Patent Application
20230404923
The present invention relates to a growth factors-enriched dry powder, and in particular to the use of a growth factors-enriched dry powder for relieving inflammation or injury.
Inflammation is the body's defense mechanism. Inflammatory reactions are often triggered in various parts of the body due to infections, traumas or hypersensitivity in order to remove harmful irritants and pathogens to promote tissue repairing. Inflammation can be divided into acute inflammation and chronic inflammation such as dermatitis, allergic rhinitis, pneumonia, hepatitis and enteritis. Dermatitis can be divided into Irritant contact dermatitis (ICD) and allergic contact dermatitis; Severe pneumonia can lead to disease such as Chronic Obstructive Pulmonary Disease (COPD) and pulmonary fibrosis; while severe hepatitis can lead to disease such as liver cirrhosis and cancer.
Platelet-Rich Plasma (PRP) is a blood-derived product with concentrations approximately 1.5 to 5 times higher than normal blood. Platelets contain platelet-related growth factors, such as Platelet-Derived Growth Factor (PDGF), Vascular Endothelial Growth Factor (VEGF), Epidermal Growth Factor (EGF), Fibroblast Growth Factor (FGF), and Transforming Growth Factor Beta 1 (TGF-β1).
Although Platelet-Rich Plasma (PRP) has anti-inflammatory properties (Reference 1, 2), clinical studies have found that Platelet-Rich Plasma (PRP) is ineffective in suppressing inflammation. The reason for this is that the low concentration and unstable quality of growth factors in Platelet-Rich Plasma (PRP), and it is not easy to preserve; moreover, in allogeneic clinical applications, Platelet-Rich Plasma (PRP) can easily trigger allergic reactions, thus increasing the risk of clinical use.
There is an urgent need in the market for a pharmaceutical composition capable of breaking through the limitation of current technological to relieve or treat inflammation or injury in various parts.
In view of the defects from the prior art, the objective of the present invention is intended to provide a growth factors-enriched dry powder that can effectively relieve or treat the degree of inflammation or injury.
Another objective of the present invention is to provide a method for preparing a growth factors-enriched dry powder as described.
A further objective of the present invention is to provide a growth factors-enriched dry powder for use in relieving or treating the degree of inflammation or injury described.
It is also an objective of the present invention to provide the preparation of a pharmaceutical composition or health composition for use in relieving or treating the degree of inflammation or injury.
A method of manufacturing a growth factors-enriched dry powder comprising: (a) taking a unit of a platelet-containing solution and performing a process of purifying platelets to obtain ultra-pure platelet solution; (b) performing an activation procedure to allow platelets activation in ultra-pure platelet solution and release growth factors to obtain a platelet growth factors-extract solution; (c) performing a procedure to remove a platelet-associated structure to obtain a growth factors-enriched solution; and (d) subjecting the growth factors-enriched solution to a drying process to obtain the growth factors-enriched dry powder.
In some embodiments, the drying process is a freeze-drying process. In some embodiments, the drying process is a freeze-drying process, and the freeze-drying process comprises several steps which freeze the multiple components of the growth factors-enriched solution and cause at least one of the frozen components to sublimate directly into a gaseous state and escape, thereby (1) forming multiple pores (e.g., multiple gas-entrapping pores or multiple gas-evacuation pores) within the growth factors-enriched dry powder, or (2) producing a change sufficient to cause an alteration in the physiological response through the process of converting a growth factors-enriched solution to a growth factors-enriched dry powder. In some embodiments, the frozen components comprise a frozen solute, a frozen platelet-related growth factors, and a plurality of other frozen components; and the frozen component that is directly sublimated into a gaseous state are (1′) frozen solute or (1″) frozen solute and at least one other frozen component. In some embodiments, the morphology of the multiple pores (e.g., multiple gas-entrapping pores or multiple gas-evacuation pores) within the growth factors-enriched dry powder is observed by electron microscopy or other means.
In some embodiments, the drying process is a freeze-drying process, a low temperature drying process, or a vacuum drying process.
In some embodiments, the process of purifying platelets in step (a) further comprises a procedure for removing white blood cells so that the number of white blood cells per milliliter (mL) of the ultra-pure platelet solution is less than 106 cells.
In some embodiments, the process of purifying platelets in step (a) further comprises a procedure for removing white blood cells so that the number of white blood cells per milliliter (mL) of the ultra-pure platelet solution is less than 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000, 100,000, 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, or 800,000 cells.
In some embodiments, the process of purifying platelets in step (a) further comprises a procedure for removing plasma so that the concentration of albumin in the ultra-pure platelet solution is less than 3 g/dL; in some embodiments, the process of purifying platelets further comprises a procedure for removing plasma so that the concentration of globulin in the ultra-pure platelet solution is less than 2 g/dL; in some embodiments, the process of purifying platelets further comprises a procedure for removing plasma so that the concentration of fibrinogen in the ultra-pure platelet solution is less than 100 μg/mL.
In some embodiments, the process of purifying platelets in step (a) further comprises a procedure for removing plasma so that the concentration of albumin in the ultra-pure platelet solution is less than 0.25, 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2, 2.25, 2.5 or 2.75 g/dL.
In some embodiments, the process of purifying platelets further comprises a procedure for removing plasma so that the concentration of globulin in the ultra-pure platelet solution is less than 0.25, 0.5, 0.75, 1, or 1.25 g/dL.
In some embodiments, the process of purifying platelets further comprises a procedure for removing plasma so that the concentration of fibrinogen in the ultra-pure platelet solution is less than 10, 20, 30, 40, 50, 60, 70, 80 or 90 μg/mL.
