Patent Application
20210369875
The present disclosure relates to an alginic acid-based injectable hydrogel system, and in particular, to an alginic acid-based injectable hydrogel system which may help to stay at the injection site for a certain period of time to accurately determine a position of a target site by an image, without diffusing a contrast agent contained in an alginic acid to the surroundings and thereby helps to treat or alleviate symptoms at the target site by slowly releasing the drug contained in alginic acid at the target site.
As one of the ‘minimal invasive surgeries’ which reduces the injury or influence on the patient's body, laparoscopic surgery is a surgery mode using surgical tools (forceps, electric scalpel, hemostasis suture, etc.) while puncturing a small hole of 0.5 to 1.5 cm and inserting endoscope (laparoscope) loaded with a special camera into the body, looking inside the abdominal cavity, instead of incising the abdomen or chest.
Laparoscopic surgery has been advanced over the past several decades with the advantages of faster recovery and shorter hospital stays as it has fewer postoperative pain and fewer incisions than a laparotomy. Recently, as robotic surgery using this laparoscopic surgery has been introduced, laparoscopic surgery has been accepted as the basis of surgical techniques. For the surgical treatment of colorectal cancer, gastric cancer, etc., a process of visually confirming the treatment site, such as the cancer site by inserting an endoscope into the stomach or the large intestine before incision and then removal of the cancer lesion in the body through laparoscopic surgery is needed. However, since the viewing angle and range obtained through the endoscope and the viewing angle and range obtained through the laparoscope are completely different in the surgery of prior art, it is difficult to accurately find the target position to be incised, and thus the accuracy and the range of the incision are inevitably different depending on the skill of the operator, and the range to be incised is inevitably increased. Therefore, in order to label the region to be removed by surgery, a method of injecting a drug such as Indian ink into the lesion identified through an endoscopic injection needle with the endoscope to label the lesion, and then performing surgery for the region labeled with Indian ink is used, while checking with the naked eye through laparoscopy at the time of surgery. However, such a labeling method has a disadvantage that complications may occur due to ink incorrectly injected into the muscle layer, and that the region to be removed by surgery is widened as ink or dye spreads around the injected area. Therefore, in order to minimize the region from which normal tissue is removed during laparoscopic surgery, a contrast agent for “marking” or “marker” is required to label an appropriate disease lesion location.
If surgical treatment is difficult for a region of a cancer or inflammatory disease, treatment and symptom relief may be achieved by injecting a drug locally, and in the case of hydrophobic drugs, since they are not easily soluble in water, local injection of the drugs is not easy and in the case of hydrophilic drugs, since they spread quickly at the injected site, the duration of the drug is very short. Therefore, there is a need for a “locally injectable” drug formulation capable of maximizing therapeutic effect or symptom relief effect at the target site by allowing the drug to be released slowly at a desired rate while allowing the drug to be evenly injected into the target lesion.
There may be various image contrast agent or image methods capable of labeling lesions, and recently, a technique for labeling lesions using near-infrared fluorescent dyes is rapidly developing. Fluorescence image technology using near-infrared fluorescent dyes, has the advantage of not only being able to detect even a labeled region located deep in a tissue from the fluorescent image, but also sensitively detecting a fluorescent signal even with a small amount of fluorescent dye. As a conventional fluorescent image contrast agent, indocyanine green, a near-infrared fluorescent dye that has been clinically approved by the FDA, has been used for image diagnosis and vascular image during surgery, but there are several limitations in its clinical application. Since there is a disadvantage that Indocyanine green decreases fluorescence brightness due to concentration-dependent aggregation phenomenon, has chemical instability in aqueous solution in vivo, and spreads rapidly to surrounding tissues to reduce image discrimination accuracy and rapidly weaken the image signal intensity, it makes the contrast agent difficult to be used. For this reason, there is a need to develop a method that can increase the labeling efficiency for lesions and increase the half-life of the contrast agent in vivo.
Next, when the drugs are injected into a living body, side effects may occur on the surrounding normal tissues by being rapidly absorbed and spreading at the site where the drug was injected, and since the drugs are decomposed by enzyme action in the living body, resulting in poor drug persistence. Thus, there is a difficulty in administering large amounts of drugs or repeatedly administering drugs in order to obtain a desired drug effect. For this reason, there is a need to develop a method capable of improving the duration of the drug effect on the target site by preventing the diffusion of the drug in vivo.
The present disclosure was conceived to solve the problems of the prior art as described above, and the present inventors have completed the invention by developing an alginic acid-based injectable hydrogel system using polyvalent ions as an ionic cross-linking agent based on alginic acid. Accordingly, the present disclosure provides a composition for preparing an injectable hydrogel, comprising a first agent comprising alginic acid and an ionic crosslinking agent; and a second agent comprising alginic acid and a crosslinking rate controller. In addition, the present disclosure provides a composition for labeling the lesion, comprising a first agent comprising alginic acid and an ionic crosslinking agent; and a second agent comprising alginic acid and a crosslinking rate controller, and a method for labeling the lesion, comprising injecting the composition to an individual.
An alginic acid-based injectable hydrogel system according to the present disclosure allows the contrast agent contained in alginic acid to stay at the injection site for a certain period of time without being diffused to the surroundings, so that the location of the target site may be accurately determined by image.
In addition, the alginic acid-based injectable hydrogel system according to the present disclosure may include a drug as well as a contrast agent into alginic acid and inject them into the target site, and in particular, if the alginic acid hydrogel according to the present disclosure including a drug such as an anticancer agent is injected to the target site, the drug may be released slowly for a long period of time from alginic acid while preventing the drug from spreading to the surroundings, so that the effect of drugs can last for a long time.
Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by an expert skilled in the art to which the present disclosure belongs. In general, the nomenclature used in this specification is well known and commonly used in the art.
