Patent Application
20240148871
The present invention relates to a spontaneous respiratory biomaterial for use in tissue engineering.
Oxygen-delivering blood accounts for about 7 to 8% of a body weight and losing at least one-fifth thereof may be life-threatening. The reason why loss of blood through various diseases such as arteriosclerosis, cerebral infarction, myocardial infarction, etc., or vascular injury makes life dangerous is due to lack of oxygen caused by loss of oxygen delivery power. In addition, an organ extracted for organ transplantation is damaged due to hypoxia, ischemia/reperfusion (I/R) damage, and the like, and viability is rapidly reduced due to the lack of oxygen at a site where cell therapies for in vivo injection are injected, and thus a large amount of cell therapies need to be injected into the body (a rapid increase in marginal cell mass). Furthermore, carbon dioxide, which is a by-product of cellular respiration, rapidly affects pH change inside and outside cells and affinity to oxygen, and thus the control of carbon dioxide in a microenvironment around the cells is also an important factor.
Until now, two materials have been developed for oxygen delivery. A first one is an inorganic chemical-based oxygen-releasing biomaterial (ORB), and a second one is an oxygen-generating biomaterial (OBB). However, the materials have a limitation in clinical application due to issues such as (1) cell damage caused by generation of active oxygen species due to initial massive oxygen release, (2) a limited duration of oxygen supply, (3) inability to control carbon dioxide, (4) biocompatibility, etc.
Transplantation of cell-based devices is generally hindered by inadequate oxygen delivery dye to an inevitable delay in angiogenesis after transplantation. Insufficient oxygen supply in the transplanted cells may lead to cell necrosis and cell death in a part of a graft, as well as a conversion into anaerobic metabolism and energy conservation.
It is particularly difficult to provide sufficient oxygen delivery to an implant including cells with high metabolic activity, such as cells including an islet, because an oxygen consumption rate of the islet is high compared to many other cell types, as well as sensitive to dysfunction even at moderate oxygen tension.
Thus, the development of a method for increasing oxygen availability in a tissue engineering implant during an initial period of engraftment may help to mitigate hypoxia-induced cell death. In situ oxygen generation is a highly desirable approach in that the generation does not require several operations and provides supplemental oxygen immediately upon implantation. These approaches highlight the potential of in situ oxygen generation to improve cell viability, but most of the approaches make transplantation difficult by increasing a size of a transplant device or by introducing a toxic by-product.
In conclusion, for the development of oxygen-generating biomaterials, it is necessary to reduce sensitivity in producing side reactions such as hydrogen peroxide intermediates or hydroxyl radicals by eliminating end products with cytotoxicity and controlled reactivity.
Thus, in order to overcome the above limitations, there is a need to develop a spontaneous respiratory material for tissue engineering, which is capable of supplying oxygen and controlling carbon dioxide and active oxygen.
One aspect is to provide a hydrogel composition including a chloroplast and a chloroplast transit peptide (CTP).
Another aspect is to provide a hydrogel composition including a chloroplast, a chloroplast transit peptide (CTP) and alginate.
A further aspect is to provide a hydrogel composition including a chloroplast, a chloroplast transit peptide (CTP), alginate and a pancreatic cell.
A yet further aspect is to provide a pharmaceutical composition for preventing or treating diabetes, including the hydrogel composition.
A still yet further aspect is to provide a microcapsule including the hydrogel composition.
A still yet further aspect is to provide a method for preparing a hydrogel composition, the method including: binding alginate and a chloroplast transit peptide (CTP); and
Another aspect is to provide a method for delivering oxygen to a cell or a tissue,
In order to overcome the limitations of the related art, the present inventors have developed a spontaneous respiratory material for tissue engineering, which is capable of supplying oxygen and controlling carbon dioxide and active oxygen.
Hereinafter, the present invention will be described in more detail.
Terms used in the present application are used only to describe a certain exemplary embodiment and are not intended to limit the present invention. All the terms used herein including technical or scientific terms have the same meaning as commonly understood by those ordinary skilled in the art, to which the present invention pertains, unless defined otherwise.
The present invention may provide a hydrogel composition including a chloroplast and a chloroplast transit peptide (CTP).
In the present specification, the term “respiratoid” may refer to a sustainable biomaterial of the present invention, which is a coined word made by combining “respirate” and a suffix of “˜oid” which means “artificiality.” The respiratoid may rapidly regenerate carbon dioxide, which is produced by cells/organs through cellular respiration, into oxygen, may increase a production rate thereof when being exposed to a specific wavelength, and may regenerate active oxygen (superoxide anion, hydrogen peroxide, etc.) generated from cells or tissues into oxygen again in the case of a temporary hypoxia state. In addition, the respiratoid according to the present invention may be highly biocompatible and thus may have little toxicity.
Respiratoid may have “excellence/differentiation” as summarized in a table below compared to existing oxygen-releasing materials (ORB) and oxygen-generating materials (OBG).
In one embodiment of the present invention, the hydrogel composition may be a respiratoid, which is a spontaneous respiratory material for tissue engineering, including a chloroplast and CTP. Thus, when the hydrogel composition is used, carbon dioxide generated by cells/organs through cellular respiration may be rapidly regenerated back into oxygen.
In one embodiment of the present invention, the hydrogel composition may use chloroplasts present in plant leaves, and alginate used for gelation may be obtained from plants such as seaweed leaves, etc. In addition, the chloroplast transit peptide having high selectivity for an outer membrane among the two layers of chloroplast composed of fatty acids is used rather than the chloroplast per se in a modification reaction, thereby having excellent biocompatibility and less influence on photoreaction of chloroplasts, Calvin-circuit reaction, etc.
Furthermore, the hydrogel composition may supply oxygen supply and control carbon dioxide, and thus may be highly likely to be widely used in the field of tissue engineering such as blood cell therapies, organ transplantation, and the like. Specifically, there may be provided very high originality and versatility, as well as utilization in the fields of (1) artificial blood development, (2) organ extraction and preserving agents, (3) large-capacity culture of microorganisms/cells with high efficiency, (4) (stem) cell therapies and tissue engineering, and the like. In addition, research on an in vivo cell-cell interaction may be possible in a biomimetic environment, so that a new function of cells may be examined in terms of physiology, and may be applied as a new technique in a field of research on stem cell therapy.
In one embodiment of the present invention, the hydrogel composition may use a stromal calvin cycle and thylakoid present in a chloroplast of plant leaves, which simultaneously controls oxygen and carbon dioxide. For this purpose, the hydrogel composition may include a chloroplast transit peptide (CTP) capable of binding or conjugating to the chloroplast. In one specific embodiment, the CTP may bind to a chloroplast outer membrane.
In the present invention, “binding” may include chemical binding and physical binding. For example, the CTP may be physically conjugated to or inserted into the chloroplast outer membrane, and the CTP may bind to the chloroplast outer membrane through covalent bonding.
In one embodiment of the present invention, the CTP may be derived from a chloroplast membrane protein. For example, the CTP may include a partial sequence of the chloroplast membrane protein, and specifically may include a partial sequence of the chloroplast outer envelope protein.
In one embodiment of the present invention, the CTP may be derived from outer envelope protein 34 (OEP34) of chloroplasts (or translocon at the outer envelope membrane of chloroplasts 34 (TOC34, Arabidopsis thaliana sp.)) or outer envelope protein 64 (OEP64) (or translocon at the outer envelope membrane of chloroplasts 64 (TOC64, Arabidopsis thaliana sp.)). In one specific embodiment, the CTP may be derived from OEP34/TOC34 or OEP64/TOC64.
In one embodiment of the present invention, the CTP may include a part of a sequence of a transmembrane domain of chloroplast outer envelope protein 34 or outer envelope protein 64. For example, the CTP may be a peptide including a part of one or more sequences selected from amino acid sequences of transmembrane domains represented by SEQ ID NO: 3 and SEQ ID NO: 4.
In one embodiment of the present invention, the CTP may be a peptide including one or more selected from amino acid sequences represented by SEQ ID NO: 1 and SEQ ID NO: 2.
In one embodiment of the present invention, the CTP may be a peptide including MFAFQYLLVM (SEQ ID NO: 1), which may be a peptide sequence derived from LI PLMFAFQYLL VMKPLV (SEQ ID NO: 3), which is a predicted sequence of a transmembrane domain of chloroplast outer envelope protein OEP34 (or TOC34).
In one embodiment of the present invention, the CTP may be a peptide including VILGLGLAGI (SEQ ID NO: 2), which may be a peptide sequence derived from SPSSQ IWVILGLGLA GIYVL (SEQ ID NO: 4), which is a predicted sequence of a transmembrane domain of chloroplast outer envelope protein OEP64 (or TOC64).
In one embodiment of the present invention, the CTP may increase oxygen generation of chloroplasts.
In one embodiment of the present invention, the hydrogel composition may further include alginate.
In one embodiment of the present invention, the hydrogel may be gelated by binding a CTP terminus and an alginate terminus. In one specific embodiment, the hydrogel may be gelated by conjugating or binding primary amine of the CTP and a carboxyl group of alginate. In addition, in one specific embodiment, the alginate may be mixed with a calcium solution and gelated through cross-linking.
In one embodiment of the present invention, the hydrogel may further include pancreatic cells. In one specific embodiment, the pancreatic cells may be encapsulated in a chloroplast-CTP-alginate hydrogel. A chloroplast-CTP-alginate hydrogel composition of the present invention, in which pancreatic cells are encapsulated, may exhibit excellent cell viability and a preventive or therapeutic effect on diabetes according to spontaneous respiration, and thus may be applied to a hydrogel-based implant for tissue engineering using the same.
In one embodiment of the present invention, the hydrogel composition may generate oxygen during light irradiation.
In one embodiment of the present invention, the light irradiation may be performed at an oxygen concentration of 101/ml to 1010/ml. In one specific embodiment, the light irradiation may be performed at an oxygen concentration of 102/ml to 108/ml.
In one embodiment of the present invention, the light irradiation may be performed at a temperature of 20° C. to 40° C. In one specific embodiment, the light irradiation may be performed at a temperature of 23° C. to 38° C.
In one embodiment of the present invention, the light irradiation may be continuously performed for a predetermined time, or may be non-continuously and alternately performed by exposing light for a predetermined time and not exposing light at predetermined time intervals.