In some embodiments, the platelet-containing solution is whole blood, and step (a) comprises: (a1) taking a unit of platelet-containing solution; (a2) performing the first sedimentation process, so that the platelet-containing solution is separated into a plasma layer, a buffy coat layer, and a red blood cells layer; (a3) Removing the buffy coat layer and the red blood cells layer to obtain a Plasma layer solution; (a4) performing a second sedimentation process, which allows platelets to settle and form a pellet; (a5) removing the supernatant and mixing the pellet with a solvent to obtain the ultra-pure platelet solution.
In some embodiments, the activation procedure is a freezing and then thawing procedure (i.e., a freeze-thaw procedure), an addition of Ca2+ procedure, an addition of thrombin procedure, an addition of collagen procedure, or an addition of ADP procedure, or any combination thereof.
In some embodiments, the activation procedure is a freezing and then thawing procedure (i.e., freeze-thaw procedure), and the freezing and then thawing procedure (i.e., freeze-thaw procedure) comprises (b1) placing a container containing the ultra-pure platelet solution in liquid nitrogen to freeze the ultra-pure platelet solution; and (b2) raising the temperature of the container to thaw the frozen ultra-pure platelet solution.
In some embodiments, the content of PDGF-BB per gram (g) of growth factors-enriched dry powder is 10 pg˜3×107 pg.
In some embodiments, the content of PDGF-BB per gram (g) of growth factors-enriched dry powder is 10, 20, 30, 40, 50, 60, 70, 80, 90, 1×102, 5×102, 1×103, 5×103, 1×104, 5×104, 1×105, 5×105, 1×106, 5×106, or 1×107 pg.
In some embodiments, the content of VEGF per gram (g) of growth factors-enriched dry powder is 2×102 pg˜3×106 pg.
In some embodiments, the content of VEGF per gram (g) of growth factors-enriched dry powder is 4×102, 8×102, 1×103, 5×103, 1×104, 5×104, 1×105, 5×105, or 1×106 pg.
In some embodiments, the content of FGF per gram (g) of growth factors-enriched dry powder is 10 pg˜1×104 pg.
In some embodiments, the content of FGF per gram (g) of growth factors-enriched dry powder is 10, 20, 30, 40, 50, 60, 70, 80, 90, 1×102, 5×102, 1×103 or 5×103 pg.
In some embodiments, the content of TGF-β1 per gram (g) of growth factors-enriched dry powder is 2×105 pg˜1.2×107 pg.
In some embodiments, the content of TGF-β1 per gram (g) of growth factors-enriched dry powder is 3×105, 5×105, 1×106, 5×106, or 1×107 pg.
In some embodiments, the content of EGF per gram (g) of growth factors-enriched dry powder is 1×102 pg˜6×105 pg.
In some embodiments, the content of EGF per gram (g) of growth factors-enriched dry powder is 2×102, 5×102, 1×103, 5×103, 1×104, 5×104, 1×105, or 5×105 pg.
In some embodiments, the freeze-drying procedure comprises lowering the temperature to less than or equal to −35° C. and lowering the pressure to less than or equal to 80 mTorr.
In some embodiments, the freeze-drying procedure comprises lowering the temperature to less than or equal to −40, −45, −50, −60, −65, or −70° C.
In some embodiments, the freeze-drying procedure comprises lowering the pressure to less than or equal to 70,60,50,40, or 30 mTorr.
In some embodiments, the platelet-containing solution is whole blood, apheresis platelet, leukocytes-reduced platelets apheresis, or Platelet-Rich Plasma (PRP), or any combination thereof; or the platelet-containing solution is derived from an autologous blood sample or an allogeneic blood sample, or a combination thereof.
In some embodiments, the solvent in step (a5) is sterile saline, water for injection, 0.45% NaCl, 4.5% hypertonic saline, sterile warm spring water, or isotonic saline; or, the platelet concentration in the ultra-pure platelet solution in step (a5) is 1×109 cells/mL 10×109 cell s/mL.
In some embodiments, the platelet concentration in the ultra-pure platelet solution in step (a5) is 2×109 cells/mL, 3×109 cells/mL 4×109 cells/mL, 5×109 cells/mL 6×109 cells/mL, 7×109 cells/mL, 8×109 cells/mL, or 9×109 cells/mL.
In some embodiments, step (c) comprises: (el) performing a sedimentation process to allow a platelet-associated structure to settle and form a platelet-associated structure pellet; (c2) removing the platelet-associated structure pellet to obtain the growth factors-enriched solution.
The present invention further provides a growth factors-enriched dry powder that is prepared by a preparation method, and the preparation method comprises: (a) taking a unit of platelet-containing solution and performing a process of purifying platelets to obtain an ultra-pure platelet solution; (b) performing an activation procedure to allow platelets activation in ultra-pure platelet solution and release growth factors to obtain a platelet growth factors-extract solution; (c) performing a platelet-associated structure removal procedure to obtain a growth factors-enriched solution; and (d) performing the freeze-drying procedure on growth factors-enriched solution in order to obtain the growth factors-enriched dry powder, wherein the freeze-drying procedure comprises lowering the temperature to less than or equal to −30° C. and lowering the pressure to less than or equal to 100 mTorr; wherein, the content of PDGF-BB per gram (g) of growth factors-enriched dry powder is 10 pg˜3×107 pg.
In some embodiments, the activation procedure is a freezing and then thawing procedure (i.e., a freeze-thaw procedure), and the freezing and then thawing procedure (i.e., a freeze-thaw procedure) comprises: (b1) placing a container containing the ultra-pure platelet solution in liquid nitrogen to freeze the ultra-pure platelet solution; and (b2) raising the temperature of the container to thaw the frozen ultra-pure platelet solution.
The invention further provides for the use of a growth factors-enriched dry powder in the preparation of a pharmaceutical composition or health composition for administrating an effective dose to a specific site in an individual to relieve the degree of inflammation or injury in the site.
In some embodiments, the individual is a human or other mammal.