The present disclosure relates to an alginic acid-based injectable hydrogel system to address the above-described technical problem. Specifically, the present disclosure provides an alginic acid-based injectable hydrogel system in the form of a composition for preparing an injectable hydrogel, comprising a first agent comprising alginic acid and an ionic crosslinking agent; and a second agent comprising alginic acid and a crosslinking rate controller. In addition, the present disclosure provides a composition for labeling the lesion, comprising a first agent comprising alginic acid and an ionic crosslinking agent; and a second agent comprising alginic acid and a crosslinking rate controller, and a method for labeling the lesion, comprising injecting said composition to an individual.
In the present disclosure, the first agent or the second agent may comprise an image contrast agent or a drug.
That is, the composition for preparing an injectable hydrogel according to the present disclosure was made into an injectable hydrogel form by mixing the first and second agents and injecting the mixture, in which an image contrast agent or a drug is incorporated into an alginic acid or alginic acid/hyaluronic acid mixture, and an ionic crosslinking agent and a crosslinking rate controller are included therein.
Accordingly, the present disclosure relates to an injectable hydrogel composition in which the first agent and the second agent are mixed together.
When an image contrast agent or a drug is topically injected into the target lesion region using such an alginic acid-based injectable hydrogel system, the injection solution becomes a gel at the labeling site, thereby preventing the contrast agent or drug from spreading, and resulting in increasing the duration. Through this, not only can the location of the target site be determined relatively accurately by image, but also in the case of a drug, the drug effect can last for a long time, so that side effects caused by the diffusion of the drug may be reduced, and the desired drug effect may be achieved at the target site.
In the present disclosure, the “ionic crosslinking agent” is included in the first agent and serves as an ion source that provides polyvalent ions for cross-linking of alginic acid to form the ionic cross-linked hydrogel. These ionic crosslinking agents may be selected from the group consisting of calcium carbonate, strontium carbonate, calcium disodium edetate (calcium EDTA), calcium citrate, calcium sulfate, calcium alginate, calcium gluconate, calcium phosphate dibasic (CaHPO4), barium carbonate, zinc carbonate, calcium chloride, calcium lactate, calcium aspartate, calcium saccharate, calcium oxovalerate, calcium lactobionate, calcium lactogluoconate, and a mixture thereof, but are not limited thereto.
In the present disclosure, the “crosslinking rate controller” is included in the second agent and serves to control the rate of alginic acid cross-linking formation. Such a crosslinking rate controller may comprise any one or more selected from the group consisting of an organic acid such as gluconic acid, acetic acid, malic acid, lactic acid, ascorbic acid, or a salt thereof or a mixture thereof, and a photoacid generator (PAG). More specifically, the crosslinking rate controller may be D-gluconic acid, but is not particularly limited thereto.
In the present disclosure, the ratio of an ionic crosslinking agent and a crosslinking rate controller is not limited, but the ionic crosslinking agent is preferable to have a molar ratio of 0.1 to 0.4× based on the number of moles (X) of a carboxylic acid group present in alginic acid. In the case of the crosslinking rate controller, it is used in an amount that makes the pH of the solution neutral.
In the present disclosure, the alginic acid may be mixed with hyaluronic acid. Since the hydrogel is well formed even under various mixing conditions in which the mixing ratio of alginic acid and hyaluronic acid is from 100:0 to 1:99, the mixing ratio of alginic acid and hyaluronic acid is not significantly limited. Hyaluronic acid is decomposed by hyaluronidase, which is an enzyme present in the human body, so the higher the mixing ratio of hyaluronic acid, the faster the decomposition rate of the hydrogel and the faster the release rate of the drug, and the mixing ratio of alginic acid and hyaluronic acid is adjusted according to the desired release rate of the contrast agent or drug.
In the present disclosure, the “image contrast agent” may be a magnetic resonance image contrast agent, a computed tomography (CT) contrast agent, or a fluorescent dye.
When the magnetic resonance image contrast agent (MRI contrast agent) is injected into the human body, it changes the relaxation rate of tissues to widen the difference in relaxation degree between tissues, and induces change in MRI signal to make the contrast between tissues more clearly. The MRI contrast agent includes ionized gadolinium (Gd) (III) complexes and neutral gadolinium (Gd)(III) complexes. More specifically, the MRI contrast agent may be a gadolinium complex, a manganese complex, a copper (II) complex, an iron oxide nanoparticle, and manganese oxide nanoparticles. In an embodiment of the present disclosure, a manganese oxide nanoparticle, and Feridex™, which is iron oxide nanoparticle contrast agent, and manganese chloride were used as MRI contrast agent, but are not limited thereto.
In the present disclosure, the CT contrast agent as a material used to clarify shading when X-rays are taken, may be made of a compound containing a metal or iodine, or nanoparticles, and Rose Bengal is used in an embodiment of the present disclosure, but is not limited thereto.