In one embodiment of the present invention, the light irradiation may be performed once to five times by continuously exposing light for 10 to 50 minutes, and then not exposing light for 10 to 50 minutes in a cycle. In one specific embodiment, the light irradiation may be performed twice to four times by continuously exposing light for 20 to 40 minutes, and then not exposing light for 20 to 40 minutes in a cycle.
In one specific embodiment, an oxygen generation rate of the hydrogel composition may be increased upon exposure to a specific wavelength, and the wavelength may be 620 nm to 700 nm, 640 nm to 680 nm, or 650 nm to 670 nm.
In addition, in one specific embodiment, the hydrogel composition may regenerate active oxygen, which is generated when surrounding cells or tissues are in a temporary hypoxia state, into oxygen.
In one embodiment of the present invention, the hydrogel may include chloroplasts at a concentration of 101/ml to 1010/ml. In one specific embodiment, the hydrogel may include chloroplasts at a concentration of 103/ml to 1010/ml, 106/ml to 1010/ml, 107/ml to 109/ml, or 103/ml to 109/ml.
In one embodiment of the present invention, the hydrogel may include insulin secreted by pancreatic cells. The pancreatic cells included in the hydrogel composition of the present invention may be present in a state of being encapsulated in the hydrogel, thereby stably secreting insulin.
In one embodiment of the present invention, the hydrogel composition may be for delivering oxygen to a cell or a tissue. In one specific embodiment, the oxygen delivery may be performed by rapidly regenerating carbon dioxide produced through cellular respiration back into oxygen, or by reproducing active oxygen, which is generated from cells or tissues in a temporary hypoxia state, back into oxygen. The hydrogel composition of the present invention may alleviate cell death by oxygen delivery, and may control an oxygen generation rate.
In addition, the present invention may provide a microcapsule including the hydrogel composition. The microcapsule may be used in a cell therapeutic agent.
In one embodiment of the present invention, the microcapsule may be a microcapsule for generating oxygen during light irradiation. In addition, in one embodiment, the microcapsule may be a microcapsule for preventing or treating metabolic diseases.
Furthermore, the present invention may provide a pharmaceutical composition for preventing or treating metabolic diseases, including the hydrogel composition.
In the present specification, the term “prevention” may refer to all the actions of inhibiting a disease or delaying the onset of the disease in an individual by administration of the pharmaceutical composition according to one aspect.
In the present specification, the term “treatment” may refer to all the actions of ameliorating or beneficially changing symptoms of a disease by administration of the pharmaceutical composition according to one aspect.
In addition, “active ingredient” or “pharmaceutically effective amount” may refer to any amount of a composition used in a course of practicing the invention provided herein, to be sufficient for alleviating, inhibiting a progression of, or preventing a disease, disorder, or condition, or one or more symptoms thereof.
According to one embodiment of the present invention, the metabolic disease may be one or more selected from the group consisting of type 1 diabetes, type 2 diabetes, impaired glucose tolerance, impaired fasting glucose, dyslipidemia, impaired lipid metabolism, obesity, fatty liver, insulin resistance, and glucose tolerance syndrome. In one specific embodiment, the metabolic disease may be diabetes, and may be type 1 diabetes or type 2 diabetes.
The pharmaceutical composition may further include a pharmaceutically acceptable carrier or diluent. Pharmaceutically acceptable carriers or diluents may be those known in the art. The carrier or diluent may be lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water (e.g., saline and sterile water), syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, Ringer's solution, buffering agent, maltodextrin solution, glycerol, ethanol, dextran, albumin, or any combination thereof. The pharmaceutical composition may further include a lubricant, humectant, a sweetener, a flavoring agent, an emulsifying agent, a suspending agent, or a preservative.
The pharmaceutical composition may be formulated using a pharmaceutically acceptable carrier and/or excipient according to methods known to those skilled in the art to prepare a unit dose form or may be incorporated into a multi-dose container. In this case, a formation may be in a form of solution, suspension, syrup, or emulsion in oil or aqueous solvent, or may be in a form of extract, powder, granule, tablet, or capsule, and may further include a dispersant or a stabilizer. The aqueous solvent may include physiological saline or PBS. The pharmaceutical composition according to one specific embodiment may be formulated into an oral or parenteral dosage form, preferably a parenteral dosage form. In the case of intramuscular, intraperitoneal, subcutaneous, and intravenous dosage forms, a sterile solution of the active ingredient may be conventionally prepared to include a buffer capable of suitably adjusting a pH of the solution, and in the case of intravenous administration, an isotonic agent may be included to impart isotonicity to the preparation.
A dosage (effective amount) of the pharmaceutical composition according to one specific embodiment may be variously prescribed depending on factors such as a formulation method, an administration method, a patient's age, weight, gender, pathologic condition, food, administration time, administration route, excretion rate and reaction sensitivity, and those skilled in the art may appropriately adjust the dosage in consideration of these factors. The administration may be performed once a day or twice or more within a range of clinically acceptable side effects, and the administration may be performed at one site or at two sites or more, and the administration may be performed daily or at intervals of two to five days with the total number of administration lasting from one day to 30 days during one treatment. If necessary, the same treatment may be repeated after an appropriate period. Non-human animals may be administered in the same dosage as that of human per kg, or may be administered in an amount obtained by converting the above dosage into, for example, a volume ratio (e.g., an average value) of a target animal and an organ (heart, etc.) of human. Possible routes of administration may include parenteral (e.g., subcutaneous, intramuscular, intraarterial, intraperitoneal, intrathecal, or intravenous), topical (including transdermal), and injection, or insertion of an implantable device or substance. Examples of the animal to be treated according to one specific embodiment may include humans and other target mammals, and specifically include humans, monkeys, mice, rats, rabbits, sheep, cattle, dogs, horses, pigs, etc.
In addition, the present invention may provide a health functional food for preventing or ameliorating metabolic diseases, including the hydrogel composition as an active ingredient.
The hydrogel composition, metabolic diseases, and prevention may be the same as described above.
In the present specification, the term “amelioration” may refer to all the actions of inhibiting a metabolic disease or delaying the onset of the metabolic disease in an individual by administration of the composition according to one aspect.
The health functional food defined in the present invention may be a health functional food which has sufficiently established the functionality and safety for the human body newly defined through the Health Functional Foods Act amended in 2008 and has been registered in the regulations on the recognition of functional raw materials for health functional foods prescribed in the Food and Drug Administration Notice No. 2008-72 of the Food and Drug Administration.
When the composition of the present invention is used by being included in a health functional food, the composition may be added per se or used together with other health functional foods or health functional food ingredients, and may be appropriately used according to a conventional method. A mixing amount of the active ingredient may be suitably determined according to a purpose of use. In general, the active ingredient constituting the composition according to the present invention may be included in an amount of 0.01 to 15% by weight, preferably 0.2 to 10% by weight, based on the total weight of the food, and when being prepared as a beverage, the active ingredient may be included at a ratio of 0.1 to 30 g, preferably 0.2 to 5 g based on 100 mL, and the entire beverage may be composed of a natural ingredient. However, in the case of long-term intake for the purpose of health control and hygiene, the amount may be equal to or less than the above range, and the active ingredient may be used in an amount equal to or greater than the above range since there is no problem in terms of safety.
The composition for health functional food according to the present invention may be formulated into a dosage form of conventional health functional food known in the art. The health functional food may be prepared, for example, in a form of powder, granule, tablet, pill, capsule, suspension, emulsion, syrup, precipitate, liquid, extract, gum, tea, jelly, drink, or the like, and preferably in a form of drink. As the sitologically acceptable carrier or additive, any carrier or additive known to be usable in the art may be used for preparing a formulation to be prepared. It may also include foods used as feed for animals.
The health functional food may further contain nutritional supplements, vitamin, electrolyte, flavoring agent, coloring agent, pectic acid and salts thereof, alginic acid and salts thereof, organic acid, protective colloidal thickener, pH adjusting agent, stabilizer, preservative, glycerin, alcohol, carbonator used in carbonated beverage, etc., according to purpose or preference. Besides, the health functional food may contain natural fruit juice and pulp for preparing fruit juice beverage and vegetable based beverage. In addition, the health functional food composition may further include a food additive, and the suitability as a “food additive” may be determined according to the specifications and standards for the relevant item in accordance with the general rules, general test methods and the like of the Food Additive Code approved by the Ministry of Food and Drug Safety, unless otherwise specified.
Furthermore, the present invention may provide a method for preparing a hydrogel composition, the method including: binding alginate and a chloroplast transit peptide (CTP); and mixing the bound alginate-CTP composite with a chloroplast isolated from an individual to gel a resulting mixture.
In one embodiment of the present invention, the preparation method may further include: encapsulating pancreatic cells inside the gelled hydrogel.
In addition, the present invention may provide a method for delivering oxygen to a cell or a tissue, the method including: treating the hydrogel composition; and irradiating the treated hydrogel composition with light.
In one embodiment of the present invention, the light irradiation may be performed at an oxygen concentration of 101/ml to 1010/ml. In one specific embodiment, the light irradiation may be performed at an oxygen concentration of 102/ml to 108/ml.
In one embodiment of the present invention, the irradiating with light may be performed at a temperature of 20° C. to 40° C. In one embodiment, the irradiating with light may be performed at a temperature of 23° C. to 38° C.
In one embodiment of the present invention, the irradiating with light may be continuously performed for a predetermined time, or may be non-continuously and alternately performed by exposing light for a predetermined time and not exposing light at predetermined time intervals.
In one embodiment of the present invention, the irradiating with light may be performed once to five times by continuously exposing light for 10 to 50 minutes, and then not exposing light for 10 to 50 minutes in a cycle. In one specific embodiment, the irradiating with light may be performed twice to four times by continuously exposing light for 20 to 40 minutes, and then not exposing light for 20 to 40 minutes in a cycle.
In one specific embodiment, an oxygen production rate according to the method may be increased upon exposure to a specific wavelength, and the wavelength may be 620 nm to 700 nm, 640 nm to 680 nm, or 650 nm to 670 nm.
In addition, the present invention may provide a method for preventing or treating a metabolic disease, the method including: administering the hydrogel composition to an individual. In one embodiment, the method may further include: irradiating the individual with light.
In addition, the present invention may provide a use of the hydrogel composition.