In some embodiments, the activation procedure is a freezing and then thawing procedure (i.e., a freeze-thaw procedure), and the freezing and then thawing procedure (i.e., a freeze-thaw procedure) comprises: (b1) place a container containing the ultra-pure platelet solution in liquid nitrogen to freeze the ultra-pure platelet solution; and (b2) raise the temperature of the container to thaw the frozen ultra-pure platelet solution.
In some embodiments, the process of purifying platelets in step (a) further comprises a procedure for removing white blood cells so that the number of white blood cells per milliliter (mL) of the ultra-pure platelet solution is less than 106 cells.
In some embodiments, the process of purifying platelets in step (a) further comprises a procedure for removing plasma so that the concentration of albumin in the ultra-pure platelet solution is less than 3 g/dL; or, the process of purifying platelets comprises a procedure to remove plasma so that the concentration of globulin in the ultra-pure platelet solution is less than 2 g/dL; or, the process of purifying platelets comprises a procedure to remove plasma so that the concentration of fibrinogen in the ultra-pure platelet solution is less than 100 μg/mL.
In some embodiments, the content of VEGF per gram (g) of growth factors-enriched dry powder is 2×102 pg˜3×106 pg.
In some embodiments, the content of FGF per gram (g) of growth factors-enriched dry powder is 10 pg˜1×104 pg.
In some embodiments, the content of TGF-β1 per gram (g) of growth factors-enriched dry powder is 2×105 pg˜1.2×107 pg.
In some embodiments, the content of EGF per gram (g) of growth factors-enriched dry powder is 1×102 pg˜6×105 pg.
In some embodiments, the growth factors-enriched dry powder in the preparation of a pharmaceutical composition or health composition to relieve the degree of inflammation or injury of a respiratory tract, skin, muscles, tendons, bones, joints, or ligaments of the individual.
The following is a detailed description of the embodiments of the present invention, as well as the technology and features of the present invention. However, this embodiment is not intended to limit the present invention, and any changes and modifications made by anyone who is familiar with the technology without departing from the spirit and scope of the present invention should be included in the scope of the patent application of the present invention.
In the present invention, the growth factors-enriched dry powder refers to the dry powder rich in platelet-related growth factors such as Platelet-Derived Growth Factor (PDGF) enriched dry powder, Vascular Endothelial Growth Factor (VEGF) enriched dry powder, Epidermal Growth Factor (EGF) enriched dry powder, Fibroblast Growth Factor (FGF) enriched dry powder, or Transforming Growth Factor Beta 1 (TGF-β1) enriched dry powder, or any combination thereof.
In the present invention, “enriched platelet-related growth factors” dry powder has a higher concentration of platelet-related growth factors than the source composition from which the dry powder is isolated.
In the present invention, the growth factors-enriched solution refers to the solution rich in platelet-related growth factors, such as Platelet-Derived Growth Factor (PDGF) enriched solution, Vascular Endothelial Growth Factor (VEGF) enriched solution, Epidermal Growth Factor (EGF) enriched solution, Fibroblast Growth Factor (FGF) enriched solution, or Transforming Growth Factor Beta 1 (TGF-β1) enriched solution, or any combination thereof.
In the present invention, an “enriched platelet-related growth factors” solution has a higher concentration of platelet-related growth factors than the source solution from which the solution is isolated.
In the present invention, “one unit” of a platelet-containing solution refers to the capacity of a container of any size for a platelet-containing solution, such as the capacity of a blood bag for a platelet-containing solution is 250 mL, the capacity of a 50 mL centrifuge tube for a platelet-containing solution is 50 mL, the capacity of a 1 mL container for a platelet-containing solution is 1 mL.
In the present invention, ultra-pure platelet solution refers to a higher purity platelet solution purified from a platelet-containing solution.
In the present invention, the platelet-associated structure refers to platelets, platelet fragments, platelet structures formed by aggregation between platelets, or any combination thereof.
In the present invention, the sedimentation process refers to the process of depositing and precipitating substances, such as the process of depositing and precipitating substances by using gravity, centrifugal force, or electromagnetic force.
In the present invention, “health composition” or “health product” refers to a nutritional supplement; a composition that can promote subject's tissues to maintain normal physiological functions after being administered to an subject, or can relieve subject's symptoms after being administered to an subject.
Please refere to
In some embodiments, the platelet-containing solution is whole blood, apheresis platelet, leukocytes-reduced platelets apheresis, or Platelet-Rich Plasma (PRP), or any combination thereof. In some embodiments, the platelet-containing solution is derived from an autologous blood sample or an allogeneic blood sample, or a combination thereof.
In some embodiments, step (S11) further comprises sub-steps:
In some embodiments, step (S112) further comprises sub-steps:
In some embodiments, the solvent is sterile saline, water for injection, 0.45% NaCl, 4.5% hypertonic saline, sterile warm spring water, or isotonic saline, or any combination thereof. In some embodiments, the platelet concentration in the ultra-pure platelet solution is 1×109/mL 10×109/mL.
In some embodiments, the lysis procedure in step (S12) is a freezing and then thawing procedure (i.e., freezing-thaw procedure), an addition of Ca′ procedure, an addition of thrombin procedure, an addition of collagen procedure, or an addition of ADP procedure, or any combination thereof.
In some embodiments, the activation procedure in step (S12) is a freezing and then thawing procedure (i.e., a freeze-thaw procedure), and step (S12) further comprises sub-steps:
In some embodiments, step (S13) further comprises sub-steps:
In some embodiments, in step (S14), the freeze-drying procedure comprises lowering the temperature to less than or equal to −30° C. and lowering the pressure to less than or equal to 100 mTorr. In some embodiments, in step (S14), the freeze-drying procedure comprises lowering the temperature to less than or equal to −35° C. and lowering the pressure to less than or equal to 80 mTorr.