In the present disclosure, the “fluorescent dye” may be fluorescein, CR110: carboxyrhodamine 110: rhodamine green (trade name), TAMRA: carboxytetramethylrhodamine: TMR, carboxyrhodamine 6G: CR6G, BODIPY FL (trade name): 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid, BODIPY 493/503 (trade name): 4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene-8-propionic acid, BODIPY R6G (trade name): 4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-propionic acid, BODIPY 558/568 (trade name): 4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s-indacene-3-propionic acid, BODIPY 564/570 (trade name): 4,4-difluoro-5-styryl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid, BODIPY 576/589 (trade name): 4,4-difluoro-5-(2-pyrrolyl)-4-bora-3a,4a-diaza-s-indacene-3-propionic acid, BODIPY 581/591 (trade name): 4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-propionic acid, EvoBlue10 (trade name), EvoBlue30 (trade name), MR121, ATTO 655 (trade name), ATTO 680 (trade name), ATTO 700 (trade name), ATTO MB2 (trade name), Alexa Fluor 350 (trade name), Alexa Fluor 405 (trade name), Alexa Fluor 430 (trade name), Alexa Fluor 488 (trade name), Alexa Fluor 532 (trade name), Alexa Fluor 546 (trade name), Alexa Fluor 555 (trade name), Alexa Fluor 568 (trade Name), Alexa Fluor 594 (trade name), Alexa Fluor 633 (trade Name), Alexa Fluor 680 (trade name), Alexa Fluor 700 (trade name), Alexa Fluor 750 (trade name), Alexa Fluor 790 (trade name), Flamma 496 (trade name), Flamma 507 (trade name), Flamma 530 (trade name), Flamma 552 (trade name), Flamma 560 (trade name), Flamma 575 (trade name), Flamma 581 (trade name), Flamma 648 (trade name), Flamma 675 (trade name), Flamma 749 (trade name), Flamma 774 (trade name), Flamma 775 (trade name), Rhodamine Red-X (trade name), Texas Red-X (trade name), 5(6)-TAMRA-X (trade name), STAMRA (trade name), cyanine-based dyes (Cy5, Cy5.5, Cy7, IR820), Indocyanine green (ICG) or ZW800, etc., but is not limited thereto. Specifically, the fluorescent dye may be a complex in which a fluorescent dye and human serum albumin are bound. The bond may be an ionic bond, a hydrophobic bond, or a covalent bond, and more specifically, a covalent bond, but is not particularly limited thereto. Typically, a BODIPY dye may form a hydrophobic bond with human serum albumin, and cyanine-based dyes and Alexa-based dyes introduced with a sulfonyl group to improve hydrophilicity may form ionic bonds.
In the present disclosure, the “drug” is not particularly limited in its kind. A drug suitable for the target disease which may be selected, may be for example, anticancer agents, anti-inflammatory agents, anesthetic agents, antiviral agents, antibacterial agents, therapeutic antibodies, antibiotics, or immunotherapeutic agents, etc.
In the present disclosure, examples of the anticancer agent may include, but are not limited to, one or more selected from the group consisting of doxorubicin, gosipol, nitrogen mustard, imatinib, oxaliplatin, rituximab, erlotinib, neratinib, lapatinib, gefitinib, vandetanib, nitrotinib, semasanib, bosutinib, akcitinib, cediranib, restaurtinib, trastuzumab, gefitinib, bortezomib, sunitinib, carboplatin, bevacizumab, cisplatin, cetuximab, viscumalbum, asparaginase, tretinoin, hydroxycarbamide, dasatinib, estramustine, gemtuzumabozogamycin, ibritumomab tiuxetan, heptaplatin, methylaminolevulinic acid, amsacrine, alemtuzumab, procarbazine, alprostadil, holmium nitrate chitosan, gemcitabine, doxyfluridine, pemetrexed, tegafur, capecitabine, gimeracin, oteracil, azacitidine, methotrexate, uracil, cytarabine, fluorouracil, fludarabine, enocitabine, flutamide, decitabine, mercaptopurine, thioguanine, cladribine, carmophor, raltitrexed, docetaxel, paclitaxel, irinotecan, belotecan, topotecan, vinorelbine, etoposide, vincristine, vinblastine, teniposide, doxorubicin, idarubicin, epirubicin, mitoxantrone, mitomycin, bleromycin, daunorubicin, dactinomycin, pyrarubicin, aclarubicin, pepromycin, temsirolimus, temozolomide, busulfan, ifosfamide, cyclophosphamide, melpharan, altrethamine, dacarbazine, thiotepa, nimustine, chlorambucil, mitolactol, leucovorin, tretonin, exmestane, aminoglutesimide, anagrelide, navelbin, padrazole, tamoxifen, toremifen, testolactone, anastrozole, letrozole, borozole, bicalutamide, lomustine and carmustine.
In the present disclosure, examples of the anti-inflammatory agent may include salicylates, ibuprofen, naproxen, fenoprofen, indomethacin, phenyltazone, methotrexate, cyclophosphamide, mechlorethamine, dexamethasone, prednisolone, celecoxib, valdecoxib, nimesulide, cortisone, or corticosteroid.
On the other hand, when an alginic acid-based hydrogel is injected into the lower mucosa for image labeling, the patient's immune system recognizes that a foreign substance has invaded into the living body and may cause an inflammatory reaction in removing the foreign substance. At this time, proteins including various enzymes, complement systems, antibodies, and immune cells are introduced into the injection site, and actions to remove alginic acid and fluorescent dyes present at the injection site occur. Therefore, there is a possibility that the rate at which the fluorescent dye contained in the hydrogel is removed due to the inflammatory reaction occurring at the injection site would be fast. As a method for reducing the inflammatory reaction and side effects, there is a method of separately orally administering an anti-inflammatory agent or injecting an anti-inflammatory agent into a target site by mixing with a hydration gel, when injecting a hydration gel. Since hydrophobic anti-inflammatory drugs do not dissolve well in water, they are loaded into microspheres, nanoparticles, liposomes or micelles, etc., to ensure good dispersion in aqueous solution and injected with a hydrogel.
The composition for preparing the injectable hydrogel according to the present disclosure may contain a suspending agent, a solubility aid, a stabilizer, an isotonic agent, a preservative, an adsorption inhibitor, a surfactant, a diluent, an excipient, a pH adjuster, a painless agent, a buffer, and a sulfur-containing reducing agent, antioxidant, etc., as appropriate, if necessary. For example, sterile water, physiological saline, conventional buffers (phosphoric acid, citric acid, other organic acids, etc.), stabilizers, salts, antioxidants (ascorbic acid, etc.), surfactants, suspending agents, isotonic agents, or preservatives, etc., may be included. As an aqueous solution for injection, for example, an isotonic solution including physiological saline, glucose or other adjuvants, such as D-sorbitol, D-mannose, D-mannitol, sodium chloride, and also buffers, for example, a phosphate buffer solution, a sodium acetate buffer solution, a painless agent such as procaine hydrochloride, a stabilizer such as benzyl alcohol, phenol, or an antioxidant may be combined. Pharmaceutically acceptable carriers and formulations suitable for the present disclosure are described in detail in Remington's Pharmaceutical Sciences, 19th ed., 1995.
