Patent Application
20140314867
Osteonecrosis, i.e., avascular necrosis of bone, is the death of bone cells caused by decreased blood flow to these cells. According to statistics, there are approximately 300,000 to 600,000 cases that occur in the US every year, and it usually affects people between 20 and 50 years of age.
In recent years, many studies have pointed out that vitamin K2 can effectively inhibit osteoclast activity, which disassembles bone tissues, and promote bone regeneration. Further; vitamin K2 can induce osteoblast cells, which synthesize bone, to differentiate bone cells at the osteonecrosis site from those at normal sites, and help repair the injured bone tissue (e.g., necrosis). It can also avoid the disadvantage of the hard-to-control growth factor activity. Therefore, vitamin K2 is useful in treating osteoporosis.
However, due to conventional in vivo delivery routes (e.g., oral), the efficacy of vitamin K2 can be maintained only for a short time as it is metabolized very fast in the body, requiring inconvenient multiple dosages in a day. Moreover, to achieve therapeutic effects, vitamin K2 is taken at a high dosage, causing various side effects.
There is a need to develop a system that can release vitamin K2 in an extended, controllable manner and at a low dose.
The invention is based on an unexpected discovery of a vitamin K2 microsphere capable of releasing vitamin K2 in a controlled manner.
One aspect of this invention relates to a vitamin K2 microsphere that contains a particle formed of a poly(lactic-co-glycolic acid) (PLGA) and vitamin K2. The PLGA, the viscosity of which can be 0.1-3 dl/g, has a molecular weight of 1000-300000 and contains lactic acid repeat units and glycolic acid repeat units. The molar ratio between the lactic acid repeat units and the glycolic acid repeat units is 1-9:9-1. Vitamin K2 is embedded in the particle and constitutes 0.005-75% by weight of the vitamin K2 microsphere.
The vitamin K2 microsphere can have a particle size of 1-150 μm and contains vitamin K2 in the amount of 0.01-0.3 mg.
Another aspect of this invention relates to a method of preparing the vitamin K2 microsphere described above. The method includes the steps of: (a) providing a vitamin K2 solution that contains vitamin K2, the PLGA mentioned above, and a first solvent; (b) providing a polyvinyl alcohol (PVA) solution that contains polyvinyl alcohol and a second solvent; (c) forming a vitamin K2 emulsion by mixing the vitamin K2 solution with the PVA solution; and (d) removing the first and second solvents to obtain vitamin K2 microspheres, each of which contains the PLGA and vitamin K2 embedded in a particle formed of the PLGA.
The first solvent is an organic solvent, e.g., dichloromethane, chloroform, tetrahydrofuran, dimethylformamide, benzene, toluene, or a combination thereof. The weight/volume ratio between vitamin K2 and the first solvent is 0.005-75%. The second solvent is water.
The vitamin K2 microspheres thus obtained can be purified via filtration or centrifugation.
Optionally, a plasticizer is added at step (c) to mix with the vitamin K2 solution and the PVA solution to form the vitamin K2 emulsion.
Also within the scope of this invention is a method of treating osteoporosis by administering to a subject in need thereof an effective amount of the above-described vitamin K2 microsphere.
Still within the scope of this invention is a pharmaceutical composition containing this vitamin K2 microsphere and a pharmaceutically acceptable carrier.
This invention further includes use of the vitamin K2 microsphere in the manufacture of a medicament for treating osteoporosis or for repairing damaged bone tissues.
The details of one or more embodiments of the invention are set forth in the description below. Other features, objects, and advantages of the invention will be apparent from the description and the figures, and from the claims.
The vitamin K2 microsphere of this invention contains a particle formed of PLGA, a biodegradable polymer, and vitamin K2 that is embedded in the particle. As the polymer degrades, vitamin K2 is slowly released in a controlled manner. The vitamin K2 microsphere is useful for bone tissue regeneration.