In this embodiment, the following steps (S11′)˜(S14′) are used to prepare growth factors-enriched dry powder:
From the above preparation procedure, the preparation procedure of the growth factors-enriched dry powder comprises the step of removing white blood cells (WBC) and plasma, and the step of stimulating platelets (e.g., by freezing and thawing), the step of removing platelet-associated structure, and step of drying (e.g., via freezing, cryogenic, or vacuum drying steps). Therefore, the growth factors-enriched dry powder comprises low concentration or even no white blood cells (WBC), plasma, and platelets.
In some embodiments, the growth factors-enriched dry powder comprises low concentration or even no albumin.
In some embodiments, the growth factors-enriched dry powder comprises low concentration or even no globulin.
In some embodiments, the growth factors-enriched dry powder comprises low concentration or even no fibrinogen.
Using an analytical balance (ME204 model, Mettler brand) to measure the weight of 3 bottles of growth factors-enriched dry powder. Mixing 3 bottles of growth factors-enriched dry powder with 1 mL of normal saline respectively to obtain 3 bottles of growth factors-enriched dry powder solution/suspension. An Automated Hematology Analyzer (XP-300 model, SYSMEX brand) is used to analyze the number of white blood cells (WBC) in each bottle of growth factors-enriched dry powder solution/suspension, and use a microscope and counting methods to detect the number of platelets in growth factors-enriched dry powder solution/suspension; then use enzyme-linked immunosorbent assay (ELISA) to analyze the concentration of various growth factors (including PDGF-BB, VEGF, EGF, FGF, TGF-β1) in growth factors-enriched dry powder solution/suspension. Subsequently, use the various growth factor concentrations, white blood cell (WBC) concentration, and platelet concentration in the growth factors-enriched dry powder solution/suspension to deduce the weight of various growth factors, the number of white blood cells (WBC), and the number of platelets per gram (g) of growth factors-enriched dry powder solution/suspension. The experimental results are shown in Table 1.
As shown in Table 1, the weight of each bottle of growth factors-enriched dry powder is 0.0030 grams (g) on average. The growth factors-enriched dry powder comprises 3,446,200 pg of PDGF-BB, 379,787.01 pg of VEGF, 253,430 pg of EGF, 4,515.05 pg of FGF, and 819,530 pg of TGF-β1 per gram (g) on average; moreover, either no white blood cells (or about 0-100 white blood cells (WBC)) and no platelets (or about 0-1,000 platelets) can be detected in growth factors-enriched dry powder per nanogram (ng) on average.
Perform the step of Experiment 1-1 (S11′) to prepare an ultra-pure platelet solution and the platelet concentration in the ultra-pure platelet solution is 1×109/mL. Then, the ultra-pure platelet solution is divided into two groups, i.e., the refrigerated group and the Frozen-thawed group.
Frozen-thawed group: take 1 mL of the ultra-pure platelet solution, and perform the steps (S12′)˜(S14′) of Embodiment 1-1 to prepare the dry powder of the frozen-thawed group (that is, the growth factors-enriched dry powder in this invention).
Refrigerated group: take 1 mL of the ultra-pure platelet solution, and not perform the activation procedure in step (S12′) of Embodiment 1-1; i.e., the ultra-pure platelet solution is not placed in liquid nitrogen to freeze and then thaw, but only refrigerated at 4° C. Then, perform the steps (S13′)˜(S14′) of Embodiment 1-1 to prepare the dry powder of the refrigerated group.
The dry powder of the refrigerated group and the dry powder of the frozen-thawed group were respectively mixed with 1 mL of water for injection to prepare the refrigerated group dry powder solution and the frozen-thawed group dry powder solution. Then, they used the enzyme-linked immunosorbent assay (ELISA) to analyze the concentration of growth factors in each group of solutions, and they used the following formula to calculate the multiples of growth factors released from each group.
The multiple of growth factor released in the frozen-thawed group=(the growth factor concentrations of the solution in the frozen-thawed group×the volume of the solution in the frozen-thawed group)÷(the growth factor concentrations of the solution in the refrigerated group×the volume of the solution in the refrigerated group)
The multiple of growth factor released in the refrigerated group=(the growth factor concentrations of the solution in the refrigerated group×the volume of the solution in the refrigerated group)÷(the growth factor concentrations of the solution in refrigerated group×the volume of the solution in the refrigerated group solution)
Please refer to the experimental results shown in Table 2. As shown in Table 2, the total amount of growth factors in the dry powder solution of the frozen-thawed group is 1.8-15 folds that of the dry powder solution in the refrigerated group. What can be seen is that the activation procedure of freezing and then thawing has an unexpected effect of significantly increase the release of growth factors by 1.8 to 15 folds.
Perform the step (S11′) of Embodiment 1-1 to prepare an ultra-pure platelet solution, and the platelet concentration in the ultra-pure platelet solution is 1×109/mL. Then, the ultra-pure platelet solution is divided into control group and 4 experimental groups, namely calcium ion group, collagen group, adenosine diphosphate group, and −196° C. (liquid nitrogen) group.
Control group: take 1 mL of the ultra-pure platelet solution, and instead of performing the activation procedure in the step (S12′) of the embodiment 1-1, the steps (S13′)˜(S14′) of the embodiment 1-1 is performed directly to prepare dry powder of growth factors without the activation procedure; and then mix with 1 mL of water for injection, which is the control group solution.
−196° C. (liquid nitrogen) group: take 1 mL of the ultra-pure platelet solution, and perform the steps (S12′)˜(S14′) of Embodiment 1-1 to prepare the −196° C. (liquid nitrogen) group dry powder (that is, the growth factors-enriched dry powder in present invention); then, mix with 1 mL of water for injection to prepare −196° C. (liquid nitrogen) group solution.