Furthermore, the present invention may provide a use of the hydrogel composition for use in preparing a medicament for the prevention or treatment of metabolic diseases.
Moreover, the present invention may provide a use of a microcapsule including the hydrogel composition.
Besides, the present invention may provide a use of a microcapsule including the hydrogel composition for use in preparing a medicament for the prevention or treatment of metabolic diseases.
The hydrogel composition including a chloroplast, a chloroplast transit peptide (CTP) and alginate of the present invention can supply oxygen to a cell or a tissue and control carbon dioxide.
The hydrogel composition including a chloroplast, a chloroplast transit peptide (CTP), alginate, and pancreatic cells of the present invention can be harmless to cells and individuals, and can exhibit an excellent effect of preventing or treating diseases such as diabetes, etc.
Hereinafter, the present invention will be described in more detail through exemplary embodiments. However, these exemplary embodiments are provided only for the purpose of illustrating the present invention, and thus the scope of the present invention is not limited thereto.
Alginate (—COOH) and CTP (—NH2) were conjugated by the following method to obtain a composite.
After preparing 10 ml of 0.1 M MES buffer (+0.5 M NaCl), pH was adjusted to 6. 0.2 mM sodium alginate (228 mg/10 ml) was dissolved in 10 ml of MES buffer prepared above, so that sodium alginate might be well loosen and might not be agglomerated. When solution was sufficiently dissolved, 4 mg/10 ml of EDC (191.7 MW) corresponding to 2 mM was dissolved. After that, 6 mg/10 ml of NHS (5 mM) was added thereto and reacted at RT for 15 minutes. The pH was raised to 7 using PBS or sodium bicarbonate (NaHCO3, sodium hydrogen carbonate). Peptide (10 mg/ml, peptide was synthesized upon request by Peptron, a peptide synthesis company) dissolved in 1% DMSO was added to solution in an amount corresponding to a molar ratio (1:1, 1:5, 1:10, 1:25, 1:50). After that, the resulting mixture was reacted at RT for two hours. The resulting mixture was subjected to dialysis for three days using a molecular weight cut-off (MWCO) dialysis membrane of 6,000 to 8,000 g/mol. The resulting product was frozen in a deep freezer, and then freeze-dried to obtain an alginate-CTP composite in the form of powder (see
An amount of CTP (protein) in an alginate-CTP composite was quantified by the following method.
Controls A to I were prepared by using a BCA assay kit. Foil was spread on ice and placed at 96-well plate, after which 24 μl of control was loaded thereon. A sample was loaded under a line with the control loaded thereon. (n=5, concentration of 2 mg/ml (RIPA solution)) After that, 200 μl of A+B solution per well was loaded on the loaded sample. At this time, solutions A and B need to be kept at room temperature (A=196 μl, B=4 μl, A:B=50:1). The control was added starting from a low concentration. After incubation for 30 minutes, an absorbance was measured at 562 nm using an instrument (see
As a result, a moral ratio of 1:19, 1:31, 1:36, 1:79 and 1:62 could be confirmed in order. Thus, it was confirmed that the synthesis is well performed, as the molar ratio value becomes less (1:1, 1:5).
A strength of hydrogel formed for each molar ratio of the alginate-CTP composite was confirmed by the following method.
To prepare an alginate or alginate-CTP solution (0.07 mM), 0.9% NaCl,
1.5% alginate or alginate-CTP were added and diluted with tertiary distilled water (500 μl of tertiary distilled water+4.5 mg of NaCl+4 mg of alginate or alginate-CTP). At this time, alginate powder was slowly added and sufficiently diluted. In order to prepare a CaCl2) solution (100 mM), 11 g of CaCl2) (110.98 g/mol) and 2.6 g of HEPES were added to 1 L of tertiary distilled water. The prepared CaCl2) solution was filtered and refrigerated. After that, 200 μl of the solution of process 1 and 500 μl of 100 mM CaCl2) solution were reacted in 48-well plate for 30 minutes to carry out gelation. The strength of hydrogel was confirmed by measuring rheology through a rheometer.
As a result, it was confirmed that capsule gel is not formed at an alginate-CTP feed molar ratio of 1:280 or more (see
In addition, as a result of a rheology test on the prepared capsule gel, it was found that alginate shows 6 kPa (G′) based on 100 mM CaCl2), which is decreased as the feed molar ratio increases, thus obtaining a value of 5.3 kPa(G′) at a molar ratio of 1:18 and a value of 1.7 kPa(G′) at a molar ratio of 1:62, and it could be confirmed that G′(storage modulus) values are greater than G′(loss modulus) in all groups (see
Chloroplast was isolated from spinach by the following method (see
The materials included 35 g of spinach leaves, scissors, a blender, gauze, a conical tube, cooling centrifuge, 1× chloroplast isolation buffer (CIB) without BSA (0.33 M sorbitol, 0.1 M tris-cl pH 7.8, 5 mM MgCl2, 10 mM NaCl, 2 mM EDTA), 1× chloroplast isolation buffer with BSA (0.1% W/V), 40% percoll (4 ml percoll and 6 ml 1×CIB buffer with BSA) and 80% percoll (8 ml percoll and 2 ml 1×CIB buffer with BSA).
All experiments were rapidly performed at a cold room or under a cold condition. 35 g of spinach leaves, excluding the leaf vein, were obtained, and the spinach leaves were cut into small pieces having a width of 1 cm using scissors which were sterilized with ethanol. 120 ml of CIB buffer containing BSA was placed in a cold blender, and blending was performed 10 times for two seconds along with spinach leaves. The above solution was poured into a beaker covered with several layers of gauze having ice therein and was subjected to filtration. The filtered solution was placed in a conical tube and subjected to centrifugation at 4° C., 2500 RPM for 70 seconds. The supernatant was transferred to a new conical tube, and then subjected to centrifugation at 4° C., 2500 RPM for seven minutes. The supernatant was discarded and a green pellet was gently loosen with finger tapping. The pellet was resuspended with 2 ml of 1×CIB buffer with BSA and was subjected to pipetting. 40%, 80% percoll solution was prepared, and 3 ml of 80% percoll, 3 ml of 40% percoll, and 6 ml of chloroplast suspension were carefully placed in 15 ml conical tube in order. The reaction mixture was subjected to centrifugation at 4000 RPM for 10 minutes. Only the solution between the 80%/40% percoll layers containing intact chloroplast was carefully taken and transferred to another conical tube. After that, the obtained pellet was mixed with 10 ml of 1×CIB buffer without BSA, and then wrapped in foil and stored in a refrigerated state.
It was evaluated whether or not DMSO (10 mg/ml) used to dissolve peptide was toxic to chloroplast (1*108/ml) for each time by the following method.
0% and 1% DMSO was prepared, diluted in 1 ml of 1*108/ml chloroplast in CIB solution, and treated for 0, 5, 10, 30 and 60 minutes. 10 μl of the solution was taken for each time, and the number of chloroplasts was counted using a hemocytometer.
As a result, there was no significant difference between a control group not treated with DMSO and a group treated with 1% DMSO, and it was confirmed that 1% DMSO does not affect chloroplast (see
Chlorophyll was measured to quantify chloroplast by the following method.
Using CIB, 2*108/ml chloroplasts were diluted from 0% to 100% with a total of 14 sections. 250 μl of 80% acetone (+5 mg/ml pH 7 EDTA) was added to 250 μl of each diluted solution. 80% acetone (+5 mg/ml pH 7 EDTA) was performed to obtain chlorophyll by crushing the chloroplast, so as to obtain a solution having a final concentration of 1*108/ml (based on 100%). UV-VIS absorbance at 645 nm, 663 nm was measured using a nanodropdrop. After that, a μg/ml value of total chlorophyll was obtained by substituting the measured value into the following Arnons equation.
<Arnons Equation>
*Chlorophyll a(μg/ml)=12.7(A663)−2.69(A645)
*Chlorophyll b(μg/ml)=22.9(A645)−4.68(A663)
*Total chlorophyll (μg/ml)=20.2(A645)+8.02(A663)
7.1. Confirmation of Binding of Chloroplast Outer Membrane to CTP-FITC Using Fluorescent Image
It was confirmed by fluorescence that peptide-FITC may be anchored on an outer membrane of chloroplast by the following method (see
Care was taken not to be exposed to light during the experiment. 500 μl of 1*108/ml chloroplast (control) and 500 μl of 1*108/ml chloroplast+CTP-FITC were prepared. At this time, CTP-FITC was obtained upon request from Anygen, a synthesis company. In 1*108/ml chloroplast+CTP-FITC, CTP-FITC was treated at a concentration of 0.6 mg/ml. At this time, if 500 μl of 1*108/ml chloroplast (in CIB) is taken and 30 μl of 10 mg/ml CTP (in DMSO) is taken and added, the final concentration may become 0.6 mg/ml. After that, 100 μl was taken and immobilized every 1, 5, 10, 30 and 60 minutes, and the solution reacted for each time was subjected to centrifugation at 13500 RPM for one minute. A pellet was obtained, immobilized in a 2.5% glutaraldehyde (0.05 M SCB) fixative (500 μl, 15 min), and washed twice with PBS to remove unreacted CTP-FITC. After that, 500 μl was filled with CIB buffer, and then mounted using slide glass/cover glass. A fluorescent photograph was taken using a confocal microscope, and sectioning was performed with the thickness of about 1 μm (green fluorescence (FITC)=488 nm, red fluorescence (chloroplast auto fluorescence)=580 nm).
7.2. Confirmation of Binding of Chloroplast Outer Membrane to CTP-FITC Using TEM
It was confirmed by TEM whether peptide-FITC may be anchored on an outer membrane of chloroplast by the following method (see
500 μl of 1*108/ml chloroplast (control) and 500 μl of 1*108/ml chloroplast+CTP-FITC, which were reacted by the same method and at the same time (1, 5, 10, 30 and 60 minutes) as in above 7.1, were immobilized in 1 ml of 2% glutaraldehyde+2% paraformaldehyde in 0.05 M sodium cacodylate buffer fixative for 15 minutes. After immobilization, in order to remove the unreacted CTP-FITC, PBS washing was repeated twice to obtain a pellet. 1 ml of 2% glutaraldehyde+2% paraformaldehyde in 0.05 M sodium cacodylate buffer was added to the obtained pellet, and reacted at 4° C. overnight. After a TEM pre-treatment process (prepared in a block with resin after immobilizing chloroplast) was performed, fine flakes were produced using an ultramicrotome, TEM was photographed and confirmed, and the TEM pre-treatment and subsequent processes were conducted at the Joint Research Institute of Seoul National University's College of Agriculture and Life.
