Preparation Method of Withaferin A Double Carrier Complex and Its Application in Anti-Osteoarthritis
The dual-carrier complex was formed by loading F127 hydrogel and PEG-PCL nanomicrobes, which solved the problem of insufficient solubility and targeting of drugs in the treatment of osteoarthritis, and achieved the efficient targeting and anti-inflammatory effects of gossamol A in bone tissue, promoting bone regeneration.
Patent Information
- Application Number
- CN202410945910.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-07-15
- Publication Date
- 2025-07-08
- Estimated Expiration
- 2044-07-15
AI Technical Summary
In the prior art, in the treatment of osteoarthritis, the solubility, bioavailability and targeting of the drug are insufficient, resulting in poor treatment effect, and the stability and circulation time of the drug in the body are short.
F127 hydrogel and PEG-PCL nanomicrobes were used to jointly load ashwagen A to form a two-carrier complex of ashwagen A, improving its solubility and bioavailability, and reducing non-specific distribution by targeting the inflammatory site in bone tissue.
Significantly improves the stability and solubility of ashwacin A in the body, prolongs its circulation time in the body, enhances the targeting of bone tissue, reduces bone destruction, promotes bone regeneration, and shows powerful anti-inflammatory effects in various inflammatory models.
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Figure CN118903156B_ABST
Abstract
Description
Technical Field
[0001] This application relates to the technical field of osteoarthritis, and specifically to a preparation method of a dual-carrier complex of withaferin A and its application in anti-osteoarthritis. Background Art
[0002] Osteoarthritis generally refers to degenerative osteoarthropathy. Degenerative osteoarthropathy, also known as osteoarthritis, degenerative arthritis, senile arthritis, hypertrophic arthritis, is a degenerative disease caused by many factors such as aging, obesity, strain, trauma, congenital joint abnormalities, and joint deformities, resulting in degradation and damage of articular cartilage and reactive hyperplasia of the joint margin and subchondral bone. The main treatment methods are to reduce joint load and excessive large-range activities to delay the progression of the disease. Obese patients should lose weight to reduce the joint load. Crutches or canes can be used when there are lesions in the lower limb joints to reduce the joint burden. Physical therapy and appropriate exercise can maintain the range of joint motion. Splints, braces, and canes can be used when necessary, which helps to control the symptoms in the acute phase. Anti-inflammatory and analgesic drugs can relieve or control the symptoms, but should be used carefully after evaluating the patient's risk factors and should not be taken for a long time. Cartilage protectants such as glucosamine sulfate have the effect of relieving symptoms and improving function, and long-term use can delay the structural progression of the disease. For advanced cases, total joint replacement is an effective method recognized to eliminate pain, correct deformities, and improve function under the condition that the general condition can tolerate surgery, which can greatly improve the quality of life of patients. Summary of the Invention
[0003] This application provides a preparation method of a dual-carrier complex of withaferin A and its application in anti-osteoarthritis.
[0004] Therefore, the embodiments of this application at least disclose the following technical solutions:
[0005] In a first aspect, an embodiment discloses a preparation method of a dual-carrier complex loaded with withaferin A, including:
[0006] Preparing an F127 hydrogel solution;
[0007] Preparing a micelle solution loaded with withaferin A; and
[0008] Mixing the F127 hydrogel solution and the micelle solution loaded with withaferin A.
[0009] In some embodiments, the preparation steps of the F127 hydrogel solution include: dissolving F127 powder in ddH2O at low temperature.
[0010] In some embodiments, the concentration of F127 in the F127 hydrogel solution is 40% (w / v).
[0011] In some embodiments, the preparation steps of the micellar solution loaded with withanolide A include:
[0012] Mix a DMSO solution containing withanolide A, a chloroform solution containing PEG-PCL, and water to form an emulsion;
[0013] Remove chloroform by rotary evaporation, dialyze in pure water using a dialysis bag with a molecular cut-off of 14000 Da to remove DMSO, ultrafiltrate and concentrate, and filter.
[0014] In some embodiments, the concentration of withanolide A in the DMSO solution containing withanolide A is 1-10 mg / mL, and the concentration of PEG-PCL in the chloroform solution containing PEG-PCL is 20-30 mg / mL.
[0015] In some embodiments, the volume ratio of the DMSO solution containing withanolide A, the chloroform solution containing PEG-PCL, and the water is 1:1.5:20.
[0016] In some embodiments, the concentration of withanolide A in the micellar solution loaded with withanolide A is 0.1-0.5 mg / mL.
[0017] In some embodiments, the concentration of withanolide A in the micellar solution loaded with withanolide A is 0.34 mg / mL.
[0018] In a second aspect, the embodiments disclose the application of the dual-carrier complex loaded with withanolide A prepared by the preparation method described in the first aspect in the preparation of a drug for treating osteoarthritis.