In the present disclosure, the “individual” may be a mammal including humans, dogs, cats, cows, horses, pigs, mice, etc., but is not particularly limited thereto.
If the region to be removed by surgery is marked in advance, or if it is necessary to mark the location of the diseased lesion for a certain period of time, an image contrast agent is locally injected into the lesion, but as it spreads to surrounding tissues with diffusing at a fast rate, there is a disadvantage that the accuracy of image discrimination is deteriorated, and the image signal strength is rapidly weakened.
The composition for preparing an alginic acid-based injectable hydrogel containing an image contrast agent according to the present disclosure allows the contrast agent contained in the gel to remain at the injected lesion site for a certain period of time without spreading to the surroundings, thereby allowing the location of the target site to be determined relatively accurately with image and to be removed surgically, if necessary.
In addition, when the drug is injected intravenously, in most cases, it affects unwanted cells or organs, causing serious side effects in many cases. Even when the drug is injected locally at the target site, the drug is rapidly absorbed and spread at the injected site, so that side effects on the surrounding normal cells cannot be completely avoided, and the duration of the drug at the injection site is also shortened.
The composition for preparing an alginic acid-based injectable hydrogel containing a drug according to the present disclosure may be sustained for a long time in drug efficacy as the drug contained in the gel at the site of the injected lesion is slowly released for a certain period of time without being diffused to the surroundings. Therefore, even if a small amount of the drug is injected, the desired drug effect may be achieved at the target site, while reducing the side effects of the drug on the surrounding tissues.
Hereinafter, preferred embodiments of the present disclosure will be described in more detail with reference to the accompanying drawings in order to describe the present disclosure in more detail. However, the present disclosure is not limited to the embodiments described herein and may be embodied in other forms. Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by an expert skilled in the art to which the present disclosure belongs. In general, the nomenclature used in this specification is well known and commonly used in the art.
1-1: Measurement of Fluorescence and Absorbance of Indocyanine Green and Indocyanine Green-Human Serum Albumin Complex
Before developing a surgical marker using indocyanine green and human serum albumin conjugate, in order to determine the appropriate concentration of indocyanine green and human serum albumin conjugate, the two substances were mixed by each concentration to observe change in fluorescence (λex=720 nm) and absorbance.
3 mL of distilled water was added to 1.5 mg of indocyanine green (ICG, purchased from Sigma-Aldrich) and dissolved by stirring to prepare a high concentration of indocyanine green solution. Distilled water was added to the high concentration of indocyanine green solution and mixed through stirring to prepared a 2 to 100 μM solution.
1 mL of the high concentration of indocyanine green solution was taken, added to 42.89 mg of human serum albumin (HSA, purchased from Sigma-Aldrich), and mixed with each other thoroughly by pipetting to obtain a high concentration of indocyanine green-human serum albumin complex. The high concentration of the indocyanine green-human serum albumin solution was added to distilled water and stirred to prepared a 2 to 100 μM of solution.
<Evaluation 1-Evaluation of Fluorescence Properties>
When only indocyanine green is in 2 to 100 μM, the maximum fluorescence intensity increased to about 20 μM, and then due to an aggregation of the fluorescent dye, the fluorescence decreased from the concentration or higher (
<Evaluation 2-Evaluation of Absorbance Properties>
As a result of observing the absorbance of the indocyanine green and the indocyanine green-human serum albumin complex bound in 1:1 in concentration range of 2 to 10 μM, it was confirmed that both showed a tendency to increase the absorbance as the concentration increased, and the absorbance of the indocyanine green-human serum albumin complex appeared stronger (
1-2: Measurement of Fluorescence and Absorbance According to Mixing Ratio of Indocyanine Green-Human Serum Albumin Complex
Before developing a surgical marker using indocyanine green-human serum albumin conjugate, in order to determine the appropriate mixed ratio of indocyanine green-human serum albumin conjugate, the two substances were mixed by each concentration to observe change in fluorescence (λex=720 nm) and absorbance.
In the same manner as in Preparation Example 1, a high concentration of indocyanine green solution was prepared. After adding 1 mL of distilled water to 42.89 mg of human serum albumin, pipetting was performed to thoroughly mix to prepare a high concentration of human serum albumin solution. The high concentration of indocyanine green solution was fixed to a final concentration of 30 μM by taking 0.125 mL, and only the human serum albumin solution was added in an amount corresponding to the molar ratio of indocyanine green of 0 to 2 to make a total of 1 mL of a solution with distilled water and stir to mix thoroughly.
<Evaluation 3-Evaluation of Fluorescence and Absorbance Properties>
Changes in fluorescence and absorbance were observed using the solution of Preparation Example 3 (
1-3: Observation of Gelation of Alginic Acid Solution Over Time after Addition of Calcium
In order to make an alginic acid hydrogel with an appropriate gelation time that may be used as a surgical marker, calcium ions of different concentrations as shown in Table 1 were added to the alginic acid solution through a dual syringe, and then the change in the alginic acid solution over time was observed (
Calcium ions form a hydrogel through cross-linking with alginic acid. Since calcium carbonate (purchased from Sigma-Aldrich) has low solubility, it was made to allow rapid cross-linking with alginic acid by regulating the dissolution rate of calcium carbonate with a mix of D-gluconic acid (purchased from Sigma-Aldrich), which is an acidic condition through a dual syringe. Through this process, alginic acid may be gelled evenly throughout. The pH of the total solution was adjusted to neutral by adding two folds of the number of moles of D-gluconic acid with respect to calcium carbonate.
