin, elastin, vitronectin, hyaluronic acid, fibrinogen, and polybranched-polymers.
The enzyme includes one or more selected from the group consisting of horseradish peroxidase, glutathione peroxidase, haloperoxidase, myeloperoxidase, catalase, hemoprotein, peroxide, peroxiredoxin, animal heme-dependent peroxidase, thyroid peroxidase, vanadium bromoperoxidase, lactoperoxidase, tyrosinase, and catechol oxidase.
A content of the polymer may be 10 to 30 wt % or 15 to 25 wt %.
A content of the hydrogen peroxide may be 0.02 to 0.08 wt %, 0.03 to 0.07 wt %, or 0.035 to 0.06 wt %.
A concentration of the enzyme may be 0.001 to 0.005 mg/ml or 0.002 to 0.004 mg/ml.
By mixing the polymer, enzyme, and hydrogen peroxide as described above, the injectable hydrogel rod manufactured according to the present disclosure exhibits a degree of crosslinking of 20% to 100%, 30% to 80%, 40% to 70%, or 45% to 60%, and thus the hydrogel rod may exhibit sustained release properties for more than three (3) months by controlling the initial release rate to 10% or less, 7% or less, or 5% or less.
In the separating from the mold, the rod-shaped hydrogel may be separated from one type of molds selected from the group consisting of gelatin, metal, and Teflon.
Specifically, in the separating from the mold, the rod-shaped hydrogel may be separated by dissolving the gelatin at a temperature of 30° C. to 40° C. to remove the gelatin mold.
Alternatively, in the separating from the mold, the hydrogel rod in the mold may be separated by pushing with a rod-shaped mold that is made of metal or Teflon materials.
The freeze-drying may be performed at a temperature of −70 to −85° C. for 12 to 24 hours to manufacture a hydrogel rod.
Hereinafter, the present disclosure will be described in more detail through examples to help understanding of the present disclosure. However, examples below are merely intended to illustrate the present invention, and the scope of the present disclosure is not limited to the following examples. Examples of the present disclosure are provided to more completely explain the present disclosure to those skilled in the art.
Temperature-sensitive gelatin was used as a mold to manufacture a rod-shaped drug delivery carrier of injectable gelatin-PEG-tyramine (GPT) hydrogel ((a) of
As shown in
In order to identify the mechanical properties in accordance with the control of the degree of crosslinking of the injectable hydrogel rod of the present disclosure, in Example 1, the final concentration of H2O2 used for crosslinking was adjusted to 0.02, 0.04, and 0.08 wt % as shown in (a) of
Following evaluation of the control of physical properties upon control of a degree of crosslinking of the injectable hydrogel rod, the evaluation of the control of bevacizumab release was carried out. While the hydrogel rod formed by the degree of crosslinking was incubated in DPBS (37° C., 100 rpm), a DPBS sample including bevacizumab released at the specific time point for 120 days was obtained, frozen, and stored, followed by addition of a new DPBS solution. The obtained samples were subjected to quantitative analysis using the enzyme linked immunosorbent assay (ELISA) technique, which enables specific quantification of bevacizumab, an anti-VEGF.
As shown in (a) of
Referring to (b) of
The comparison was made on drug release behaviors of the hydrogel rod (pre-crosslinked hydrogel rod) of Examples and the in situ forming hydrogel of Comparative Example. Polymers under the same condition (GPT 20 wt %) and crosslinking conditions (H2O2 0.04 wt %) were used, and 1500 g of bevacizumab was loaded, followed by evaluation of the release. Referring to (a) and (b) of
As a result of conducting comparative evaluation with the in situ forming hydrogel that is not in the form of a rod form of Comparative Example as above, the in situ forming hydrogel of Comparative Example is a substance that undergoes phase transition after minimally invasive injection and also quickly releases the loaded drug at an initial stage, but the hydrogel rod of the present disclosure may enable minimally invasive injection to be applied in a crosslinked form to a pre-targeted degree, such that it was determined that the loaded drug is prevented from being released quickly at an early stage and exhibits sustained release for more than four (4) months.
The long-term efficacy and biological stability of drugs released were evaluated through culture of human umbilical vein endothelial cells (HUVECs), which are cells that are involved in angiogenesis. After culturing HUVECs, they were prepared in 2×104 cells/well in a 48-well plate and treated with the long-term released bevacizumab drug samples diluted in 1:1 ratio in a cell culture medium (EBM media containing EGM supplements and 1% P/S) and fresh bevacizumab drug samples by concentration so as to observe cell growth inhibition and safety. For the control of fresh bevacizumab drug samples by concentration, preparation was performed at final concentrations of 1, 10, and 100 g/mL, diluted in cell culture media.
(a) of
Thereby, it is noticeable that the in situ forming hydrogel of Comparative Example shows the rapid release of more than 40% for the initially loaded drug to lead to excessive release, thereby showing the high growth inhibition of cells at the early stage, whereas the hydrogel rod of Example exhibits the drug efficacy through cell growth inhibition for a long period of time because the initial release rate is controlled to exhibit the sustained release for more than four (4) months.
As shown in
After animal sacrifice and ocular separation by date, pharmacokinetic analysis of vitreous, retina, and aqueous tissues was performed via ELISA. Referring to (a) of
Referring to (b) of
Referring to (c) of
(d) of
As shown above, it was found that the in situ forming hydrogel of Comparative Example exhibited high initial burst release and elimination to the surrounding tissue, with difficulty in maintaining the drug efficacy for a long time. On the other hand, it was found that the initial burst release was suppressed upon injection of the hydrogel rod of Example to cause sustained release above the concentration for drug efficacy for 4 months, and elimination to the surrounding tissues thereby was greatly reduced. Based on this, it is possible to overcome the issue concerning side effects such as systemic side-effects caused by excessive elimination as well as inflammation caused by frequent injections for drug efficacy.
After intraocular injection of the hydrogel rod, ultrasonic analysis was conducted to analyze the conformational stability and safety for each day. As shown in
It is difficult for the hydrogel of Comparative Example to maintain its shape because it is diluted with the surrounding tissue fluid before the complete phase transition after injection, with a risk of causing side effects through adhesion with the surrounding tissue during the phase transition, but it was found that the hydrogel rod of Example is injected in the form of a crosslinked rod to ensure a stable conformation and safety in vivo for 4 months.
While a specific part of the present disclosure has been described in detail above, it is clear for those skilled in the art that this specific description is merely preferred example embodiments, and the scope of the present disclosure is not limited thereby. In other words, the substantial scope of the present disclosure is defined by the attached claims and their equivalents.
Number | Date | Country | Kind |
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10-2022-0015609 | Feb 2022 | KR | national |
Filing Document | Filing Date | Country | Kind |
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PCT/KR2022/017200 | 11/4/2022 | WO |