7.3. Sub-Conclusion
As a result of the above experiment, it could be confirmed that the CTP is sufficiently coated by anchoring on an outer membrane of chloroplast starting from a reaction time of five minutes or more, and then the reaction time of the CTP and the chloroplast was set to five minutes.
8.1. Alginate-CTP Hydrogel Gelation and Rheology Test (CIB)
A degree of gelation of alginate-CTP and CaCl2) solution in the CIB was confirmed by the following method to determine whether the use of the CIB has no effect when the alginate-CTP and chloroplast is anchored. At this time, it was assumed that gelation may not occur.
Alginate (control) and alginate-CTP (1:50) were prepared, and a 100 mM CaCl2) solution, which was a positive control for confirming the existing gelation function, and Ca2+, Mg2+-free PBS, which was a negative control, and the CIB, which was an experimental group where gelation was not expected were prepared. 4 mg of alginate or alginate-CTP, 4.5 mg of NaCl, and 500 μl of tertiary distilled water were well mixed. 250 μl of the solution of process 3 was reacted with the CIB in 48-well plate for 30 minutes. The strength of gel was confirmed (about having the properties of gel) by measuring rheology through a rheometer.
8.2. Alginate-CTP-Chloroplast Hydrogel Gelation and Rheology Test
The following method was performed to determine whether gelation occurs by reacting the alginate-CTP composite with chloroplast, and to measure the strength of the hydrogel thus produced.
Alginate (control) and alginate-CTP (a molar ratio of 1:1, 1:5, 1:10, 1:25 and 1:50) were prepared, and 4 mg of alginate or alginate-CTP, 4.5 mg of NaCl, and 500 μl of tertiary distilled water were well mixed. Chloroplast isolated from spinach on the same day was prepared at a concentration of 1*108/ml. 250 μl of the solution of process 2 and 250 μl of the solution of process 3 were well mixed in 48-well plate, and then reacted with 100 mM CaCl2) for 30 minutes. The strength of hydrogel was confirmed (about having the properties of hydrogel) by measuring rheology through a rheometer.
9.1. Quantification of Chloroplast Using Residual Solution of Alginate-CTP-Chloroplast Hydrogel
After the removal of alginate-CTP-chloroplast hydrogel, a residual solution was used to quantify chloroplast in hydrogel by the following method.
Alginate-CTP-chloroplast hydrogel was prepared as in the method of above 9.2, and all solutions excluding hydrogel were recovered and subjected to centrifugation at 13,500 RPM for one minute. A pellet was dissolved in 250 μl of 80% acetone after being suspended (repeated three times of vortexing & stay). After measuring absorbance at 663 nm and 645 nm using a nanodropdrop, the measured value was used to calculate an amount of total chlorophyll (μg/ml) according to the following Arnons equation.
<Arnons Equation>
*Chlorophyll a(μg/ml)=12.7(A663)−2.69(A645)
*Chlorophyll b(μg/ml)=22.9(A645)−4.68(A663)
*Total chlorophyll (μg/ml)=20.2(A645)+8.02(A663))
9.2. Quantification of Chloroplast Present in Alginate-CTP-Chloroplast Hydrogel
The chloroplast contained in the alginate-CTP-chloroplast hydrogel was quantified by the following method.
EDTA was added at 5 mg/ml to PBS, and the pH was increased to 7. An alginate lyase was placed in an EDTA solution, and the vortexing & stay was repeated three times.
When hydrogel was loosen, centrifugation was performed at 13500 RPM for one minute, and a pellet was suspended in 250 μl of 80% acetone. After that, vortexing & stay was repeated three times to perform dissolution, and absorbance was measured at 663 nm and 645 nm using a nanodrop drop. The measured absorbance was substituted into Arnons equation below to calculate an amount of total chlorophyll (μg/ml).
<Arnons Equation>
Chlorophyll a(μg/ml)=12.7(A663)−2.69(A645)
Chlorophyll b(μg/ml)=22.9(A645)−4.68(A663)
Total chlorophyll (μg/ml)=20.2(A645)+8.02(A663)
9.3. Sub-Conclusion
As a result of the above experiment, it was confirmed that the hydrogel efficiency of alginate-CTP having a feed molar ratio of 1:1 and 1:5 is very good with an encapsulation rate of about 99%. Thus, it was confirmed that alginate-CTP having a feed molar ratio of 1:1 and 1:5 may effectively bind to almost all chloroplasts (see
It was confirmed whether the alginate-CTP-chloroplast hydrogel is loosen over time by the following method.
An alginate-CTP-chloroplast hydrogel was prepared according to the method of above Example 9. After that, a rheology test was performed through a rheometer to confirm whether hydrogel is loosen for four weeks to confirm the strength of hydrogel.
In order to determine whether chloroplast affects the cells, CCK-8 assay was performed using HEK 293 cells, which are a general cell line, so as to confirm the results.
HEK 293 T cells (3.5*104/well) stabilized at 96-well plate for 24 hours were prepared (n=5). Each of the prepared groups was treated with 100 μl of the prepared chloroplast solution at each concentration. After incubation for 24 hours, the chloroplast solution was suctioned. The resulting product was washed twice with PBS and treated with 100 μl of media (DMEM)+10 μl of CCK solution. After incubation by wrapping the foil for two hours, absorbance was measured at 450 nm using a reader. At this time, 50 μl of only the supernatant was taken to reduce the effect of chloroplast absorbance.
As a result, even if the concentration of chloroplast is increased, it was confirmed that there is no toxicity to the cells (see
12.1. Measurement of Amount of Oxygen Generated from Chloroplast Depending on Presence or Absence of Light
An amount of oxygen generated from chloroplast depending on the presence or absence of light was confirmed by the following method.
The chloroplast was diluted in 96-well plate in accordance with the group by using the CIB, and 300 μl was added thereto. An amount of oxygen generated from chloroplast depending on the presence or absence of light (660 nm) was measured using an oxygen sensor. At this time, OxyLite (Oxford Optronix, U.K.) was used as the oxygen sensor. A distance between material and light was set to 5 cm. An amount of oxygen generated was measured in 0, 30, 60 and 120 min units using the oxygen sensor (reading time: 3 min).
As a result, the group (no light) not exposed to light (660 nm) showed a level of oxygen generation up to 6.5% higher, but the control group showed the same level of oxygen generation as that of visible light under the light (660 nm) condition, whereas the group having chloroplast inserted showed a level of oxygen generated up to 9.3% higher. It was confirmed that oxygen generation is rather reduced at a high concentration of chloroplast than that of the control group (see
12.2. Measurement of Amount of Oxygen Generated from Alginate-CTP-Chloroplast Hydrogel Depending on Presence or Absence of Light
An amount of oxygen generated from alginate-CTP-chloroplast hydrogel depending on the presence or absence of light was confirmed by the following method.
An alginate-CTP-chloroplast hydrogel was prepared by adding chloroplast to 96-well plate in accordance with the group. An amount of oxygen generated from chloroplast depending on the presence or absence of light (660 nm) was measured using an oxygen sensor. At this time, OxyLite (Oxford Optronix, U.K.) was used as the oxygen sensor. A distance between material and light was set to 5 cm. An amount of oxygen generated was measured in 0, 30, 60 and 120 min units using the oxygen sensor (reading time: 3 min).
As a result, it was confirmed that an amount of oxygen generated under a partial pressure increases to about 105% as time passed, and an amount of oxygen generated increases under the light (660 nm) condition. On contrary, when not exposed to light (660 nm), an amount of oxygen generated appeared to increase in the beginning, but appeared to decrease in 120 minutes later. It was confirmed that hydrogel containing 108/ml of chloroplast shows a decrease up to about 95% under the light (660 nm) condition (see
It represents an increase compared to under the condition for measuring an oxygen concentration of chloroplast per se, not hydrogel (about 55%).
13.1. Measurement of Amount of Oxygen Generated from Alginate-CTP-Chloroplast Hydrogel in Normoxic and Hypoxic States
An amount of oxygen generated from alginate-CTP-chloroplast hydrogel according to exposure to light in normoxic and hypoxic states was confirmed by the following method.
An alginate-CTP-chloroplast hydrogel was prepared by adding chloroplast to 96-well plate in accordance with the group. The 96-well plate was implemented in normoxic and hypoxic states under the following conditions.
After that, an amount of oxygen generated from chloroplast according to light exposure in the normoxic and hypoxic states was measured using an oxygen sensor. At this time, OxyLite (Oxford Optronix, U.K.) was used as the oxygen sensor. A distance between material and light was set to 15 cm, and an amount of oxygen generated was measured in 0, 30, 60 and 120 min units using the oxygen sensor (reading time: 3 min).
As a result, it was confirmed that an amount of oxygen generated in the group having chloroplast inserted is higher than that of the control group (about 8% increase), and that a level of oxygen generated in the hypoxic state is more effective than that of the normoxic state. It was confirmed that an amount of oxygen generated in the group of chloroplast at a high concentration is decreased both in normoxic and hypoxic conditions (see
13.2. Measurement of Amount of Oxygen Generated from Each Group According to Normoxic and Hypoxic States
An amount of oxygen generated from each group (free chloroplast, alginate-chloroplast hydrogel, and alginate-CTP-chloroplast hydrogel) according to exposure to light in normoxic and hypoxic states was confirmed by the following method.
Free chloroplast, alginate-chloroplast hydrogel, and alginate-CTP-chloroplast hydrogel were prepared by adding chloroplast to 96-well plate in accordance with the concentration. The 96-well plate prepared for each experimental group was implemented in normoxic and hypoxic states as follows.
An amount of oxygen generated from chloroplast according to light exposure in the normoxic and hypoxic states was measured using an oxygen sensor. At this time, OxyLite (Oxford Optronix, U.K.) was used as the oxygen sensor.
A distance between material and light was set to 5 cm, and an amount of oxygen generated was measured for six days using the oxygen sensor (at an interval of 24 hours). On the 5th day of the measurement, an amount of oxygen generated was measured by changing into a normoxic environment.