[0019] In this application, F127 hydrogel and PEG-PCL nanomicelles are used to co-load and encapsulate withanolide A to form a nanocomplex, which can improve the solubility and bioavailability of withanolide A, making it more effectively target the inflammatory sites in bone tissue. This dual-carrier complex has a significant effect on inhibiting bone destruction and promoting bone regeneration, and may play a role by reducing the infiltration of inflammatory cells in the bone marrow and decreasing the release of inflammatory mediators.
[0020] Moreover, in other types of inflammation (such as arthritis, muscle inflammation), this dual-carrier complex also improves the bioavailability of withanolide A.
[0021] In addition, this dual-carrier complex can significantly improve the stability and solubility of withanolide A in vivo and prolong its circulation time in vivo. This dual-carrier complex has excellent targeting properties, can act more precisely on bone tissue, and reduce the non-specific distribution of the drug in other tissues.
[0022] Moreover, in various inflammation models, this dual-vector complex exhibits strong anti-inflammatory effects and can effectively reduce the levels of inflammatory mediators. The targeting of this dual-vector complex may vary in different tissues, being particularly prominent in bone inflammation, while in other inflammations, its targeting effect depends on the inflammation site and the properties of the nanomicelles. Brief Description of the Drawings
[0023] Figure 1 Storage modulus (G') and loss modulus (G") of 20%, 30%, and 40% (w / v) F127 hydrogels provided for the examples.
[0024] Figure 2 Average swelling rate of 20%, 30%, 40% (w / v) F127 hydrogels provided for the examples.
[0025] Figure 3 Average degradation rate of 20%, 30%, 40% (w / v) F127 hydrogels provided for the examples.
[0026] Figure 4 Stained section images of the joint tissues of SD rats in the Nomal group, OA-Model group, F127+PEG-PLC group, and F127+PEG-PLC+WithaferinA group provided for the examples. Figure 4 A is the HE staining image, Figure 4 B is the immunofluorescence image, Figure 4 C are the safranin-fast green and alcian blue staining images respectively. Detailed Description of the Embodiments
[0027] In order to make the objectives, technical solutions, and advantages of the present application clearer and more understandable, the present application will be further described in detail below with reference to the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application. The reagents not specifically described in detail in the present application are all conventional reagents and can be obtained from commercial sources; the methods not specifically described in detail are all conventional experimental methods and can be learned from the prior art.
[0028] F127 hydrogel
[0029] 1. Preparation of F127 Hydrogel
[0030] F127 (Cat. No. 60318ES60, Yeasen Biotechnology (Shanghai) Co., Ltd.), also known as Poloxamer 407, is a multifunctional non-ionic surfactant composed of a block copolymer of ethylene oxide and propylene oxide. This white powder or granular substance is known for its unique thermosensitivity, being liquid at low temperatures and turning into a gel state after heating. Weigh 200 mg, 300 mg, and 400 mg of F127 powder respectively, dissolve them in 1 mL of ddH2O, and fully dissolve them overnight at 4°C to obtain 20%, 30%, and 40% (w / v) F127 hydrogels.
[0031] 2. Rheological properties and modulus
[0032] Measure the storage modulus (G') and loss modulus (G") of the F127 hydrogel on a DHR-2 rheometer using the strain sweep mode, with a strain range from 1% to 1000% and a fixed frequency of 1 rad / s.
[0033] As Figure 1 shown, the changes in the storage modulus (G') and loss modulus (G") of F127 hydrogels with three mass fractions were tested with the strain sweep mode of the rheometer as the strain varied from 0.1% to 1000%. As the strain increased, the G' of the hydrogel continuously decreased, the G" continuously increased, and the G' and G" curves would intersect, and the structure of the hydrogel reached a critical state. After the intersection point, G" was always greater than G', indicating that the network structure of the hydrogel was destroyed and the hydrogel presented a sol state. The 40% F127 hydrogel had stronger rheological properties due to its higher cross-linking density.
[0034] 3. Analysis of the swelling and degradation properties of F127 hydrogels
[0035] Freeze-dry the hydrogel, weigh a certain amount of the hydrogel and label it as M0. Immerse the freeze-dried sample in deionized water, and after reaching swelling equilibrium at 37°C, blot the water on the surface of the hydrogel with absorbent paper and weigh it, label it as M1. The swelling ratio S of the hydrogel = (M1 - M0) / M0; perform 3 parallel experiments for each group and take the average value.
[0036] Weigh the hydrogel, record the mass as W1, then immerse it in PBS with pH = 7.4, place it in a water bath at 37°C, take out the hydrogel sample every other day, remove the excess salts and dry it, and record the mass as W2. Evaluate the degradation behavior of the hydrogel by calculating the remaining mass ratio of the hydrogel (100% × W2 / W1) through the following formula.