(The mixing condition of the alginic acid hydrogel through a dual syringe; X means the number of moles of the carboxy group contained in 1.0 w/v % of alginic acid.)
0.036 g of sodium alginate (purchased from Kimika) was dissolved in 3 mL of distilled water to prepare a 1.2 wt % solution of alginic acid. 0.25 mL of distilled water was added to 1.25 mL of a 1.2 wt % alginic acid solution to prepare a 1.0 wt % alginic acid solution. 1 mL of a 1.0 wt % alginic acid solution was added to 1.14 mg of calcium carbonate and mixed by pipetting or vortexing to prepare a first agent solution. 0.007 mL of a D-gluconic acid solution was added to 0.833 mL of 1.2 wt % of alginic acid solution, and made to a total of 1 mL with distilled water, followed by mixing completely through vortexing and stirring to prepare a second agent solution. Calcium carbonate and D-gluconic acid were used 0.1 and 0.2 times, respectively, based on the number of moles of carboxyl groups contained in 1.0 w/v % of alginic acid.
A solution was prepared in the same manner as in Preparation Example 4, except that the calcium carbonate of the first agent was 2.29 mg and the D-gluconic acid solution of the second agent was 0.015 mL. Calcium carbonate and D-gluconic acid were used 0.2 and 0.4 times, respectively, based on the number of moles of carboxyl groups contained in 1.0 w/v % of alginic acid.
A solution was prepared in the same manner as in Preparation Example 4, except that the calcium carbonate of the first agent was 3.43 mg and the D-gluconic acid solution of the second agent was 0.022 mL. Calcium carbonate and D-gluconic acid were used 0.3 and 0.6 times, respectively, based on the number of moles of carboxyl groups contained in 1.0 w/v % of alginic acid.
<Evaluation 4-Evaluation of Gelation of Aqueous Alginic Acid Solution According to Amount of Calcium Ions>
After injecting the solution of the first agent and the second agent of Preparation Examples 4 to 6 to be a total volume of 0.5 mL in a glass bottle through a dual syringe, the formation of gel over time was observed by turning the glass bottle over. As shown in Table 1 and
In the 0.1× calcium condition, the solution was in a liquid state (sol) flowing immediately after injection, and then changed to a solid state (gel) after about 46 minutes. In the 0.2× calcium condition, the solution was in a liquid state (sol) slightly flowing immediately after injection, and then changed to a solid state (gel) after about 2 minutes. Finally, in the 0.3× calcium condition, the solution exists in a state that hardly flow immediately after injection, and then changed to a solid state (gel) after about 1 minute. This gelation time is plotted on a graph according to the concentration of calcium ions used in
It was observed that the gelation time of alginic acid decreased as the concentration of calcium ions added to alginic acid increased through 0.1 to 0.3× calcium conditions. Especially, among them, in the case of a calcium condition of 0.3×, it existed in a solid state (gel) that hardly flows immediately after injection. In this experiment, in order to effectively prevent the diffusion of the indocyanine green-human serum albumin complex, a subsequent experiment was conducted by selecting a calcium condition of 0.3×, which becomes a gel immediately after injection.
1-4: Fluorescence Measurement of Alginic Acid Hydrogel Containing Indocyanine Green-Human Serum Albumin Complex
In order to prevent the easy diffusion of indocyanine green in the living body, the indocyanine green-human serum albumin complex was incorporated into alginic acid, and then cross-linked with calcium ions through a dual syringe to form an injectable hydrogel, and fluorescence (λex=720 nm) and fluorescence images (λex=780 nm, λem=845 nm) were confirmed under in vitro conditions.
A high concentration of indocyanine green solution was prepared in the same manner as in Preparation Example 1. Distilled water was added to 0.139 mL of the high concentration of indocyanine green solution to make a total of 1.5 mL, followed by a mix with stirring to prepare the first agent solution. Distilled water was used as the second agent solution. The final solution of the first agent and the second agent was prepared so that the concentration of the indocyanine green was 30 μM.
A high concentration of indocyanine green-human serum albumin solution was prepared in the same manner as in Preparation Example 2. Distilled water was added to 0.139 mL of the high concentration of indocyanine green-human serum albumin to make a total of 1.5 mL, followed by a mix with stirring to prepare the first agent solution. Distilled water was used as the second agent solution. The final solution mixed with the first agent and the second agent was prepared so that the concentration of indocyanine green-human serum albumin complex was 30 μM.
A high concentration of indocyanine green-human serum albumin solution was prepared in the same manner as in Preparation Example 2. The first agent and the second agent solutions were prepared in the same method as in Preparation Example 6, except that 0.139 mL of the high concentration of indocyanine green-human serum albumin solution was added to 1.25 mL of a 1.2 wt % of alginic acid solution, and then the total volume was made to 1.5 mL with distilled water.
<Evaluation 5-Fluorescence Evaluation>
The solutions of Preparation Examples 7 to 9 were injected into 96 wells, respectively so that the total volume was 200 μL using a dual syringe. The fluorescence graph and fluorescence image of the injected gel were confirmed over time.
As shown in
It was confirmed through the fluorescence image of
1-5: Fluorescence Measurement of Alginic Acid Hydrogel Containing Indocyanine Green-Human Serum Albumin in Animal Model
In order to see the fluorescence persistence of the alginic acid hydrogel containing indocyanine green-human serum albumin in a nude mouse, it was injected in the form of a gel subcutaneously to the nude mouse through a dual syringe, and then the labeling performance over time was photographed with a fluorescent image device (IVIS Lumina XR, Xenogen Corporation-Caliper, CA, USA). Three solutions to be injected into the animal model were prepared in the same manner as in Preparation Examples 7 to 9.