The particle size distribution of the vitamin K2 microsphere can be between 1 μm and 150 μm, for example, between 2 μm and 100 μm. The particle size of the vitamin K2 microsphere varies depending on the amount of encapsulated vitamin K2. Generally, the particle size increases when the concentration of vitamin K2 increases. The microsphere slowly releases vitamin K2 into the bone cell growth environment to promote bone formation and help bone tissue reconstruction. Accordingly, to achieve delayed release, it is preferred that the particle size of the microsphere be controlled in an appropriate range. When the particle size is too small, microspheres may not stay at a target site. When the particle size is too large, controlled drug release may not be achieved. As such, the particle size of the microsphere is critical. The vitamin K2 microsphere of this invention can be prepared by a nonaqueous phase separation method. Known nonaqueous phase separation methods include non-solvent phase precipitation, temperature dropping, solvent distillation, and a combination thereof. See Gast et al., J. of Colloid and Interface Science 1983, 96, 251-67. Non-solvent phase precipitation and solvent distillation can be used in combination to prepare the vitamin K2 microsphere.
The vitamin K2 microsphere can contain vitamin K2 in the amount of 0.01-0.3 mg. The viscosity of the PLGA can be 0.1-3 dl/g (e.g., 0.14-0.22 dl/g).
More specifically, vitamin K2 (e.g., 0.001-0.1 g) and PLGA (e.g., 0.0013-200 g) are dissolved in a solvent (e.g., dichloromethane) by stirring in an ice bath. The weight/volume ratio of the vitamin K2 and PLGA can be 0.005-75% (e.g., 0.01-1%).
Subsequently, in step 24 shown in
Next, in step 26 shown in
In Step 28 shown in
Not to be bound by any of the theory, PLGA can encapsulate different amounts of vitamin K2, which is then released in a controllable manner into a target site at a desired concentration. By encapsulating different amounts of vitamin K2, the size of the microspheres can be tuned, along with the drug release rates. A skilled person in the art can determine the amount of vitamin K2 and the size of the microsphere for different applications in various bone healing situations.
Also within the scope of this invention is a pharmaceutical composition that contains the vitamin K2 microsphere described above and a pharmaceutically acceptable carrier including water, ethanol, and glycerol. The weight/volume ratio of the vitamin K2 microsphere and the pharmaceutical acceptable carrier can be 0.005-75%.
The carrier in the pharmaceutical composition must be “acceptable” in the sense that it is compatible with the vitamin K2 microsphere (and preferably, capable of stabilizing the microsphere) and not deleterious to the subject to be treated. One or more solubilizing agents can be utilized as pharmaceutical excipients for delivery of the vitamin K2 microsphere.
Further disclosed is use of the vitamin K2 microsphere thus prepared for the manufacture of a medicament to treat osteoporosis or repair damaged bone tissue.
Moreover, this invention covers a method of administering an effective amount of the vitamin K2 microspheres described above to a patient in need thereof. “An effective amount” refers to the amount of the vitamin K2 microspheres that is required to confer a therapeutic effect on the treated subject. Effective amounts, as recognized by those skilled in the art, depend upon the diseases to be treated, the route of administration, the excipient, and the possibility of co-usage with other therapeutic treatment.
To practice the method of the present invention, a composition having the above-described vitamin K2 microspheres can be administered parenterally. The term “parenteral” as used herein refers to subcutaneous, intracutaneous, intravenous, intrmuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional, or intracranial injection, as well as any suitable infusion technique.
A sterile injectable composition can be a solution or suspension in a non-toxic parenterally acceptable diluent or solvent, such as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that can be employed are mannitol, water, Ringer's solution, and isotonic sodium chloride solution. In addition, fixed oils are conventionally employed as a solvent or suspending medium (e.g., synthetic mono- or diglycerides). Fatty acid, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions can also contain a long chain alcohol diluent or dispersant, carboxymethyl cellulose, or similar dispersing agents. Other commonly used surfactants such as Tweens or Spans or other similar emulsifying agents or bioavailability enhancers which are commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms can also be used for the purpose of formulation.
The specific examples below are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. Without further elaboration, it is believed that one skilled in the art can, based on the description herein, utilize the present invention to its fullest extent. All publications cited herein are hereby incorporated by reference in their entirety.
Examples below illustrate surface morphology observations, particle size distributions, physical and chemical property analysis, and drug release tests of different concentrations of vitamin K2 microspheres (VK2MSs). Further, MG-63 cells are co-cultured with cell culture medium containing different concentrations of vitamin K2 microspheres (VK2MSs). The effects on the cell activity of vitamin K2 microspheres (VK2MSs) are observed by methods including cell viability assay (MTT assay), alkaline phosphatase activity assay (ALP activity assay) and immunohistochemical staining to find the most suitable vitamin K2 microspheres (VK2MSs) for applying to bone tissue repair engineering.