As a result, it was found that the group of free chloroplast maintains an oxygen concentration higher than a severe hypoxia level of 40 mmHg (69.5 μM/L) for five days. In addition, as a result of exposure to a normal oxygen concentration for 24 hours on the 5th day of the measurement, it was confirmed that most of the free chloroplast group is recovered to an initial oxygen concentration. However, as in the previous experimental results, it was found that a low level of oxygen concentration is measured in the control and the group of high concentration (108/ml). In particular, it was confirmed that the group of high concentration is not recovered to an oxygen concentration in the normoxic state on the 5th day of the measurement (see
A reason why an amount of oxygen generated is decreased in the group of high-concentration chloroplast was confirmed by the following method.
The materials included (1) ABTS (548.68 g/mol), (2) HRP (peroxidase, 250 units/ml), (3) H202 solution (for standard curve, 8.943 M(9M/L)), and (4) chloroplast (0-3×108/ml).
Free chloroplast and alginate-CTP-chloroplast hydrogel were prepared by adding chloroplast to 96-well plate in accordance with the concentration.
After that, a sample required for ABTS assay was prepared as follows.
To prepare a standard curve, 1 ml of HRP and 1 ml of H2O2 at each concentration were added, and then 1 ml of ABTS was added. HRP:H2O2:ABTS=1:1:1 was reacted, and ABTS was finally added. 200 μl of the solution was taken and transferred to 96-well plate, reacted for five minutes, and put into a plate reader to confirm absorbance at 405 nm. In order to confirm the H2O2 generated from chloroplast, 1 ml of chloroplast and 1 ml of HRP were added to a brown epen tube at each concentration, and then 1 ml of ABTS was added thereto. In this case, the brown tube was used because chloroplast is sensitive to light. After reacting for three minutes, the reaction mixture was subjected to centrifugation at 13500 RPM for two minutes. After that, 200 μl of the supernatant was taken, transferred to 96-well plate, and put into a plate reader to confirm absorbance at 405 nm. Due to a potential effect of absorbance caused by a color of chloroplast per se, only the supernatant was obtained.
As a result of confirming an amount of hydrogen peroxide generated by the above method, both free chloroplast and ACC groups showed a significantly high level of hydrogen peroxide generated at a high concentration of chloroplast. Thus, it was confirmed that when a high concentration of chloroplast is treated, hydrogen peroxide is excessively generated to rather reduce an amount of oxygen generated (see
15.1. Evaluation of Toxicity of Alginate-CTP to INS-1 Cell Through CCK-8 Assay
It was confirmed whether the alginate-CTP solution is toxic to INS-1 cells by the following method.
INS-1 cells (3*104/well) stabilized at 96-well plate for 24 hours were prepared (n=5). Each of the groups was treated with 100 μl of the alginate-CTP solution prepared at each concentration. After incubation for 24 hours, the alginate-CTP solution was suctioned. The resulting product was washed twice with PBS, treated with 100 μl of media (RPMI)+10 μl of CCK solution, and incubated by wrapping the foil for two hours, after which absorbance was measured at 450 nm using a reader.
As a result, 8 mg/ml, the concentration used in this study, did not show a significant difference from the viability of the control group (see
15.2. Evaluation of Toxicity of Alginate-CTP to INS-1 Cell Through Live & Dead Assay
It was confirmed whether the alginate-CTP solution is toxic to INS-1 cell line by the following method.
INS-1 cells (3*104/well) stabilized at 96-well plate for 24 hours were prepared (n=5). Each of the groups was treated with 100 μl of the alginate-CTP solution prepared at each concentration and incubated for 24 hours, after which the alginate-CTP solution was suctioned. After that, the resulting product was washed twice with PBS, after which each well was treated with 100 μl of a reagent prepared with 1 μM calcein-AM and 1 μM ethidium homodimer-1 (EthD-1), and incubated for 10 minutes. Then, live cells (green fluorescence) and dead cells (red fluorescence) were confirmed using a fluorescence microscope, and a photograph was taken.
As a result, a similar level of green signal was confirmed. Thus, it was confirmed that the present concentration is not toxic to single cells (see
16.1. Isolation of Pancreatic Cell from Rat
Pancreatic cells secreting insulin were isolated from the pancreas of a rat by the following method.
The materials included (1) rat of strain SD (6-7 weeks old, male), (2) surgical tool (micro-scissors, forceps, etc.), (3) 1 mg/ml of collagenase (diluted with HBSS buffer at pH 7.8), and (4) 10 ml syringe.
The rat was euthanized by injecting 100% CO2 gas for 5-10 minutes, and it was confirmed that breathing or heartbreak was stopped. The outer skin and inner skin of the abdomen were removed, the liver was lifted to find the pancreas, and the pancreas was spread out with tongs to find duct (blood vessel), which is a part connected to the liver. After pulling the duct taut, collagenase, from which the extracellular matrix (ECM) may be removed by slightly scratching with scissors without breaking, was injected into a syringe at 10 ml per mouse through the duct. Swollen pancreas was isolated from the intestine, liver, stomach, and spleen using surgical tools. High-purity pancreatic cells were obtained from the pancreas through physical and chemical purification. Besides the pancreatic cells, various debris or impurities were removed through an optical microscope. For recovery and stabilization of the pancreatic cells, incubation was performed for 24 hours.
16.2. Preparation of Alginate-CTP-Chloroplast Hydrogel with Pancreatic Cell Encapsulated Therein
16.2.1. Preparation of Alginate-CTP-Chloroplast Hydrogel Encapsulating Pancreatic Cell with Alginate-CTP Composite Treated at Each Concentration
The isolated pancreatic cells were encapsulated in alginate-CTP-chloroplast hydrogel as follows.
The isolated pancreatic cells, which were stabilized for 24 hours, were washed twice with media (RPMI). Chloroplast solution isolated from spinach on the same day was prepared at a concentration of 4*106/ml. For a cell experiment, media (RPMI+10% FBS) was used when diluting chloroplast. 20 pancreatic cells (20IEQ) were well mixed with 250 μl of the prepared solution.
An alginate-CTP composite prepared at a concentration of 0, 8 and 16 mg/ml was prepared, mixed with the solution, and added into 100 mM CaCl2) solution to react for 30 minutes. Each of the prepared alginate-CTP-chloroplast hydrogel was placed in each well of 96-well plate (RPMI+10% FBS+1% PS, 200 μl) and incubated for 24 hours.
16.2.2. Preparation of Alginate-CTP-Chloroplast Hydrogel Encapsulating Pancreatic Cell with Chloroplast Treated at Each Concentration
The pancreatic cells were encapsulated in alginate-CTP-chloroplast hydrogel as follows.
The pancreatic cells, which were stabilized for 24 hours, were washed twice with media (RPMI). Chloroplast isolated from spinach on the same day was prepared at a concentration of 4*106/ml. For a cell experiment, media (RPMI+10% FBS) was used when diluting chloroplast. 20 pancreatic cells (20IEQ) were well mixed with 250 μl of the isolated chloroplast solution. An alginate-CTP composite prepared at a concentration of 8 mg/ml was prepared. For a cell experiment, media (RPMI+10% FBS) was used when dissolving alginate-CTP. The above solution and the alginate-CTP composite were mixed, added in a 100 mM CaCl2) solution, and reacted for 30 minutes. Each of the prepared alginate-CTP-chloroplast hydrogel was placed in each well of 96-well plate (RPMI+10% FBS+1% PS, 200 μl) and incubated for 24 hours.
16.2.3. Preparation of Alginate-CTP-Chloroplast Hydrogel Encapsulating Pancreatic Cells with Chloroplast Treated at Each Concentration in Hypoxic Environment
The pancreatic cells were encapsulated in alginate-CTP-chloroplast hydrogel in a hypoxic environment as follows.
The pancreatic cells, which were isolated from an experiment animal (rat), were washed twice with media (RPMI). Chloroplast isolated from spinach on the same day was prepared at each concentration. For a cell experiment, media (RPMI+10% FBS) was used when diluting chloroplast. 20 pancreatic cells (20IEQ) were well mixed with 250 μl of the solution of the process 2. An alginate-CTP composite prepared at a concentration of 8 mg/ml was prepared. For a cell experiment, media (RPMI+10% FBS) was used when dissolving alginate-CTP. The above solutions were mixed and added to a 100 mM CaCl2) solution, and reacted for 30 minutes. Each of the prepared alginate-CTP-chloroplast hydrogel at each concentration was placed in each well of 6-well plate (RPMI+10% FBS+1% PS, 1 ml). Above groups (1), (2), (3) and (4) were added to each well of 6-well plate (RPMI+10% FBS+1% PS, 200 μl), and the 6-well plate was implemented in normoxic and hypoxic states as follows and incubated for 24 hours.
17.1. Evaluation of Toxicity of Pancreatic Cell Encapsulated in Alginate-CTP-Chloroplast Hydrogel with Alginate-CTP Composite Treated at Each Concentration
17.1.1. Evaluation of Toxicity Through CCK-8 Assay
Viability of pancreatic cells encapsulated in alginate-CTP-chloroplast hydrogel with alginate-CTP composite treated at each concentration was confirmed by the following method.
Alginate-CTP-chloroplast hydrogel, in which pancreatic cells incubated in 96-well plate for 24 hours were encapsulated and alginate-CTP was treated at each concentration, was prepared. All hydrogel was washed twice with PBS and treated with 100 μl of media (RPMI)+10 μl of CCK solution. After incubation by wrapping the foil for two hours, absorbance was measured at 450 nm using a reader.
As a result, the concentration used in this study, 8 mg/ml, did not show a significant difference from the viability of the control group (see
17.1.2. Evaluation of Toxicity Through Live & Dead Assay
Viability of pancreatic cells encapsulated in alginate-CTP-chloroplast hydrogel with alginate-CTP composite treated at each concentration was confirmed by the following method.
Alginate-CTP-chloroplast hydrogel, in which pancreatic cells incubated in 96-well plate for 24 hours were encapsulated and alginate-CTP was treated at each concentration, was prepared. All hydrogel was washed twice with PBS, and then each well was treated with 100 μl of a reagent prepared with 1 μM calcein-AM and 1 μM ethidium homodimer-1 (EthD-1), and incubated for 30 minutes. Live cells (green fluorescence) and dead cells (red fluorescence) were confirmed using a fluorescence microscope, and then a photograph was taken.