<Evaluation 6-Evaluation of the Persistence of Fluorescence of Gel in Animal Model>
The solutions of Preparation Examples 7 to 9 were injected subcutaneously to a nude mouse, respectively so that the total volume was 100 μL using a dual syringe.
In
In
In
Taking these results together, it was confirmed that the alginic acid hydrogel covered the fluorescent dye and prevented it from spreading at the target site under in vivo conditions, thereby effectively increasing the fluorescence duration.
1-6: Human Serum Albumin-ZW800-1C Incorporation Test of Alginic Acid Hydrogel
Human serum albumin-ZW800-1C fluorescent dye, a covalently bound complex, was added to the alginic acid hydrogel to confirm the persistence of fluorescence under in vivo conditions.
Human serum albumin (10 mg/mL) was dissolved in a phosphate buffer, and then reacted with ZW800-1C NHS ester (
In order to analyze the ratio of fluorescent dye bound to human serum albumin, human serum albumin-ZW800-1C conjugate was dissolved in a denatured phosphate buffer (including 1% SDS/1 mM NaOH) and absorbance was measured. The concentration of human serum albumin protein was calculated using the molar extinction coefficient (34,445 M−1cm−1) of human serum albumin at 280 nm, and the fluorescent dye was used with a known molar extinction coefficient (125,000 M−1cm−1 at 779 nm in DMSO). As a result of the analysis, it was calculated that ZW800-1C was attached with 0.26 per one human serum albumin.
The degree of absorption and fluorescence of the human serum albumin-fluorescent dye conjugate was measured by diluting in a phosphate buffer at a concentration of 5 μM (based on the fluorescent dye concentration) (
The first agent and the second agent were prepared in the same manner as in Preparation Example 9, except that the fluorescent dye covalently bound to 30 μM of human serum albumin-ZW800-1C was used in place of the indocyanine green-human serum albumin complex in the first agent.
<Evaluation 7-Evaluation of Persistence of Fluorescence of Gel in Animal Model>
The solutions of Preparation Example 11 were injected subcutaneously to a nude mouse, so that the total volume was 100 μL using a dual syringe. In the in vivo condition, it was confirmed that even in the case of the fluorescent dye covalently bound to human serum albumin-ZW800-1C, the fluorescence duration was effectively increased by preventing diffusion at the target site (
Doxorubicin (purchased from Sigma-Aldrich) as an anticancer agent, was prepared as a drug to be incorporated into the alginic acid hydrogel.
1.5 mg of doxorubicin was dissolved in 0.5 mL of distilled water to prepare a high concentration of doxorubicin solution. The first agent and the second agent solutions were prepared in the same method as in Preparation Example 6, except that 0.125 mL of the high concentration of doxorubicin was added to 1.25 mL of a 1.2 wt % of alginic acid solution, and then the total volume was made to 1.5 mL with distilled water.
<Evaluation 8-Doxorubicin Incorporation in Alginic Acid Hydrogel and Fluorescence Image Confirmation>
The solution of Preparation Example 12 was injected into the ep-tube so that the total volume was 0.5 mL using a dual syringe. Thereafter, it was confirmed that the alginic acid hydrogel formed by turning the ep-tube over as shown in
As a result of examining the fluorescence image (λex=420 nm, λem=570 nm) of the hydrogel containing doxorubicin using a fluorescence image device (IVIS) as shown in
A manganese chloride solution (manganese chloride, purchased from Sigma-Aldrich, 1M) and Ferdex I.V. (Feridex, purchased from Taejun Pharmaceutical co., Ltd., 11.2 mg Fe/mL) as the magnetic resonance image contrast agent were used as they are, as commercial products themselves are dispersed in aqueous solution.
A manganese chloride solution was diluted to 1/100 by adding 990 μL of distilled water to 10 μL of a manganese chloride solution. After adding 10, 20, and 40 μL of the diluted manganese chloride solution to 1.25 mL of a 1.2 wt % of alginic acid solution, respectively, the first agent and the second agent solutions were prepared in the same manner as in Preparation Example 6, except for using solutions of three concentrations (final concentrations 0.05, 0.1, 0.2 mM) made to 1.5 mL with distilled water.
Feridex solution was diluted to 1/20 by adding 950 μL of distilled water to 50 μL of Feridex solution. After adding 5, 10, and 20 μL of the diluted Feridex solution to 1.25 mL of a 1.2 wt % of alginic acid solution, respectively, the first agent and the second agent solutions were prepared in the same manner as in Preparation Example 6, except for using solutions of three concentrations (final concentrations 25, 50, 100 μM) made to 1.5 mL with distilled water.
<Evaluation 9-Magnetic Resonance Image Contrast Agent Incorporation in Alginic Acid Hydrogel and Magnetic Resonance Image Confirmation>
In the solutions of Preparation Examples 13 and 14, 0.2 mM of manganese chloride and 100 μM of Feridex solutions were injected into ep-tubes, respectively so that the total volume was 0.5 mL using a dual syringe. Thereafter, it was confirmed that the alginic acid hydrogel formed by turning the ep-tube over as shown in
Three concentrations of the manganese chloride solutions of Preparation Example 13 were injected into the micro-tube so that the total volume was 0.25 mL using a dual syringe. Thereafter, as shown in
Three concentrations of the Feridex solutions of Preparation Example 14 were injected into the micro-tube so that the total volume was 0.25 mL using a dual syringe. Thereafter, as shown in
It was confirmed whether the CT contrast agent may be incorporated into the alginic acid hydrogel using Rose bengal (purchased from Sigma-Aldrich) as a CT contrast agent.
100 mg of Rose bengal was dissolved in 1 mL of distilled water to prepare a high concentration of Rose bengal solution. The first agent and the second agent solutions were prepared in the same manner as in Preparation Example 6, except for using a solution dissolved with stirring by adding 0.729 mL of a high concentration of Rose bengal and 0.771 mL of distilled water to 0.015 g of sodium alginate.