Microspheres were prepared by the oil-in-water (O/W) emulsion nonaqueous phase separation method. 1.2 g of PVA was added to 60 ml of water and stirred under 100° C. for 30 minutes to obtain a 2% PVA solution. 0.001 g, 0.01 g, and 0.1 g of Vitamin K2 (VK2) and 0.2 g of PLGA were dissolved in 10 ml of dichloromethane and stirred in an ice bath at 1000 rpm for 10 minutes to form 0.01%, 0.1%, and 1% of VK2 solutions.
PVA solution was poured into a 100 ml beaker in an ice bath. 10 ml of the VK2 solution was slowly dropped into the PVA solution, stirred at 3200 rpm by a homogenizer, and then stirred at 5000 rpm for 10 minutes. Stirring was conducted in the hood at room temperature for 24 hours to remove dichloromethane. Then, larger microspheres were filtered by a cell sieve with 100 μm pore size. The obtained filtrate was poured into a 50 ml centrifuge tube and centrifuged at 2500 rpm for 10 minutes. After that, fresh deionized water was added.
The washing step using deionized water was repeated 4 times. Then, the VK2 solution was poured into a microcentrifuge tube, rapidly cooled down by liquid nitrogen (N2(I)), and dried by a freeze dryer for 24 hours. The obtained microspheres encapsulating VK2 were abbreviated as VK2MS. The product was weighed by an electronic microbalance to calculate the yield, stored in a drying cabinet and prepared for use.
After the microspheres prepared by the oil in water (O/W) emulsion nonaqueous phase separation method were weighed by an electronic microbalance, an optical microscope (OM) and scanning electron microscope (SEM) were used to observe the surface morphology of the microspheres. Attenuated total reflectance-fourier transform infrared spectrometry (ATR-FTIR) was used to determine whether VK2 was embedded in PLGA, A laser scattering particle size distribution analyzer (LS) was used to measure the particle size distribution. An ultraviolet-visible spectrophotometer (UV/Vis) was used to analyze the different encapsulation effects of the microspheres with different dosages embedded.
Surface Morphology Observation the of the Microspheres
After dichloromethane was removed, 0.5 ml of a solution containing microspheres was added into a microcentrifuge tube, and then 20 μl of that was dropped onto a hemocytometer and observed with an optical microscope (OM). The VK2MS containing different concentrations of VK2 were spherical, and uniformly dispersed without aggregation. Therefore, microspheres of uniform size were successfully formed by the oil in water (O/W) emulsion solvent distilling method.
The freeze-dried microspheres were gently placed on a conductive tape, and those that did not adhere to the conductive tape were removed by a blowing ball. Then, after the surface of microspheres was platinized by an ion sputter by 15 mA for 3 minutes, the surface morphology was observed with a scanning electron microscope (SEM), as shown in
Qualitative Analysis of the Microspheres
To determine whether the VK2 was successfully embedded in PLGA and whether the surfactant of PVA was removed clearly, attenuated total reflectance-fourier transform infrared spectroscopy (ATR-FTIR) was used to confirm the existence of functional groups of VK2 and PVA in the VK2MS. The freeze-dried microspheres were observed by ATR-FTIR to observe the absorption spectrum of VK2MS. (Number of scans: 128, Resolution: 8, Wavenumber: 4000-650 cm−1)
Generally, PLGA has —OH stretching vibration at 3200-3500 cm−1, —CH stretching at 2850-3000 cm−1, —C═O stretching vibration at 1700-1800 cm—1, and C—O stretching at 1050-1250 cm−1. PVA has a broader O—H stretching vibration at 3100-3400 cm−1, and C—H stretching vibration at 2930 cm−1. VK2 has C═C stretching vibration at 1500-1600 cm−1, C═O stretching at 1690-1760 cm−1, and C—H stretching vibration at 3010-3100 cm−1.