As a result, a similar level of green signal was confirmed even through live & dead assay (see
17.2. Evaluation of Toxicity of Pancreatic Cell Encapsulated in Alginate-CTP-Chloroplast Hydrogel with Chloroplast Treated at Each Concentration
17.2.1. Evaluation of Toxicity Through CCK-8 Assay
Viability of pancreatic cells encapsulated in alginate-CTP-chloroplast hydrogel with chloroplast treated at each concentration was confirmed as follows.
Alginate-CTP-chloroplast hydrogel, in which pancreatic cells incubated in 96-well plate for 24 hours were encapsulated and chloroplast was treated at each concentration, was prepared. All hydrogel was washed twice with PBS and treated with 100 μl of media (RPMI)+10 μl of CCK solution. After incubation by wrapping the foil for two hours, absorbance was measured at 450 nm using a reader.
As a result, the viability of all concentration groups was not significantly different from that of the control group regardless of the concentration of chloroplast (see
17.2.2. Evaluation of Toxicity Through Live & Dead Assay
Viability of pancreatic cells encapsulated in alginate-CTP-chloroplast hydrogel with chloroplast treated at each concentration was confirmed as follows.
Alginate-CTP-chloroplast hydrogel, in which pancreatic cells incubated in 96-well plate for 24 hours were encapsulated and chloroplast was treated at each concentration, was prepared. All hydrogel was washed twice with PBS, and then each well was treated with 100 μl of a reagent prepared with 1 μM calcein-AM and 1 μM ethidium homodimer-1 (EthD-1). Incubation was performed for 30 minutes, live cells (green fluorescence) and dead cells (red fluorescence) were confirmed using a fluorescence microscope, and then a photograph was taken.
As a result, a strong green signal was confirmed through live & dead assay (see
17.3. Evaluation of Toxicity of Pancreatic Cell Encapsulated in Alginate-CTP-Chloroplast Hydrogel with Chloroplast Treated at Each Concentration in Hypoxic Environment (CCK-8 Assay)
It was performed to confirm the viability of pancreatic cells encapsulated in alginate-CTP-chloroplast hydrogel with chloroplast treated at each concentration in a hypoxic environment as follows.
Alginate-CTP-chloroplast hydrogel, in which pancreatic cells incubated in normoxic and hypoxic environments for 24 hours were encapsulated and chloroplast was treated at each concentration, was prepared for each group. All groups were washed twice with PBS, treated with 100 μl of media (RPMI)+10 μl of CCK solution, and incubated by wrapping the foil for two hours, after which absorbance was measured at 450 nm using a reader. After that, an absorbance value was corrected through an amount of DNA for each experimental group using Quant-iT™ PicoGreen™ dsDNA Assay Kits.
As a result, due to exposure of pancreatic cells to a hypoxic environment for a long time, it can be confirmed that viability of pancreatic cells may not be significantly decreased compared to the IN group except for the IH group. It could be confirmed that viability of pancreatic cells is decreased about 18% in the IH group due to exposure to the hypoxic environment for 24 hours, representing that the hypoxic environment is not appropriate for culturing the pancreatic cells. It was confirmed that alginate-CTP-chloroplast (groups 4, 6) has a lower cell viability than that of the IN group, representing an insignificant result. With regard to alginate-CTP-chloroplast hydrogel, it means that the alginate-CTP-chloroplast material is not toxic to pancreatic cells regardless of a concentration of chloroplast. In addition, it was found that groups 0, 4 and 6 show higher cell viability than that of the IH group in a hypoxic environment, representing that pancreatic cells encapsulated in alginate-CTP-chloroplast hydrogel may have an appropriate condition for culturing in a hypoxic environment (see
An amount of oxygen generated from alginate-CTP-chloroplast hydrogel with pancreatic cells encapsulated therein and with chloroplast treated at each concentration was confirmed.
Alginate-CTP-chloroplast hydrogel with pancreatic cells encapsulated therein and with chloroplast treated at each concentration was prepared. One hydrogel of process 1 was added to each well of an oxygen plate reader well plate (24 well), and an amount of oxygen generated was measured using an oxygen sensor at 0, 6, 12, 18, 24 and 30 hours while incubation was performed for 30 hours. SDR SensorDish (PreSens, Germany) was used as the oxygen sensor, and SDR SensorDish-dedicated well plate was used as an oxygen plate reader well plate (24 well).
As a result, an amount of oxygen generated was steadily increased with the passage of time, and a higher level of oxygen generated was observed in a group having chloroplast inserted than in a control group without chloroplast (see
19.1. Confirmation of Functionality of Pancreatic Cell Encapsulated in Alginate-CTP-Chloroplast Hydrogel with Chloroplast Treated at Each Concentration
19.1.1. Confirmation Through Glucose-Stimulated Insulin Secretion (GSIS) Assay
An insulin secretion function of pancreatic cells encapsulated in alginate-CTP-chloroplast hydrogel with chloroplast treated at each concentration was confirmed as follows.
The materials included (1) low glucose (2.8 mM, diluted with Krebs Ringer buffered HEPES (pH 7.4)) and (2) high glucose (20.2 mM, diluted with Krebs Ringer buffered HEPES (pH 7.4)).
Alginate-CTP-chloroplast hydrogel, in which pancreatic cells incubated in 96-well plate for 24 hours were encapsulated and chloroplast was treated at each concentration, was prepared. All hydrogel was washed twice with PBS, and then low glucose (2.8 mM) was dispensed by 200 ul in each well and incubated for 30 minutes. Low glucose (2.8 mM) was again dispensed by 200 μl to each well and incubated for two hours, and then the present low glucose solution was recovered. In addition, high glucose (20.2 mM) was dispensed by 200 μl to each well and incubated for two hours, and then the high glucose solution present was recovered. An amount of insulin secretion was measured and calculated based on low glucose and high glucose solutions recovered using insulin enzyme-linked immunosorbent (ELISA) assay.
As a result, the pancreatic cells encapsulated in alginate-CTP-chloroplast hydrogel showed an amount of insulin secretion similar to that of the control group (intact islet) regardless of the concentration of chloroplast (see
19.1.2. Confirmation Through dsDNA Assay
An amount of insulin secreted from pancreatic cells encapsulated in alginate-CTP-chloroplast hydrogel with chloroplast treated at each concentration was confirmed as follows.
Alginate-CTP-chloroplast hydrogel, in which pancreatic cells incubated in 96-well plate for 24 hours were encapsulated and chloroplast was treated at each concentration, was prepared. All hydrogel was washed twice with PBS, and 0.1 M EDTA (pH 7.8) solution was dispensed by 200 μl into each well and hydrogel was crushed through sufficient pipetting. At this time, it was performed to take out the pancreatic cells present in hydrogel. Each of the solution was recovered into 1.5 ml tube and subjected to centrifugation at 1800 RPM for two minutes. The supernatant was removed and then pellets (pancreatic cells) were treated with 1 ml of RIPA buffer each. An amount of insulin secreted by pancreatic cells present in each well (alginate-CTP-chloroplast hydrogel) was corrected through dsDNA assay.
19.2. Confirmation of Functionality of Pancreatic Cell Encapsulated in Alginate-CTP-Chloroplast Hydrogel with Chloroplast Treated at Each Concentration in Hypoxic Environment
19.2.1. Confirmation Through Glucose-Stimulated Insulin Secretion (GSIS) Assay
An insulin secretion function of pancreatic cells encapsulated in alginate-CTP-chloroplast hydrogel with chloroplast treated at each concentration in a hypoxic environment was confirmed as follows.
Alginate-CTP-chloroplast hydrogel, in which pancreatic cells incubated in normoxic and hypoxic environments for 24 hours were encapsulated and chloroplast was treated at each concentration, was prepared for each group. All groups were washed twice with PBS, and then low glucose (2.8 mM) was dispensed by 200 ul in each well and incubated for 30 minutes. Low glucose (2.8 mM) was again dispensed by 200 μl to each well and incubated for two hours, and then the present low glucose solution was recovered. High glucose (20.2 mM) was dispensed by 200 μl to each well and incubated for two hours, and then the high glucose solution present was recovered. An amount of insulin was measured and calculated based on low glucose and high glucose solutions recovered using insulin enzyme-linked immunosorbent (ELISA) assay.
As a result, it can be confirmed that an insulin secretion ability of pancreatic cells may not be significantly decreased compared to the IN group except for the IH group. It is confirmed that the IH group has a reduced insulin secretion ability in pancreatic cells due to exposure to a hypoxic environment for 24 hours. Alginate-CTP-chloroplast group shows a glucose stimulation index (SI) of pancreatic cells similar to that of the IN group, representing that insulin secretion ability was not reduced even though alginate-CTP-chloroplast group (groups 4, 6) was exposed to a hypoxic environment. In addition, it was found that groups 0, 4, and 6 show a significantly higher insulin secretion ability than that of the IH group in a hypoxic environment, representing that pancreatic cells encapsulated in alginate-CTP-chloroplast hydrogel did not have a reduced insulin secretion ability even though the pancreatic cells were cultured in a hypoxic environment, and may have an appropriate condition for culturing in a hypoxic environment.
19.2.2. Confirmation Through dsDNA Assay
An amount of insulin secreted from pancreatic cells encapsulated in alginate-CTP-chloroplast hydrogel with chloroplast treated at each concentration in a hypoxic environment was corrected with a size of pancreatic cells as follows.
Alginate-CTP-chloroplast hydrogel, in which pancreatic cells incubated in normoxic and hypoxic environments for 24 hours were encapsulated and chloroplast was treated at each concentration, was prepared for each group. All groups were washed twice with PBS, and 0.1 M EDTA (pH 7.8) solution was dispensed by 200 μl into each well and cells and hydrogel were crushed through sufficient pipetting. (It was performed to take out the pancreatic cells present in hydrogel.) Each of the solution was recovered into 1.5 ml tube and subjected to centrifugation at 1800 RPM for two minutes. The supernatant was removed and then pellets (pancreatic cells) were treated with 1 ml of RIPA buffer each. An amount of insulin secreted by pancreatic cells present in each well (alginate-CTP-chloroplast) was corrected through dsDNA assay.
20.1. Measurement of Amount of Oxygen Generated Using Hypoxia Chamber
An amount of oxygen generated from chloroplast at each concentration in a hypoxic environment depending on the presence or absence of light (660 nm) was confirmed as follows.