<Evaluation 10-Rose Bengal Incorporation in Alginic Acid Hydrogel and CT Image Conformation>
The solution of Preparation Example 15 was injected into the ep-tube so that the total volume was 0.5 mL using a dual syringe. Thereafter, it was confirmed that the alginic acid hydrogel formed by turning the ep-tube over as shown in
As shown in
It was confirmed whether a hydrogel was formed under the conditions wherein alginic acid and hyaluronic acid were mixed at 50:50 and 1:99.
0.06 g of sodium hyaluronate (purchased from Lifecore Biomedical, Inc.) was dissolved in 2 mL of distilled water to prepare a 3 wt % of hyaluronic acid solution.
0.036 g of sodium alginate was dissolved in 3 mL of distilled water to prepare a 1.2 wt % of alginic acid solution. In the manner as in Preparation Example 1, a high concentration of indocyanine green solution was prepared.
0.334 mL of a 3 wt % of hyaluronic acid solution and 0.093 mL of a high concentration of indocyanine green solution were added to 0.417 mL of a 1.2 wt % of alginic acid solution, and the total volume was made 1 mL with distilled water.
1 mL of the mixed alginic acid and hyaluronic acid solution was added to 3.43 mg of calcium carbonate and vortexed to prepare the first agent solution. After adding 0.334 mL of a 3 wt % of hyaluronic acid solution and 0.022 mL of D-gluconic acid to 0.417 mL of a 1.2 wt % of alginic acid solution, the total volume was made 1 mL with distilled water, and then the solution of the second agent was prepared by mixing with vortexing and stirring.
In the same manner as in Preparation Example 16, a 3 wt % of hyaluronic acid solution, a 1.2 wt % of alginic acid solution, and a high concentration of indocyanine green solution were prepared.
0.66 mL of a 3 wt % of hyaluronic acid solution and 0.093 mL of a high concentration of indocyanine green solution were added to 0.005 mL of a 1.2 wt % of alginic acid solution, and the total volume was made 1 mL with distilled water. 1 mL of the mixed alginic acid and hyaluronic acid solution was added to 3.43 mg of calcium carbonate and vortexed to prepare the first agent solution.
After adding 0.66 mL of a 3 wt % of hyaluronic acid solution and 0.022 mL of D-gluconic acid to 0.005 mL of a 1.2 wt % of alginic acid solution, the total volume was made 1 mL with distilled water, and then the solution of the second agent was prepared by mixing with vortexing and stirring.
<Evaluation 11-Evaluation of Mixed Hydrogel of Alginic Acid and Hyaluronic Acid>
The solution of Preparation Examples 16 and 17 was injected into the ep-tube, respectively so that the total volume was 0.5 mL using a dual syringe. Thereafter, as shown in
Pigs (mini-pigs) have similar metabolic processes, organ structures, and sizes to those of the human body, making it easy to perform experimental treatment and follow-up using an endoscope. In order to confirm the fluorescent labeling performance of alginic acid hydrogel containing indocyanine green-human serum albumin, which is a fluorescent surgical marker in pigs, alginic acid hydrogel containing indocyanine green-human serum albumin was injected in the inner submucosal layer of the stomach of two pigs, and on the injected day and 3 days after the injection of the hydrogel, the fluorescent signal generated from the fluorescent dye injected into the stomach was detected as an image through a fluorescent laparoscope to evaluate whether the labeled location could be identified.
A high concentration of indocyanine green-human serum albumin solution was prepared in the same manner as in Preparation Example 2. After adding 0.558 mL of the high concentration of indocyanine green-human serum albumin solution to 8.4 mL of a 1 wt % of alginic acid solution, distilled water was further added to make the total volume to 12 mL, thereby preparing a fluorescent surgical marker solution. In this test, an experiment was conducted without adding an ionic crosslinking agent.
<Evaluation 12-Confirmation of Fluorescent Labeling Performance Using Fluorescent Laparoscope on Pigs>
Two pigs (pig #1, pig #2) were fasted for 48 hours before injecting the prepared alginic acid hydrogel, and the experiment was conducted after systemic anesthesia.
On the first day of the test, an endoscope and a catheter (Clear-Jet injector, 23G, Finemedix Co., Ltd.) were inserted through the mouth of test group 1 (pig #1), and the indocyanine green-human serum albumin-containing alginic acid hydrogel of Preparation Example 18 was injected by 1 mL into each of the three lower mucous layers in the pig's stomach. In order to confirm the injected location, the hemostasis clip was clipped right next to the hydrogel injected location. Three days after injection, a fluorescent laparoscopy system was installed in the pig's abdominal cavity, and the fluorescent signal generated from a fluorescent surgical marker located inside the stomach was detected through a fluorescent laparoscope located outside the stomach, and thereby it was evaluated whether it can be confirmed with real-time detection of the location where the surgical marker was labeled.
On the day of the fluorescent laparoscopy evaluation of test group 1, fluorescent surgical markers were injected into the three submucosal layers in the stomach in the same manner as performed in test group 1 for test group 2 (pig #2). In order to confirm the injected location, the hemostasis clip was clipped right next to the injected location. After injection of the hydrogel, a fluorescent laparoscopy system was installed in the pig's abdominal cavity, and the fluorescent signal generated from a fluorescent surgical marker located inside the stomach was detected through a fluorescent laparoscope located outside the stomach, and thereby it was evaluated whether it can be confirmed with real-time detection of the location where the surgical marker was labeled.