As shown in
Since cell experiments have to be operated under sterility, the materials were co-cultured with cells after sterilization by UV light. However, to prevent chemical reactions from occurring after the UV irradiation, the materials irradiated by UV light were analyzed by ATR-FTIR to determine whether the positions of the functional groups had changed, as shown in
According to
Particle Size Analysis of the Microspheres
5 mg of microspheres were added into 5 ml of deionized water and oscillated in the ultrasonic oscillator to uniformly disperse the microspheres. Then, a laser scattering particle size distribution analyzer (LS) was used to analyze the particle size distribution of the microspheres and calculate the span.
As illustrated in
According to Table 1, it seems that the particle size is related to the content of VK2. The average particle size increased with the increasing encapsulated amount of vitamin K2 in the microspheres. The largest particle size was 5.9±4.0 μm of 1.0%
VK2MS, which was consistent to the observation of SEM. In this particle size range, the microspheres neither fall out of the bracket when placed in tissue engineering scaffolds since the particle size is too small nor affect the uniform drug release to result in difficulty controlling the drug release due to the particle size being too large.
Preparation of Phosphate Buffer Saline (PBS)
8 g of NaCl, 0.2 g of KCl, 2.16 g of Na2HPO4, 0.2 g of KH2PO4, and 1000 ml of water were added into a bottle and stirred until completely dissolved. Then, the pH value of a solution was adjusted to 7.4. The bottle was autoclaved at 115° C. for 30 minutes and then cooled down under room temperature.
Degradation Experiments of the Microspheres
0.01 g of freeze-dried 0%, 0.01%, 0.1%, and 1.0% VK2MS were added into 15 ml centrifuge tubes, 3 ml PBS was added, then the centrifuge tubes were placed in a 37° C. water bath for drug release for 0, 14, 28, 42, 56, and 70 days. 30 minutes before the sampling time, the centrifuge tube rack was taken from the water bath and left to stand for 30 minutes to precipitate the microspheres. PBS was removed from the centrifuge tubes and the microspheres were placed into microcentrifuge tubes and solidified in a −20° C. refrigerator for 2 hours. After being dried by a freeze dryer for 24 hours, the product was weighed by an electronic microbalance.
In the present disclosure, VK2 drug was dispersed into a biodegradable polymer matrix, and could diffuse from the matrix or be released by polymer dissolution. Accordingly, the degradation rate of polymer had a great impact on the drug release rate.
In
VK2 Drug Release
0.01 g of freeze-dried 0%, 0.01%, 0.1%, and 1.0% VK2MS were added into 15 ml centrifuge tubes, 3 ml PBS was added, then the centrifuge tubes were placed in a 37° C. water bath for drug release for 0, 1, 3, 7, 14, 21, 28, 35, 42, 49, 56, 63, and 70 days. 30 minutes before the sampling time, the centrifuge tube rack was taken from the water bath and standing for 30 minutes to precipitate the microspheres. Then, 2.5 ml of the supernatant was suctioned from the centrifuge tube to a 20 ml glass vial and 2.5 ml of fresh PBS was supplied into the centrifuge tube to maintain the solution volume at 3 ml. Then, the centrifuge tubes were placed back to the 37° C. water bath.
20 ml of glass vial containing 2.5 ml of different supernatants were placed in a −20° C. refrigerator to solidify for 2 hours. After dried by a freeze dryer for 24 hours, 5 ml of dichloromethane was added into a 20 ml glass vial and stirred at 600 rpm for 10 minutes. Then, the sample was suctioned by a glass syringe to a 0.45 μm-syringe filter. The obtained filtrate was observed by an ultraviolet-visible spectrophotometer (UVIVis) at 320 nm to analyze the content of VK2.
The cumulative release amount of VK2MS encapsulated with different concentrations of VK2 is illustrated in
The cumulative release percentage can be obtained by dividing the cumulative drug release amount by the theoretical drug-coated amount, as shown in
Preparation of High Glucose-Dulbecco's Modified Eagle Medium (H-DMEM) Culture Medium
Bottles containing deionized water, beakers, and stir bars were autoclaved at 115° C. for 30 minutes for sterilization. 3.75 g of sodium bicarbonate was fine weighed and prepared for use. H-DMWM powder was poured in a 1000 ml beaker containing 900 ml deionized water in a sterile hood. After the solution was uniformly stirred by a stir bar, 3.75 g of sodium bicarbonate was added and stirred until completely dissolved.