Chloroplast at each concentration was dispensed by 1 ml each to an oxygen plate reader (24 wells) in accordance with each group, and the plate was implemented in a hypoxic state as follows.
A distance between material and light was set to 5 cm, which was applied only to a light exposure group and a light non-exposure group was performed without light. After that, an amount of oxygen generated from chloroplast depending on the presence or absence of light was measured using an oxygen sensor (reading time: 30 min). At this time, SDR SensorDish (PreSens, Germany) was used as the oxygen sensor.
As a result, it can be confirmed that an initial oxygen concentration of chloroplast with a hypoxic environment implemented is different according to a concentration of chloroplast for each group, representing that an amount of oxygen generated at the concentration of chloroplast is different (see
20.2. Measurement of amount of oxygen generated using temperature-controlled desiccator
An amount of oxygen generated from chloroplast in a hypoxic environment depending on the presence or absence of light (660 nm) was confirmed as follows.
Chloroplast at each concentration was dispensed by 1 ml each to an oxygen plate reader (24 wells) in accordance with each group. The plate was implemented in a hypoxic state as follows.
A distance between material and light was set to 5 cm, which was applied only to a light exposure group and a light non-exposure group was performed without light. An amount of oxygen generated from chloroplast depending on the presence or absence of light was measured using an oxygen sensor for 20 hours. (reading time: 30 min) At this time, SDR SensorDish (PreSens, Germany) was used as the oxygen sensor.
As a result, it could be confirmed that an oxygen concentration is significantly higher in chloroplast (108/ml) compared to the control media (RPMI+1% PS, 10% FBS) (see
21.1. Measurement of Amount of Oxygen Generated According to Temperature Condition
An amount of oxygen generated from chloroplast in a hypoxic environment according to a temperature was confirmed as follows.
Chloroplast, of which a temperature was adjusted in advance, was prepared and dispensed by 1 ml each to an oxygen plate reader (24 wells) in accordance with each group. The plate was implemented in a hypoxic state as follows.
An amount of oxygen generated from chloroplast according to a temperature was measured using an oxygen sensor. At this time, SDR SensorDish (PreSens, Germany) was used as the oxygen sensor.
As a result, it was confirmed that 25° C. chloroplast shows a small amount of change in the oxygen concentration over time, representing that it takes a long time to reach the same concentration as that of 37° C. chloroplast (see
Furthermore, when comparing a degree of decrease in the oxygen concentration for each time (0, 5, 10 and 20 hours) in the graph with an initial oxygen concentration in each group, it was confirmed that a degree of decrease in 25° C. chloroplast might be lower than that at 37° C. chloroplast (see
From the above results, it was confirmed that chloroplast may generate oxygen to the maximum when a surrounding environment is given as 25° C. However, referring to [The structural integrity of the islet in vitro: the effect of incubation temperature; A Ilieva, S Yuan], it was confirmed that pancreatic cells cultured at 24° C. lose morphology thereof and about 40% of the cells are killed when cultured for 12 days. In addition, with regard to an insulin immune activity at 24° C., it was confirmed that the homogeneity of distribution is gradually decreased, and on the 12th day, an insulin immune active region is remarkably decreased to 19±9% of the total pancreatic cells.
As a result, considering that an insulin secretion ability of the pancreatic cells used in a future experiment would be reduced, a subsequent experiment was performed in the environment of 37° C.
21.2. Measurement of Amount of Oxygen Generated According to Light Condition
21.2.1. Measurement of Amount of Oxygen Generated According to Intensity (Lux) of Light (660 nm)
An amount of oxygen generated from chloroplast in a hypoxic environment according to intensity (lux) of light (660 nm) was confirmed as follows.
Chloroplast at a concentration of 1*108/ml was dispensed by 1 ml each to an oxygen plate reader (24 wells). The plate was implemented in a hypoxic state as follows.
A distance between material and light was set to 5 cm, 10 cm and 15 cm, and an amount of oxygen generated from chloroplast according to an intensity of light was measured using an oxygen sensor. The intensity of light reaching the material may be changed to 1500 lux, 1000 lux and 500 lux according to the distance between the material and the light, which is 5 cm, 10 cm and 15 cm. At this time, SDR SensorDish (PreSens, Germany) was used as the oxygen sensor.
As a result, it can be confirmed that the initial oxygen concentration is increased as the intensity of light is decreased, and then tends to be almost constantly maintained for 20 h (see
21.2.2. Measurement of Amount of Oxygen Generated According to Exposure Time of Light (660 nm)
An amount of oxygen generated from chloroplast in a hypoxic environment according to an exposure time of light (660 nm) was confirmed as follows.
Chloroplast at a concentration of 1*108/ml was dispensed by 1 ml each to an oxygen plate reader (24 wells). The plate was implemented in a hypoxic state as follows.
A distance between material and light was set to 15 cm, and an intensity of light reaching the material was set to 500 lux. An amount of oxygen generated from chloroplast according to an exposure time of light was measured using an oxygen sensor. At this time, SDR SensorDish (PreSens, Germany) was used as the oxygen sensor.
As a result, it can be confirmed that the oxygen concentration decreases in the two groups as the exposure time of light increases (see
21.2.3. Measurement of Amount of Oxygen Generated According to an Exposure Time Interval of Light (660 nm)
An amount of oxygen generated from chloroplast in a hypoxic environment according to an exposure time interval of light (660 nm) was confirmed as follows.
Chloroplast at a concentration of 1*108/ml was dispensed by 1 ml each to an oxygen plate reader (24 wells). The plate was implemented in a hypoxic state as follows.
A distance between material and light was set to 15 cm, and an intensity of light reaching the material was set to 500 lux. After that, light was exposed three times for 30 minutes each and an amount of oxygen generated from chloroplast was measured using an oxygen sensor. At this time, SDR SensorDish (PreSens, Germany) was used as the oxygen sensor.
As a result of measuring an amount of oxygen generated for 20 hours after exposing chloroplast to light three times at a time interval of 30 minutes, it was found that the oxygen concentration is highly maintained at about 70-80 mmHg compared to the group not exposed to light (see
An alginate-CTP-chloroplast microcapsule was prepared as follows.
Chloroplast diluted in CIB was prepared at a concentration (2.5*109/ml), and 1.5% alginate and alginate-CTP solution were prepared. 3% alginate solution and tertiary distilled water were mixed at a ratio of 1:1, 1.75 ml of chloroplast and 1.75 ml of 3% (w/v) alginate solution were added thereto and mixed, and then 1.75 ml of chloroplast and 1.75 ml of 3% (w/v) alginate-CTP solution were added thereto and mixed. After that, microcapsules for each group were prepared using an encapsulator (Nozzle size: 450 μm, Frequency: 800 Hz, Electrode: 500V, Flow rate: 15 ml/min), added to a gelation bath containing 30 ml of 100 mM CaCl2), and crosslinked by stirring for 15 minutes (stirring rate: 20 rpm). After that, the CaCl2) solution was removed, and the microcapsules were washed with 10 ml of media (RPMI+10% FBS+1% PS). 500 μl of microcapsules (about 500 units) for each group as well as 500 μl of media were placed in 1 well of 24-well plate.
As a result, it was confirmed that microcapsules are well formed in all groups.
The degradation and mechanical strength of alginate-CTP-chloroplast microcapsule were evaluated as follows.
Microcapsules for each group were prepared by being incubated in 24-well plate for 120 hours. A degree of decomposition was confirmed by examining an overall shape and size of microcapsules with the naked eye, and the strength of microcapsules for each group was confirmed by measuring rheology through a rheometer.
As a result of evaluating the degree of degradation for each group, it was confirmed that the microcapsules are well maintained even after 120 hours in all groups, representing that the microcapsules may be maintained for at least five days (see
In addition, as a result of evaluating a mechanical strength for each group, it was confirmed that an elastic modulus (storage modulus, G) in the alginate-chloroplast group is 6.40 kPa, and an elastic modulus in the alginate-CTP-chloroplast group is 9.80 kPa (see
An amount of oxygen generated from alginate-CTP-chloroplast hydrogel microcapsule was confirmed as follows.
Chloroplast solution, alginate-chloroplast microcapsule, and alginate-CTP-chloroplast for each concentration corresponding to each group were prepared. Each group was put into an oxygen plate reader well plate (24 well) as follows, and an amount of oxygen generated was measured using an oxygen sensor while incubation was performed at 24-well plate for 48 hours. At this time, SDR SensorDish (PreSens, Germany) was used as the oxygen sensor.
As a result, it was confirmed that an oxygen generation rate is the highest in the group of 108/ml having a high concentration of chloroplast in all groups, and it was confirmed that CTP has a relatively higher amount of oxygen generated in the group with the CTP synthesized, representing that the CTP has an effect of helping oxygen generation (see
The pancreatic cells were encapsulated in alginate-CTP-chloroplast microcapsule as follows.
The pancreatic cells, which were isolated from an experiment animal (rat), were washed twice with media (RPMI). Chloroplast diluted in CIB was prepared at a concentration (2.5*109/ml), and 1.5% alginate and alginate-CTP solution were prepared. After that, 3% alginate solution and tertiary distilled water were mixed at a ratio of 1:1, 1.75 ml of chloroplast and 1.75 ml of 3% (w/v) alginate solution were added thereto and mixed, and then 1.75 ml of chloroplast and 1.75 ml of 3% (w/v) alginate-CTP solution were added thereto and mixed. Each 500 IEQ of extracted pancreatic cells was added to the mixed solution and mixed well. After that, microcapsules for each group were prepared using an encapsulator (* Nozzle size: 450 μm, * Frequency: 800 Hz, Electrode: 500V, Flow rate: 15 ml/min), added to a gelation bath containing 30 ml of 100 mM CaCl2), and crosslinked by stirring for 15 minutes (stirring rate: 20 rpm). After that, the CaCl2) solution was removed, and the microcapsules were washed with 10 ml of media (RPMI++10% FBS+1% PS). 500 μl of microcapsules (about 500 units) for each group as well as 500 μl of media were placed in 1 well of 24-well plate,
26.1. Evaluation of Toxicity Through CCK-8 Assay
It was performed to confirm the viability of pancreatic cells encapsulated in alginate-CTP-chloroplast microcapsule in a hypoxic environment as follows.