As a result of observing the location where alginic acid hydrogel containing indocyanine green-human serum albumin was injected, from an image using a fluorescent laparoscopic system, the locations where the hydration gel was injected could be confirmed from the fluorescence image both in test group 1 where a fluorescent marker was injected prior to 3 days (
Subsequently, in order to confirm that the location identified from the fluorescence image was the location where the hydrogel was actually injected, the pig was euthanized and the pig's stomach was incised. It was confirmed that a fluorescent signal was generated next to the location where a haemostasis clip clipped right next to the location where the hydrogel was injected, and from this result, it was found that the fluorescence confirmed from the fluorescence laparoscopic image of the pig experiment was a fluorescence signal generated from the actual hydrogel (
Taking these results together, it can be seen that the alginic acid gel containing indocyanine green-human serum albumin had excellent performance as an image marker for the area to be removed by surgery.
When an alginic acid hydrogel is injected into the lower mucosa for image labeling, the patient's immune system recognizes that a foreign substance has invaded into the living body and may cause an inflammatory reaction in removing the foreign substance. At this time, proteins including various enzymes, complement systems, antibodies, and immune cells are introduced into the injection site, and actions to remove alginic acid and fluorescent dyes present at the injection site occur. Therefore, there is a possibility that the rate at which the fluorescent dye contained in the hydrogel is removed due to the inflammatory reaction occurring at the injection site would be fast.
Therefore, in the Example, biodegradable microspheres loaded with an anti-inflammatory agent was prepared and injected into a living body together with a hydrogel to suppress the inflammatory reaction at the injection site, and accordingly, a strength of the fluorescence image signal at the injected site was evaluated whether the strength could also be maintained higher. Celecoxib was used as an anti-inflammatory agent, and drug-loaded microspheres were prepared using a biodegradable polymer, poly(lactic-co-glycolic acid) (PLGA).
7-1. Preparation and Analysis of Biodegradable Microspheres Loaded with Anti-Inflammatory Agents.
135 mg of PLGA (50:50, Mw 38,000-54,000, purchased from Sigma-Aldrich) and 13.5 mg of celecoxib (purchased from Sigma-Aldrich) were dissolved in 4.5 mL of dichloromethane. 0.5 g of polyvinyl alcohol (PVA, Mw 13,000-23,000, purchased from Sigma-Aldrich) was added to mL of distilled water, heated to 65° C. to dissolve completely, and then the solution was cooled at 25° C. A beaker containing an aqueous solution wherein polyvinyl alcohol as a surfactant is dissolved, was immersed in ice, and a dichloromethane solution wherein celecoxib and PLGA are dissolved, was added dropwise, while a homogenizer (Homogenizer PT 3100 Polytron, Kinematica AG, Littau-Lucerne, Switzerland) was stirred vigorously at 5,000 rpm for 5 minutes to prepare an oil-in-water emulsion solution. To remove the organic solvent, dichloromethane, the prepared emulsion solution was mixed with 100 mL of distilled water, and stirred at 300 rpm for 8 hours under a temperature condition of 25° C. Centrifugation and washing were repeated 3 times at 3,000 rpm for 5 minutes in order to remove PVA, which is a surfactant present on the surface of the prepared microspheres. Thereafter, the aqueous solution was freeze-dried, and the obtained microspheres were stored at 4° C.
<Evaluation 13-Analysis of Microspheres Loaded with Prepared Anti-Inflammatory Agent>
After dispersing the PLGA microspheres loaded with celecoxib in distilled water at a concentration of 0.5 mg/mL, a few drops ware added on a slide glass and the shape and size was observed by using an optical microscope (Axio Imager M2 microscope, Zeiss, Germany) (
<Evaluation 14-Analysis of Amount of Drug Loaded in Microspheres>
After dissolving 1 mg of celecoxib in 10 mL of acetonitrile, a standard solution of concentrations of 0.625, 1.25, 2.5, 5, 10, 12.5, and 25 μg/mL was prepared while diluting with acetonitrile. Each solution was measured for absorbance at a wavelength of 251 nm to obtain a standard curve for celecoxib (
5 mg of PLGA microspheres loaded with celecoxib were completely dissolved in 2.5 mL of acetonitrile, diluted 50 times with acetonitrile solution, absorbance was measured at a wavelength of 251 nm, and the amount of drug contained in the microspheres was analyzed by comparison with the standard curve (
7-2. Animal Test Evaluation
When alginic acid hydrogel containing indocyanine green-human serum albumin was mixed with celecoxib-loaded microspheres to suppress the inflammatory reaction, it was evaluated through animal experiments whether the fluorescent dye was maintained at the injected site for a longer time and the fluorescence intensity.
Indocyanine green-human serum albumin solution was prepared in the same manner as in Preparation Example 2. After adding 0.093 mL of an indocyanine green-human serum albumin solution to 1.167 mL of a 1.2 wt % of alginic acid solution, distilled water was further added to make the total volume of 2 mL, followed by stirring.
In order to prepare an alginic acid hydrogel containing an anti-inflammatory agent, 0.136 mL of a distilled water solution wherein PLGA microspheres loaded with celecoxib were dispersed at a concentration of 9 mg/mL was prepared. 2 mL of an alginic acid aqueous solution containing indocyanine green-human serum albumin was prepared as described above, and 0.136 mL of an aqueous microsphere solution and 2 mL of an alginic acid solution were mixed and stirred. The final concentration of celecoxib drug contained in the alginic acid hydrogel was 600 μg/mL.
<Evaluation 15-Evaluation of Persistence of Fluorescence of Gel in Animal Model>
Using a syringe, each of 100 μL of the solutions of Preparation Examples 20 and 21 was subcutaneously injected to a white mouse.
As described above, a specific part of the content of the present disclosure has been described in detail, and for one skilled in this art, it will be obvious that this specific technology is only a preferred embodiment, and the scope of the present disclosure is not limited thereby. Therefore, it will be said that the substantial scope of the present disclosure is defined by the appended claims and their equivalents.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2018-0135161 | Nov 2018 | KR | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/KR2019/014682 | 11/1/2019 | WO | 00 |