A pH meter was used to measure the pH value of the solution, and the pH value was adjusted to 7.26 by accessing CO2. H-DMEM solution was filtered by 0.22 μm sterile filtration equipment and poured into the sterilized bottle. Then, 100 ml of fetal bovine serum (FBS), which was deactivated at 56° C. for 30 minutes and 10 ml of PSA, were added. The sealed bottle was kept in a 4° C. refrigerator. The product was H-DMEM culture medium containing 10% FBS.
MG-63 Cells Culture and Sub-Culture
A human osteosarcoma cell line (osteoblast-like cell line) MG-63, purchased from Food Industry Research and Development Institute (FRIDI) was used in the present disclosure.
H-DMEM containing 10% FBS was used to adjust the concentration of MG-63 cell solution to 1×105 cells/ml. 5 ml of cell solution was added into a T-25 flask and incubated in an incubator at 37° C., 5% CO2, and 95% R.H. H-DMEM containing 10% FBS was changed every two days. An inverted microscope (Olympus, CKX31) was used to observe the growth of MG-63 cells. When MG-63 cells were about 80%-full in the flask, MG-63 cells could be sub-cultured. The cell passage of MG-63 cells was 8-24.
Preparation of Cell Culture Medium with Different VK2 Concentration
0.0001 g, 0.0005 g, 0.005 g, and 0.05 g of VK2 was fine weighed and sterilized by UV light in a sterile hood overnight, then added into 50 ml centrifuge tubes. 50 ml of H-DMEM containing 10% FBS was then added to prepare cell culture medium with different VK2 concentrations: 0 mg/mL, 0.002 mg/mL, 0.01 mg/mL, 0.1 mg/mL, and 1 mg/mL.
Co-Culture Cells and Cell Culture Medium Containing Different VK2 Concentration
H-DMEM containing 10% FBS was used to adjust the concentration of MG-63 cell solution to 1×104 cells/ml. 1 ml/well of cell solution was seeded to a 24-well plate and incubated in an incubator for 1 day to make cells adhere. Then, the culture medium was removed, 1 ml/well of cell culture medium with different VK2 concentration were added respectively and incubated for 1, 3, and 7 days. Cell viability analysis (MTT) and alkaline phosphatase activity assay (ALP) were conducted. Cell morphology was observed by an inverted microscope and recorded in photographs.
Preparation of Cell Culture Medium with Different VK2MS
0.01 g of 0%, 0.01%, 0.1%, and 1.0% VK2MS was fine weighed and sterilized by UV light in a sterile hood overnight, then added into 50 ml centrifuge tubes. 50 ml of H-DMEM containing 10% FBS was then added to prepare cell culture medium with different VK2 concentrations.
Implant Cell Culture Medium Containing VK2MS with Different VK2 Concentration Into Cells
H-DMEM containing 10% FBS was used to adjust the concentration of MG-63 cell solution to 1×104 cells/ml. 1 ml/well of cell solution was seeded to a 24-well plate and incubated in an incubator for 1 day to make the cells adhere. Then, the culture medium was removed, 1 ml/well of cell culture medium with different VK2 concentration were added respectively and incubated for 1, 3, and 7 days. In addition, the cell culture medium without microspheres (MS) was added 1 ml/well and incubated for 1, 3, and 7 days as control groups. Subsequently, cell viability analyse (MTT) and alkaline phosphatase activity assays (ALP) were conducted. Cell morphology was observed by an inverted microscope and recorded by photographs.
Moreover, H-DMEM was used to adjust the concentration of MG-63 cell solution to 1×104 cells/ml. 1 ml/well of cell solution was seeded to a 24-well plate and incubated in an incubator for 1 day to make cells adhere. Then, the culture medium was removed, 1 ml/well of cell culture medium containing microspheres with different VK2 concentration were added respectively and incubated for 1, 3, and 7 days. Then, histochemical staining analysis was conducted by H&E, Von Kossa, and Alizarin red.