Calcium ion group: take 1 mL of the ultra-pure platelet solution, and not perform the activation procedure in the step (S12′) of the Embodiment 1-1; that is, the ultra-pure platelet solution is not placed in liquid nitrogen to freeze and then thaw, but mix with calcium chloride to make the calcium ion concentration in the mixture reach 4.54 mg/mL, and let it stand for 20-30 minutes (the activation time can be expanded to 5-120 minutes, and the standing time is the same as that of the −196° C. (liquid nitrogen) group when performing the step (S121′)). Subsequently, perform the steps (S13′) and (S14′) of Embodiment 1-1 to prepare the calcium ion group dry powder, and then mix it with 1 mL of water for injection to prepare the calcium ion group solution.
Collagen group: take 1 mL of the ultra-pure platelet solution, and not perform the activation procedure in the step (S12′) of the Embodiment 1-1; that is, the ultra-pure platelet solution is not placed in liquid nitrogen to freeze and then thaw; instead, mix with collagen (C7661, Sigma-Aldrich brand) to make the collagen concentration in the mixture reach 25 ug/mL, and let it stand for 20-30 minutes (the activation time can be expanded to 5-120 minutes, and the standing time is the same as that of the −196° C. (liquid nitrogen) group when performing the step (S121′)). Subsequently, perform the steps (S13′) and (S14′) of Embodiment 1-1 to prepare the collagen group dry powder, and then mix it with 1 mL of water for injection to prepare the collagen group solution.
Adenosine diphosphate (ADP) group: take 1 mL of the ultra-pure platelet solution, and not perform the activation procedure in the step (S12′) of the Embodiment 1-1; that is, the ultra-pure platelet solution is not placed in liquid nitrogen to freeze and then thaw; instead, mix with adenosine diphosphate (ADP) to make the adenosine diphosphate (ADP) concentration in the mixture reaches 0.8 mM, and let it stand for 20-30 minutes, (the activation time can be expanded to 5-120 minutes, and the standing time is the same as that of the −196° C. (liquid nitrogen) group when performing the step (S121′)). Subsequently, perform the steps (S13′) and (S14′) of Embodiment 1-1 to prepare the adenosine diphosphate (ADP) group dry powder, and then mix it with 1 mL of water for injection to prepare the adenosine diphosphate (ADP) group solution.
The enzyme-linked immunosorbent assay (ELISA) is used to analyze the concentration of growth factors in each group of solutions, and the following formula is used to calculate the multiples of growth factors released from each group.
The multiple of growth factor released in the control group=(the growth factor concentrations of the solution in the control group×the volume of the solution in the control group)÷(the growth factor concentrations of the solution in the control group×the volume of the solution in the control group).
The multiple of growth factor released in the experimental group=(the growth factor concentrations of the solution in the experimental group×the volume of the solution in the experimental group)÷(the growth factor concentrations of the solution in the control group×the volume of the solution in the control group).
The experimental results are shown in Table 3 and
Please refer to
As the results are shown in Table 3 and
Perform the step (S11′) of Embodiment 1-1 to prepare an ultra-pure platelet solution, and the platelet concentration in the ultra-pure platelet solution is 1×109/mL. Then, the ultra-pure platelet solution is divided into a control group and 5 experimental groups, namely −20° C. group, −30° C. group, −40° C. group, −80° C. group, and −196° C. (liquid nitrogen) group.
Control group: take 1 mL of the ultra-pure platelet solution, and instead of performing the activation procedure in the step (S12′) of the embodiment 1-1, the steps (S13′)˜(S14′) of the embodiment 1-1 is performed directly to prepare dry powder of growth factors without the activation procedure; and then mix with 1 mL of water for injection, which is the control group solution.
−196° C. (liquid nitrogen) group: take 1 mL of the ultra-pure platelet solution, and perform the steps (S12′)˜(S14′) of Embodiment 1-1 to prepare the −196° C. (liquid nitrogen) group dry powder (that is, the growth factors-enriched dry powder in this invention).
−20° C. group: take 1 mL of the ultra-pure platelet solution, and not perform the activation procedure in the step (S12′) of the Embodiment 1-1; that is, the ultra-pure platelet solution is not placed in liquid nitrogen to freeze and then thaw. Instead, it is placed in a −20° C. refugirater to freeze and than thaw. Then, perform the steps (S13′) and (S14′) of Embodiment 1-1 to prepare the −20° C. dry powder.
−30° C. group: take 1 mL of the ultra-pure platelet solution, and not perform the activation procedure in the step (S12′) of the Embodiment 1-1; that is, the ultra-pure platelet solution is not placed in liquid nitrogen to freeze and then thaw. Instead, it is placed in a −30° C. refugirater to freeze and than thaw. Then, perform the steps (S13′) and (S14′) of Embodiment 1-1 to prepare the −30° C. dry powder.
−40° C. group: take 1 mL of the ultra-pure platelet solution, and not perform the activation procedure in the step (S12′) of the Embodiment 1-1; that is, the ultra-pure platelet solution is not placed in liquid nitrogen to freeze and then thaw. Instead, it is placed in a −40° C. refrigerator to freeze and than thaw. Then, perform the steps (S13′) and (S14′) of Embodiment 1-1 to prepare the −40° C. dry powder.
−80° C. group: take 1 mL of the ultra-pure platelet solution, and not perform the activation procedure in the step (S12′) of the Embodiment 1-1; that is, the ultra-pure platelet solution is not placed in liquid nitrogen to freeze and then thaw. Instead, it is placed in a −80° C. refrigerator to freeze and then thaw. Then, perform the steps (S13′) and (S14′) of Embodiment 1-1 to prepare the −80° C. dry powder.