Pancreatic cells for each group, which were incubated in 24-well plate in normoxic and hypoxic states for 24 hours as well as microcapsules with the pancreatic cells encapsulated therein were prepared as follows.
All the pancreatic cells and microcapsules were washed twice with PBS, treated with 1 ml of media (RPMI)+100 μl of CCK solution, and incubated by wrapping the foil for two hours, after which absorbance was measured at 450 nm using a reader.
As a result, due to exposure of pancreatic cells to a hypoxic environment for a long time, viability of pancreatic cells was significantly reduced to about 22% compared to pancreatic cells in a normoxic environment (see
26.2. Evaluation of Toxicity Through Live & Dead Assay
The viability of pancreatic cells encapsulated in alginate-CTP-chloroplast microcapsule in a hypoxic environment was confirmed as follows.
Pancreatic cells for each group, which were incubated in 24-well plate in the following normoxic and hypoxic states for 24 hours as well as microcapsules with the pancreatic cells encapsulated therein were prepared.
All the pancreatic cells and microcapsules were washed twice with PBS, after which each well was treated with 1 ml of a reagent prepared with 5 μM calcein-AM and 20 μM ethidium homodimer-1 (EthD-1), and incubated for 40 minutes. At this time, groups (3) and (4) were performed for 15 minutes. After that, live cells (green fluorescence) and dead cells (red fluorescence) were confirmed using a fluorescence microscope, and then a photograph was taken.
As a result, it was confirmed that a green signal appears to be relatively strong in the alginate-CTP-chloroplast microcapsule group compared to other groups in a hypoxic environment, and a yellow signal appears to be the weakest as a result of the combination of green and red (see
27.1. Confirmation of Functionality Through Glucose-Stimulated Insulin Secretion (GSIS) Assay
An insulin secretion function of pancreatic cells encapsulated in alginate-CTP-chloroplast microcapsule in a hypoxic environment was confirmed as follows.
The materials included (1) low glucose (2.8 mM, diluted with Krebs Ringer buffered HEPES (pH 7.4)) and (2) high glucose (20.2 mM, diluted with Krebs Ringer buffered HEPES (pH 7.4)).
Pancreatic cells for each group, which were incubated in 24-well plate in normoxic and hypoxic states for 24 hours as well as microcapsules with the pancreatic cells encapsulated therein were prepared as follows.
All the pancreatic cells and microcapsules were washed twice with PBS, and then low glucose (2.8 mM) was dispensed by 1 ml in each well and incubated for 30 minutes. Low glucose (2.8 mM) was again dispensed by 1 ml to each well and incubated for two hours, and then the present low glucose solution was recovered. After that, high glucose (20.2 mM) was dispensed by 1 ml to each well and incubated for two hours, and then the high glucose solution present was recovered. An amount of insulin was measured and calculated based on low glucose and high glucose solutions recovered using insulin enzyme-linked immunosorbent (ELISA) assay.
Then, as a result of comparing the pancreatic cells in a normoxic environment with the pancreatic cells in a normoxic environment, it was confirmed that an insulin secretion ability of the pancreatic cells is affected by the surrounding environment. It means that a hypoxic environment is not an environment appropriate for pancreatic cells to survive. It was confirmed that the pancreatic cells encapsulated in alginate-CTP-chloroplast microcapsules secrete insulin at a level similar to that of pancreatic cells of normoxia at both low and high glucose concentrations, representing that the alginate-CTP-chloroplast microcapsule material does not affect the insulin secretion ability of encapsulated pancreatic cells. It was confirmed that the pancreatic cells encapsulated in alginate-CTP-chloroplast microcapsules have an insulin secretion ability at the most similar level to that of pancreatic cells of normoxia among hypoxic environment experimental groups, representing that the alginate-CTP-chloroplast microcapsule material may best maintain an insulin secretion ability of encapsulated pancreatic cells. It was confirmed that the pancreatic cells encapsulated in the alginate-CTP-chloroplast microcapsule have a significantly higher glycemic control function than that of the pancreatic cells encapsulated in the alginate-chloroplast microcapsule, and the pancreatic cells encapsulated in the alginate-CTP-chloroplast microcapsule maintain a glycemic control function compared to the pancreatic cells encapsulated in the alginate-chloroplast microcapsule. The pancreatic cells encapsulated in the alginate-CTP-chloroplast microcapsules have the highest ability to secrete insulin among respective groups in a hypoxic environment, representing that the alginate-CTP-chloroplast microcapsule material has the potential to increase the viability of pancreatic cells and the potential to maintain the ability thereof from a hypoxic environment. An amount of insulin secreted from the pancreatic cells encapsulated in the alginate-CTP-chloroplast microcapsule was lower than that of the pancreatic cells in the normoxic environment, but this is probably affected by metabolites (metabolome) or reactive oxygen (ROS) of the pancreatic cells present in the alginate-CTP-chloroplast microcapsule. Accordingly, in order to compensate for this problem in subsequent experiments, an experiment was performed by synthesizing catalase (CAT) or superoxide dismutase (SOD) enzyme.
27.2. Confirmation of Functionality Through dsDNA Assay
An amount of insulin secreted from the pancreatic cells encapsulated in alginate-CTP-chloroplast microcapsule in a hypoxic environment was corrected with a size of pancreatic cells as follows.
Pancreatic cells for each group, which were incubated in 24-well plate in normoxic and hypoxic states for 24 hours as well as microcapsules with the pancreatic cells encapsulated therein were prepared as follows.
All the pancreatic cells and microcapsules were washed twice with PBS, and 0.1 M EDTA (pH 7.8) solution was dispensed by 1 ml into each well and the pancreatic cells and microcapsules were crushed through sufficient pipetting (in order to take out the pancreatic cells present in microcapsules). Each of the solution containing the crushed pancreatic cells and microcapsules was recovered into 1.5 ml tube and subjected to centrifugation at 1800 RPM for two minutes. The supernatant was removed and then pellets (pancreatic cells) were treated with 1 ml of RIPA buffer each. An amount of insulin secreted by pancreatic cells present in each well (alginate-CTP-chloroplast) was corrected through dsDNA assay.
It was confirmed by fluorescence whether CTP(VILGLGLAGI)-FITC may be also anchored on an outer membrane of chloroplast in addition to CTP(MFAFQYLLVM) by the following method. Care was taken not to be exposed to light during the experiment.
500 μl of 1*108/ml chloroplast (control) and 500 μl of 1*108/ml chloroplast+CTP-FITC were prepared (CTP-FITC was obtained upon request from Peptron, a synthesis company. In 1*108/ml chloroplast+CTP-FITC, CTP-FITC was treated at a concentration of 0.6 mg/ml. If 500 μl of 1*108/ml chloroplast (in CIB) is taken and 30 μl of 10 mg/ml CTP (in DMSO) is taken and added, the final concentration may become 0.6 mg/ml. After that, 100 μl was taken and immobilized every 1, 5, 10, 30 and 60 minutes, and the solution reacted for each time was subjected to centrifugation at 13500 RPM for one minute. A pellet was obtained, immobilized in a 2.5% glutaraldehyde (0.05 M SCB) fixative (500 μl, 15 min), and washed twice with PBS to remove unreacted CTP-FITC. After that, 500 μl was filled with CIB buffer, and then mounted using slide glass/cover glass. A fluorescent photograph was taken using a confocal microscope, and sectioning was performed with the thickness of about 1 μm (green fluorescence (FITC)=488 nm, red fluorescence (chloroplast auto fluorescence)=580 nm).
As a result, it could be confirmed that CTP(V-) is anchored onto an outer membrane of chloroplast from a reaction time of five minutes or more to be sufficiently coated. In addition, it was confirmed that anchoring is possible up to an inner membrane, and it was confirmed that CTP(V-) stably surrounds chloroplast (see
Carboxylate (—COOH) group of alginate and primary amine (—NH2) of CTP(V-) were synthesized by the following method. At this time, CTP(VILGLGLAGI) was used instead of CTP(MFAFQYLLVM). A carbodiimide coupling reaction was used to form an amide bond with carboxylate (—COOH) group of alginate and primary amine of CTP(V-) to prepare a conjugate. EDC/NHS was used to increase the efficiency of the carbodiimide coupling reaction (see
After preparing 10 ml of 0.1 M MES buffer (+0.5 M NaCl), pH was adjusted to 6. 0.088 mM sodium alginate (10 mg/ml) was dissolved in 10 ml of the prepared MES buffer, so that sodium alginate might be well loosen and might not be agglomerated. When the solution was sufficiently dissolved, 4 mg/10 ml EDC (191.7 M.W) corresponding to 2 mM was dissolved. After that, 6 mg/10 ml NHS (5 mM) was added thereto, reacted at RT for 15 minutes, and titrated to pH 7 with PBS or sodium bicarbonate (NaHCO3, sodium hydrogen carbonate). Peptide (10 mg/ml, peptide was synthesized upon request by Peptron, a synthesis company) dissolved in 1% DMSO was added to the solution in an amount corresponding to a molar ratio (1:1.9, 1:3.8, 1:19) and reacted at RT for two hours. The resulting mixture was subjected to dialysis for three days using a dialysis membrane with a molecular weight cut-off (MWCO) of 6,000 to 8,000. After that, the resulting product was frozen in a deep freezer, and then freeze-dried to obtain an alginate-CTP(VILGLGLAGI) composite in the form of powder (see
29.2. Confirmation of Alginate-CTP (VILGLGLAGI) Conjugation
29.2.1. CONFIRMATION THROUGH FT-IR
A conjugate having a ratio of alginate and peptide at 1:19 well showed the properties of peptide because alginate (0.1 mg/ml) was the least contained (see
29.2.2. Confirmation Through 1H-NMR
A conjugate having a ratio of alginate and peptide at 1:19 well show the properties of peptide because alginate (0.1 mg/ml) is the least contained (see
The above description of the present invention is for illustration, and those skilled in the art to which the present invention pertains will understand that the present invention can be easily modified into other specific forms without departing from the technical idea or essential features of the present invention. Thus, it should be understood that the exemplary embodiments described above are illustrative in all aspects and are not contrived to limit the scope of the present invention.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2021-0035982 | Mar 2021 | KR | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/KR2022/003838 | 3/18/2022 | WO |