Cell Viability Analysis
MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) is a tetrazolium salt which reveals mitochondrial dehydrogenase of live cells and capable of reducing mitochondrial dehydrogenase of live cells to blue formazan crystals. When the cell number increases or the cell proliferation is good, the vigorous function of cellular mitochondria results in increasing mitochondrial dehydrogenase. Accordingly, blue formazan crystals formed after a reaction with MTT also increases, which can be a quantitative basis of the cellular mitochondria activity.
First, MTT was prepared as a 5 mg/mL reaction solution by PBS, then filtered by a 0.22 μm filter. 10% MTT reagent was prepared by H-DMEM, and then stored in the dark. After being cultured for 1 day, the cell culture medium was removed from the 24-well cell culture plate. The 24-well cell culture plate was washed twice by PBS. After the PBS was removed, 1 ml/well of 10% MTT reagent was added to the culture plate and reacted in an incubator (37° C., 5% CO2) in the dark for 4-5 hours. Purple crystal particles were produced at the bottom of culture plate after the reaction. The reactive solution was removed and 350 μl/well of DMSO solution was added. After being mixed, 1000/well of dissolved solution was suctioned to a 96-well plate and the absorbance at 570 nm was detected by an ELISA reader. The reference wavelength was set as 650 nm.
A cell standard curve can be made to reckon the cell number. The cell solution was adjusted to have a concentration of 5000, 10000, 25000, 50000, 75000, and 100000 cells/ml by H-DMEM containing 10% FBS then seeded into a 24-well plate, respectively. After 1 day, after the cells were adhered, an MTT cell viability analysis was conducted. Absorbance and cell numbers were used to make a standard curve, such that the cell number could be reckoned from the absorbance.
A growth curve of MG-63 cells was made first, and then the cell growth rate could be obtained by dividing the cell viability by that of the control group which was detected by the MTT assay. The result shown in
Similarly, the result in
Alkaline Phosphatase Activity Test
Alkaline phosphatase (ALP) is a glycoprotein compiled by many gene groups. Many scholars believe that ALP facilitates hydrolysis of phosphomonoesters and releases phosphate ions, which in turn induce the mineralization of matrix outside osteoblasts, i.e., bone-forming cells. As such, the activity of alkaline phosphatase (ALP) is used as a biological indicator for the activity of osteoblasts and the basis of bone cell differentiation.
The measurement of ALP is described below. After being cultured for 1 day, the cell culture medium was removed from the 24-well cell culture plate. Then, the 24-well cell culture plate was washed twice by PBS. After the PBS was removed, 200 μl/well of ALP extraction reagent pNPP65 (p-Nitrophenylphosphate) was added to the 24-well culture plate and reacted in an incubator (37° C., 5% CO2) in the dark for 30 minutes. Then, 50 μl of 1N sodium hydroxide was added to stop the reaction. 250 μl/well of supernatant was suctioned to a 96-well plate to detect the OD value at 405 nm by an ELISA reader. Activity of alkaline phosphatase (ALP) can be calculated by the formula below:
In this formula, A is the absorbance of a sample at 405 nm; Vt is the total reaction volume, 0.25 ml; Vs is the sample volume, 0.05 ml; t is the reaction time after adding pNPP65 (p-Nitrophenylphosphate), 30 minutes; c is mmol extinction coefficient of pNPP65 (p-Nitrophenylphosphate), 18.6 mM−1 cm−1; 1 is the optical path of the cuvette, 1 cm; 1000 is to convert the U/ml to U/l.
Alkaline phosphatase (ALP) is regarded as biological indicators of the activity of osteoblasts and the basis of bone cell differentiation. According to
Alkaline phosphatase activity of a single cell can be obtained by dividing the detected ALP activity by the cell number, as shown in
In
Similarly, alkaline phosphatase activity of a single cell can be obtained by dividing the ALP activity by the cell number, as shown in
To sum up the above, the concentration of VK2 released from VK2MS will affect the MG-63 cell growth rate and the differentiation of the activity of ALP. The higher concentration of VK2 released, the more obvious that cell growth rate is inhibited; however, the activity of differentiating ALP of cells is enhanced. Consequently, the growth characteristics of cells are affected by the changes of VK2 concentration released to the culture medium by degradation of VK2MS.
Alizarin Red S Stain
Alizarin red S, which appears bright red when combined with calcium, is a red dye usually used in histochemical staining to determine whether calcium deposits. Also, it is usually used as a basis for determining the mineral deposition of mineralization nodules formed by accumulation of osteoblasts.