Mix −20° C. dry powder, −30° C. dry powder, −40° C. dry powder, −80° C. dry powder, and −196° C. (liquid nitrogen) dry powder with 1 mL of water for injection respectively to prepare −20° C. dry powder solution, −30° C. dry powder solution, −40° C. dry powder solution, −80° C. dry powder solution, and −196° C. (liquid nitrogen) dry powder solution. Then the enzyme-linked immunosorbent assay (ELISA) is used to analyze the concentration of growth factors in each group of solutions, and the following formula is used to calculate the multiples of growth factors released from each group.
The multiple of growth factor released in the control group=(the growth factor concentrations of the solution in the control group×the volume of the solution in the control group)÷(the growth factor concentrations of the solution in the control group×the volume of the solution in the control group).
The multiple of growth factor released in the experimental group=(the growth factor concentrations of the solution in the experimental group×the volume of the solution in the experimental group)÷(the growth factor concentrations of the solution in the control group×the volume of the solution in the control group).
The experimental results are shown in Table 4 and
Please refer to
As the results are shown in Table 4 and
Human dermal fibroblast (HDF) (purchased from ScienCell Research Laboratories, Inc., product number 2320) is cultured in Dulbecco's Modified Eagle medium (DMEM) (Corning 10-013CV) containing 1% human platelet lysate (HPL), and then replaced with fresh medium for later use. The above-mentioned human dermal fibroblast (HDF) is adherent cell.
The 96-well plate is divided into untreated wells (control wells) and growth factors-enriched dry powder wells (RD2201 wells) that are prepared according to Experiment 1-1, with 2 replicates each. Human dermal fibroblasts (HDF) are added to untreated wells (control group wells) and RD2201 wells so that each well contains the same number of cells, and place in an incubator at 37° C. and 5% carbon dioxide (CO2) for 24 hours.
Aspirate the used medium. Mix 1 bottle of growth factors-enriched dry powder described in Embodiment 1-2 (each bottle contains 0.0030 grams (g) of growth factors-enriched dry powder on average) with 1 mL of DMEM culture medium (containing 1% HPL) to obtain a high-concentration mixed solution; and then mix the high-concentrated mixed solution with DMEM culture medium (containing 1% HPL) (the volume ratio of the high-concentration mixed solution to DMEM culture medium is 1:19) to prepare growth factors-enriched dry powder medium. Suspend the cells in RD2201 wells with the growth factors-enriched dry powder medium, and suspend the cells in untreated wells (control wells) with DMEM medium (containing 1% HPL). Incubate in an incubator with 5% carbon dioxide (CO2) at 37° C. for 24 hours.
Aspirate the used medium. Add 90 μL DMEM and 10 μL CCK-8 buffer (DOJINDO, product number CK04) to each well. Incubate in an incubator with 5% carbon dioxide (CO2) at 37° C. for 3 hours. The analysis is performed using a multimode microplate reader (model Varioskan LUX, Thermo Scientific brand) with an absorbance value set at 450 nm. The regression curve of absorbance value and cell number is established according to CCK-8 buffer product manual, and then the absorbance value is converted into the number of surviving cells.
Data are presented as mean±SD, and statistical analysis is performed by T TEST. Groups marked with “*” indicates statistical differences (p<0.05) from untreated wells (control group wells).
Please refer to
Utilize the same human dermal fibroblast (HDF) as Embodiment 4-1 to conduct the experiment. Human dermal fibroblast (HDF) is cultured in Dulbecco's Modified Eagle medium (DMEM) (Corning 10-013CV) containing 2% fetal bovine serum (FBS), and then replaced with fresh medium for later use.
A 96-well plate is divided into untreated wells (control group wells) and growth factors-enriched dry powder wells (RD2201 wells) with 3 replicates each; wherein the specimens of 3 replicates are derived from whole blood of three different human individuals. Human dermal fibroblasts (HDF) are added to untreated wells (control wells) and RD2201 wells, so that each well contains the same number of cells; then incubate in an incubator with 5% carbon dioxide (CO2) at 37° C. for 24 hours.
Aspirate the used medium. Mix one bottle of growth factors-enriched dry powder described in Embodiment 1-2 (each bottle contains 0.0030 grams (g) of growth factors-enriched dry powder on average) with 1 mL DMEM culture medium (containing 2% FBS) to obtain a high-concentration mixed solution; and then mix the high-concentrated mixed solution with DMEM culture medium (containing 2% FBS) (the volume ratio of the high-concentration mixed solution to DMEM culture medium is 1:19) to prepare growth factors-enriched dry powder medium. Suspend the cells in RD2201 wells with the growth factors-enriched dry powder medium, and suspend the cells in untreated wells (control wells) with DMEM culture medium (containing 2% FBS). Incubate in an incubator with 5% carbon dioxide (CO2) at 37° C. for 24 hours.
Transfer the cell culture medium from untreated wells (control group wells) and RD2201-treated wells into separate microcentrifuge tubes. Use interleukin-6 (IL-6, an inflammatory factor) ELISA kit (Human IL-6 ELISA Kit) (product number ab178013, Abcam brand) to detect the concentration of human interleukin-6 (Human IL-6) in the cell culture medium. Then the analysis is performed using a multimode microplate reader (model Varioskan LUX, Thermo Scientific brand) with an absorbance value set at 450 nm. The regression curve of the absorbance value and the concentration of the inflammatory factor IL-6 is established according to the product manual of the interleukin-6 (IL-6) ELISA kit, and then the absorbance value is converted into the concentration of the inflammatory factor IL-6; and use the following formula to calculate the release percentage of inflammatory factors in each group.
The release percentage of inflammatory factors in the control group=(the concentration of inflammatory factor IL-6 in the control group÷the concentration of inflammatory factor IL-6 in the control group)×100%.
The release percentage of inflammatory factors in the RD2201 group=(the concentration of the inflammatory factor IL-6 in the RD2201 group÷the concentration of the inflammatory factor IL-6 in the control group)×100%.
Data are presented as mean±SD, and statistical analysis is performed by T TEST. Groups marked with “*” indicates statistical differences (p<0.05) from untreated wells (control group wells).
Please refer to Table 5 and
Human dermal fibroblasts (HDF) require for this experiment are prepared according to the experimental procedure of Embodiment 4-2. A 96-well plate is divided into untreated wells (control group wells), growth factors-enriched dry powder wells (RD2201 wells), and plasma and RD2201 mixture wells with 3 replicates each; wherein the specimens of 3 replicates are derived from whole blood of three different human individuals. Adding human dermal fibroblasts (HDF) to untreated wells (control wells), RD2201 wells, and plasma and RD2201 mixture wells, so that each well contains the same number of cells; and incubate in an incubator with 5% carbon dioxide (CO2) at 37° C. for 24 hours.
Aspirate the used medium. Take a blood sample and perform the steps of embodiment 1-1 to obtain 2 bottles of growth factors-enriched dry powder with the same weight. Mix one bottle of growth factors-enriched dry powder with 1 mL of plasma (that is, the solution of plasma and plasma protein layer obtained in step S1123′) to prepare a growth factors-enriched plasma solution. Mix another bottle of growth factors-enriched dry powder with 1 mL of DMEM culture medium (containing 2% FBS) to prepare growth factors-enriched medium. Then each group then perform the following steps individually.
Untreated wells (control group wells): The cells in untreated wells (control wells) are suspended in DMEM culture medium (containing 2% FBS), and cultured in an incubator with 5% carbon dioxide (CO2) at 37° C. for 24 hours.
RD2201 wells: the growth factors-enriched culture medium is mixed with DMEM culture medium (containing 2% FBS) (the volume ratio of the growth factors-enriched culture medium to DMEM culture medium is 1:19) to obtain growth factors-enriched dry powder culture medium (the growth factors-enriched dry powder concentration is 1.5×10−4 g/mL), and the cells in the RD2201 wells are suspended with the growth factors-enriched dry powder culture medium, and the cells are cultured in an incubator with 5% carbon dioxide (CO2) at 37° C. for 24 hours.
Plasma and RD2201 mixture wells: the growth factors-enriched plasma solution is mixed with DMEM culture medium (containing 2% FBS) (the volume ratio of the growth factors-enriched plasma solution to DMEM culture medium is 1:19) to obtain growth factors-enriched dry powder and plasma culture medium (the dry powder concentration of growth factor is 1.5×10−4 g/mL), and use to suspend the cells in the plasma and RD2201 mixture wells; then culture in an incubator with 5% carbon dioxide (CO2) at 37° C. for 24 hours.
Transfer the cell culture medium from untreated wells (control group wells), RD2201 wells, and the plasma and RD2201 mixture wells into separate microcentrifuge tubes. Use interleukin-6 (IL-6, an inflammatory factor) ELISA kit (Human IL-6 ELISA Kit) (product number ab178013, Abcam brand) to detect the concentration of human interleukin-6 (Human IL-6) in the cell culture medium. Then the analysis is performed using a multimode microplate reader) (model Varioskan LUX, Thermo Scientific brand) with an absorbance value set at 450 nm. The regression curve of the absorbance value and the concentration of the inflammatory factor IL-6 is established according to the product manual of the interleukin-6 (IL-6) ELISA kit, and then the absorbance value is converted into the concentration of the inflammatory factor IL-6; and use the following formula to calculate the release percentage of inflammation in each group.
The release percentage of inflammatory factors in the control group=(the concentration of inflammatory factor IL-6 in the control group±the concentration of inflammatory factor IL-6 in the control group)×100%.
Release percentage of inflammatory factors in RD2201 group=(concentration of inflammatory factor IL-6 in RD2201 group±concentration of inflammatory factor IL-6 in control group)×100%.
The release percentage of inflammatory factors in the plasma and RD2201 mixture group=(the concentration of inflammatory factor IL-6 in the plasma and RD2201 mixture group÷the concentration of inflammatory factor IL-6 in the control group)×100%.
Data are presented as mean±SD, and statistical analysis is performed by T TEST. Group marked with “+” indicates statistical difference (p<0.05) from the growth factors-enriched dry powder (RD2201) well, and the group marked with “*” indicates statistical difference (p<0.05) from the untreated well (control group well).
Please refer to
The inventor expects to use stimulants (such as LPS) to induce inflammatory responses in other human tissues before conducting experiments, and similar results will be obtained; i.e., the step of removing plasma and plasma protein in present invention can make the product greatly relieve the induced inflammatory reaction, and then relieve the inflammation of the tissue.
Human dermal fibroblasts require for this experiment are prepared according to the experimental procedure of Embodiment 4-2. The 96-well plate is divided into untreated wells (control group wells), growth factors-enriched dry powder wells (RD2201 wells), and growth factors-enriched solution wells (RD2201 without freeze-drying wells) with 3 replicates each; wherein the specimens of 3 replicates are derived from the whole blood of three different human individuals. Human dermal fibroblasts (HDF) are added to untreated wells (control group), RD2201 wells, and RD2201 without freeze-drying wells, so that each well contained the same number of cells, and incubate in an incubator with 5% carbon dioxide (CO2) at 37° C. for 24 hours.
Aspirate the used medium. The steps (S11′)˜(S13′) of Embodiment 1-1 are performed to obtain a growth factors-enriched solution. Each group then perform the following steps individually.
Untreated wells (control group wells): Suspend the cells in untreated wells (control wells) with DMEM culture medium (containing 2% FBS), and culture them in an incubator with 5% carbon dioxide (CO2) at 37° C. for 24 hours.