First, 50 mL of deionized water was added into a 50 mL centrifuge tube containing 2 g of paraformaldehyde to prepare a 4% (w/v) paraformaldehyde solution. 50 mL of deionized water was added into a 50 ml centrifuge tube containing 1 g of alizarin red S to prepare a 2% (w/v) alizarin red S solution.
A cell culture medium in wells was suctioned. The wells were washed three times by PBS. After the PBS was suctioned, a 4% paraformaldehyde solution was added and reacted for 30 minutes to fix cells and then was suctioned. The wells were washed three times for 5 minutes each time with deionized water and then suctioned. 2% alizarin red S solution was added for 10 minutes and then suctioned. Washed three times by deionized water, 5 minutes for each time, then deionized water was suctioned. The results of the staining were observed under an inverted microscope and saved in photographs.
It can be found from the experimental results that when MG-63 cells were co-cultured with VK2MS, more bright red calcium was deposited. Moreover, the calcium deposition increased not only with the increasing loading of VK2, but also with increasing days.
Hematoxylin & Eosin Stain
Hematoxylin & Eosin stain is a routine staining, which uses two colouring agents of hematoxylin and eosin to distinguish cytoplasm and cell nucleus. Hematoxylin is a basic dye that specifically used to stain cell nucleus and basophilic cells, and appears violet after combined with nucleic acid in the cell nucleus. Eosin is an acid dye that specifically used to stain cytoplasm and acidophil cells, and appears pink after combined with protein in the cytoplasm.
First, 50 ml of deionized water was added into a 50 ml centrifuge tube containing 0.25 g of eosin to prepare a 0.5% (w/v) eosin solution. Every time before used, the pH value of solution has to be adjusted to 4.1-4.3 by glacial acetic acid.
A cell culture medium in wells was suctioned. The wells were washed three times by PBS. After the PBS was suctioned, a 4% paraformaldehyde solution was added and reacted for 30 minutes and then was suctioned. The wells were washed three times by deionized water for 5 minutes each time and then deionized water was suctioned. Harris alum hematoxylin was added for 5 minutes and then suctioned. Sample was washed by deionized water until bright violet appeared, then deionized water was suctioned. Then, sample was soaked in 0.5% eosin solution for 5 minutes (dipped in alcohol when the color was too dark). Washed three times by deionized water for 5 minutes each time and then deionized water was suctioned. The results of the staining were observed under an inverted microscope and saved in photographs.
It can be found from the experimental results that the cell number of control group is higher with more violet cell nucleus and pink cytoplasm. Moreover, the number of color increased with time. With respect to the co-culture of MG-63 cells and VK2MS, the cell number decreased with the increasing concentration of VK2. In addition, the cell number of 1.0% VK2MS group was apparently fewer on day 7.
Von Kossa Stain
Since most of the calcium ions within tissues present in the form of calcium phosphate or calcium carbonate, this method replaces calcium ions with silver ions from a silver nitrate solution to form silver phosphate or silver carbonate. By the reduction reaction of silver ions, silver phosphate or silver carbonate appears to have a brownish-black color, such that the existence of calcium phosphate or calcium carbonate can be proven. Cells appear to have a pink color.
50 ml of deionized water was added into a 50 ml centrifuge tube containing 2.5 g of AgNO3 to prepare a 5% (w/v) silver nitrate solution. 50 ml of deionized water was added into a 50 ml centrifuge tube containing 2.5 g of sodium thiosulfate to prepare a 5% (w/v) sodium thiosulfate solution.
A cell culture medium in wells was suctioned. The wells were washed three times by PBS. After the PBS was suctioned, a 4% paraformaldehyde solution was added and reacted for 30 minutes and then suctioned. The wells were washed three times by deionized water for 5 minutes each time and then deionized water was suctioned. After 5% sodium thiosulfate solution was added, sample was irradiated by UV light for 1 hour to convert Ca2+ to Ag+. Washed three times by deionized water. 5% sodium thiosulfate solution was added for 5 minutes to remove the remaining silver nitrate. After being washed three times by deionized water, nuclear fast red was added for 5 minutes as a comparison. Washed three times by deionized water for 5 minutes each time and then deionized water was suctioned. The results of the staining were observed under an inverted microscope and saved in photographs.
It can be found from the experimental results that when MG-63 cells were co-cultured with VK2MS, more brownish-black calcium was deposited. Moreover, the number of color increased with time, which was consistent with the results shown in
The results shown in histochemical staining are the same as that of alkaline phosphatase activity assay. For example, using the alizarin red S stain, more bright red calcium ions were deposited in cells when VK2MS was added; using an H&E stain, when VK2MS was added, cell proliferation was inhibited and the number of violet cell nuclei and pink cytoplasms decreased; using a Von Kossa stain, more brownish black calcium was deposited in cells when VK2MS was added, while the number of pink-cell nuclei apparently decreased.
The present disclosure successfully prepares VK2MS with 0%, 0.01%, 0.1%, and 1.0% of VK2 encapsulated by biodegradable polymer PLGA which is formed by the oil in water (O/W) emulsion nonaqueous phase separation method. The VK2MS has a sleek spherical appearance without aggregation. The highest production of 1.0 VK2MS reaches 80.8±6.9%, and the best encapsulation efficacy reaches 92.8±5.2%. The microspheres have an uniform particle-size distribution and an average particle size between 1 μm and 150 μm.
By the in vitro drug release experiment, it was found that the release curve of 0.01% VK2MS complies with the zero order kinetics mode, which is beneficial in delaying release and stably controlling the drug concentration released to the outside.
0.01% VK2MS completely releases VK2 after 35 days, which is consistent with the faster degradation rate of 0.01% VK2MS found in the degradation experiment. Moreover, it was found that a higher encapsulated amount of VK2 will hinder the hydrolysis of PLGA and decrease its degradation rate, causing a lower drug release rate.
In the cell culture tests, it was found that the growth of MG-63 cells is inhibited by VK2, and an inhibition effect becomes obvious with the increasing concentration of VK2. However, 0.02 mg/mL can enhance the ALP activity of cells and result in a higher ALP activity in a single cell. Proliferation of MG-63 cells is also inhibited by VK2MS; however, the effect is less than that of VK2, representing a delayed release effect of VK2MS. VK2MS especially can efficiently enhance the ALP activity of a single cell.
Since the in vitro drug release rate of VK2MS is affected by the degradation rate, the in vitro cell experiment was also interfered with by the same factor. It takes a short time, one week, for the in vitro cell tests in the present invention, while it takes 3-4 months to repair bone tissue, and 0.01% VK2MS has already completely released at this time. Thus, it is preferable to use 0.1% VK2MS or 1.0% VK2MS, which have a longer release time. However, in the co-culture experiment of MG-63 cells and VK2MS, it was observed that 1.0% VK2MS has a more significant cell growth inhibition effect than that of 0.1% VK2MS. Therefore, the desired effect can be achieved by using a smaller amount of 1.0% VK2MS.
The present disclosure provides a delayed VK2 drug release system, vitamin K2 microsphere (VK2MS). Compared to VK2, VK2MS is not only capable of decreasing the cell growth rate inhibition of VK2, but also increasing the differentiation of cell ALP activity. Moreover, the diffusion drug release control technique of a polymer matrix was selected in this system to reduce the risk of requiring several surgeries. Furthermore, VK2 can not only inhibit the activity of osteoclasts but also induce osteoblasts to differentiate to bone cells. Therefore, this technique has a very high value in future medical research and application. Combining the technique and tissue engineering scaffolds in vivo to treat osteoporosis or repair damaged bone tissue, and the expectation that it can be applied to bone tissue repair engineering would benefit all mankind.
All of the features disclosed in this specification can be combined in any combination. Each feature disclosed in this specification can be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.
From the above description, one skilled in the art can easily ascertain the essential characteristics of the present invention, and, without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. As such, other embodiments are also within the claims.
The application claims priority to both U.S. Provisional Application No. 61/814,801, filed on Apr. 22, 2013 and a subsequently filed Taiwanese patent application entitled “Vitamin K2 microsphere, manufacture method, use, and drug thereof”, the contents of which are hereby incorporated by reference in their entirety.
| Number | Date | Country | |
|---|---|---|---|
| 61814801 | Apr 2013 | US |