RD2201 wells: Take 1 mL of the growth factors-enriched solution, and perform the step (S14′) of Embodiment 1-1 to obtain a growth factors-enriched dry powder. Mix the growth factors-enriched dry powder with 1 mL of water for injection to prepare a growth factors-enriched dry powder solution. Mix the growth factors-enriched dry powder solution with DMEM culture medium (containing 2% FBS) to obtain a growth factors-enriched dry powder culture medium (the dry powder concentration of the growth factor is 1.5×10−4 g/mL), and suspend the cells in RD2201 wells with the growth factors-enriched dry powder culture medium, then culture them in an incubator with 5% carbon dioxide (CO2) at 37° C. for 24 hours.
RD2201 without freeze-drying wells: Mix 1 mL of growth factors-enriched solution with DMEM culture medium (containing 2% FBS) to obtain a mixture (the dry powder concentration of the growth factor is 1.5×10−4 g/mL), and suspend the cells in the RD2201 without freeze-drying wells are cultured in an incubator with 5% carbon dioxide (CO2) at 37° C. for 24 hours.
Transfer the cell culture medium in untreated wells (control group wells), RD2201 wells, and RD2201 without freeze-drying wells into microcentrifuge tubes respectively. Use interleukin-6 (IL-6, an inflammatory factor) ELISA kit (Human IL-6 ELISA Kit) (product number ab178013, Abcam brand) to detect the concentration of human interleukin-6 (Human IL-6) in the cell culture medium. Then the analysis is performed using a multimode microplate reader (model Varioskan LUX, Thermo Scientific brand) with an absorbance value set at 450 nm. The regression curve of the absorbance value and the concentration of the inflammatory factor IL-6 is established according to the product manual of the interleukin-6 (IL-6) ELISA kit, and then the absorbance value is converted into the concentration of the inflammatory factor IL-6; and use the following formula to calculate the release percentage of inflammatory factors in each group.
The release percentage of inflammatory factors in the control group=(the concentration of inflammatory factor IL-6 in the control group÷the concentration of inflammatory factor IL-6 in the control group)×100%.
The release percentage of inflammatory factors in RD2201 group=(concentration of inflammatory factor IL-6 in RD2201 group÷concentration of inflammatory factor IL-6 in control group)×100%.
The release percentage of inflammatory factors in the RD2201 without freeze-drying group=(the concentration of inflammatory factor IL-6 in the RD2201 without freeze-drying group÷the concentration of inflammatory factor IL-6 in the control group)×100%.
Data are presented as mean±SD, and statistical analysis is performed by T TEST. Groups marked with “*” indicates statistical differences (p<0.05) from untreated wells (control group wells).
Please refer to Table 7 and
The inventor expects to use stimulants (such as LPS) to induce inflammatory responses in other human tissues before conducting experiments, and similar results will be obtained; i.e., the step of freeze-drying in present invention can make the product greatly relieve the induced inflammatory response, thereby relieving the inflammation of the tissue.
In this embodiment, the platelet concentration is divided into low concentration group, medium concentration group and high concentration group. Performed the step (S11′) of Embodiment 1-1 to prepare a ultra-pure platelet solution, and the platelet concentration in the ultra-pure platelet solution is 1×109/mL. Then, each group of dry powders is prepared in the following steps separately.
High concentration group: Take 10 mL of the ultra-pure platelet solution, and the ultra-pure platelet solution is centrifuged (1,000˜2,500 g, 5 minutes) using a centrifuge to allow platelets to settle and form a pellet. Remove the supernatant, and then add 1 mL of water for injection to obtain a ultra-pure platelet solution of the high concentration group (platelet concentration is 10×109/mL). Then, perform the steps (S12′)˜(S14′) of Embodiment 1-1 to prepare a high concentration group dry powder.
Medium concentration group: Take 10 mL of the ultra-pure platelet solution, and the ultra-pure platelet solution is centrifuged (1,000˜2,500 g, 5 minutes) using a centrifuge to allow platelets to settle and form a pellet. Aspirate the supernatant, and then add 2 mL of water for injection to obtain a ultra-pure platelet solution of the medium concentration group (platelet concentration is 5×109/mL). Then, perform the steps (S12′)˜(S14′) of Embodiment 1-1 to prepare a medium concentration group dry powder.
Low concentration group: Take 10 mL of the ultra-pure platelet solution, and the ultra-pure platelet solution is centrifuged (1,000˜2,500 g, 5 minutes) using a centrifuge to allow platelets to settle and form a pellet. Aspirate the supernatant, and then add 3 mL of water for injection to obtain a ultra-pure platelet solution of the low concentration group (platelet concentration is 3.3×109/mL). Then, perform the steps (S12′)˜(S14′) of Embodiment 1-1 to prepare a low concentration group dry powder.
Mix the high concentration group dry powder, the medium concentration group dry powder, and the low concentration group dry powder with 1, 2 and 3 mL of water for injection respectively to obtain a high concentration group solution, a medium concentration group solution, and a low concentration group solution. Then, the concentration of growth factors in the three groups are analyzed by enzyme-linked immunosorbent assay (ELISA), and the total amount of growth factors released in each group is calculated by the following formula.
The total amount of growth factors released in the high concentration group=the concentration of growth factors in the high concentration group solution×the volume of the high concentration group solution.
The total amount of growth factors released in the medium concentration group=the concentration of growth factor in the medium concentration group solution×the volume of the medium concentration group solution.
The total amount of growth factors released in the low concentration group=the concentration of growth factors in the low concentration group solution×the volume of the low concentration group solution.
Please refer to
The above description is only a preferred embodiment of the present invention and is not intended to limit the scope of the present invention. Therefore, all other changes or modifications shall be included within the scope of the patent application of the present invention without departing from the spirit of the invention as disclosed herein.
| Number | Date | Country | Kind |
|---|---|---|---|
| 22179457.1 | Jun 2022 | EP | regional |
| 111150685 | Dec 2022 | TW | national |