[0037] As Figure 2As shown, the average swelling rates of 20%, 30%, and 40% (w / v) F127 hydrogels are 198.5%, 211.3%, and 258.5%, respectively, and they can absorb water exceeding their own mass. This is because a large number of pores and a large number of hydrogen bonds can lock in moisture very well. As the content of F127 increases from 20% to 40%, the hydrogen bond content continues to increase, causing the swelling rate to continue to increase. This result shows that the hydrogel has excellent water absorption properties, can effectively absorb the exudate of wound tissue, and maintain a moist environment for the wound, which is conducive to the rapid healing of wound tissue.
[0038] like Figure 3 As shown in the figure, the degradation performance test results show that F127 hydrogel has certain degradation characteristics. As the content of F127 increases from 20% to 40%, the hydrogel shows a gradually decreasing degradation rate. 40% F127 can still retain more than 50% of its original weight after 7 days. This is mainly because the increasing amount of F127 increases the cross-linking density, making the three-dimensional network structure of the hydrogel more difficult to destroy, which in turn leads to the slow degradation of the hydrogel.
[0039] Dual-carrier complex loaded with withaferin A and release of withaferin A
[0040] 1. Withaferin A-loaded micelles and dual-carrier complexes loaded with Withaferin A
[0041] The preparation method of micelles loaded with withaferin A includes: dissolving 5 mg of withaferin A (product number W133732-1, Shanghai Baishun Biotechnology Co., Ltd.) in 1 mL of DMSO, adding the solution to 1.5 mL of chloroform solution containing 40 mg of PEG-PCL (polyethylene glycol-polycaprolactone, product number Q-0092061, Xi'an Qiyue Biology), mixing evenly, slowly dropping the solution into 20 mL of ultrapure water under ultrasonic action to form an emulsion, removing the chloroform by rotary evaporation, adding the solution to a dialysis bag with a molecular cutoff of 14000 Da, and dialyzing the solution in pure water for more than 24 hours, during which the dialysate is replaced several times (more than 3 times) to completely remove DMSO, concentrating by ultrafiltration, and removing aggregates by filtration (0.22 μm aqueous phase syringe filter), and storing the product at 4° C. for later use.
[0042] The preparation method of the dual-carrier complex loaded with Withaferin A includes: First, mix 1 mL of 40% F127 hydrogel solution (40%, w / v) and 0.2 mL of PEG-PCL micelle solution loaded with Withaferin A (5 mg / mL, with the concentration of Withaferin A being 0.34 mg / mL) to form a gel. Rotate and evaporate to remove chloroform, add it to a dialysis bag with a molecular cut-off of 14,000 Da and dialyze in pure water for more than 24 h. During this period, change the dialysis fluid multiple times (more than 3 times) to completely remove DMSO. Ultrafilter and concentrate, filter through a 0.22 μm aqueous phase needle filter to remove aggregates, and store the product at 4 °C for later use, then the dual-carrier complex loaded with Withaferin A can be obtained.
[0043] 2. Particle size and potential analysis
[0044] Respectively take the micelle loaded with Withaferin A and the suspension of the dual-carrier complex loaded with Withaferin A. After diluting with a small amount of pure water, use a Malvern laser particle size analyzer to measure the average particle size, particle size distribution, and Zeta potential.
[0045] As shown in Table 1, the average particle size of the dual-carrier complex loaded with Withaferin A does not increase significantly compared with that of the micelle loaded with Withaferin A. Its particle size distribution is relatively uniform, and its Zeta potential also indicates that the stability of the dual-carrier complex loaded with Withaferin A does not decrease compared with that of the micelle loaded with Withaferin A.
[0046] Table 1
[0047] Micelles loaded with withaferin A Dual-carrier complex loaded with withaferin A Average particle size (nm) 162.59±6.36 168.25±8.20 Particle size PDI 0.11 0.13 Zeta potential (mV) 7.97±0.65 7.98±0.60
[0048] 3. Loading rate of Withaferin A
[0049] Detect the content of Withaferin A in the dialysis fluid obtained during the preparation process of the micelle loaded with Withaferin A, and calculate the mass percentage of the difference between the input amount of Withaferin A and the input amount of PEG-PCL, which is the loading rate of the micelle for Withaferin A.
[0050] Detect the content of Withaferin A in the dialysis fluid obtained during the preparation process of the dual-carrier complex loaded with Withaferin A, and calculate the mass percentage of the difference between the input amount of Withaferin A and the input amount of the PEG-PCL and F127 hydrogel, which is the loading rate of the dual-carrier complex for Withaferin A.
[0051] As shown in Table 1, there is no significant difference in the loading rate of the dual-carrier complex for Withaferin A and the loading rate of the micelle for Withaferin A.
[0052] Table 1
[0053] Micelles loaded with withaferin A Dual-carrier complex loaded with withaferin A Amount of carrier input (mg) 40 40 Loading rate (%) 6.80 6.82
[0054] 4. Release of Withaferin A
[0055] The solutions of the micelles loaded with withaferin A and the dual-carrier complexes loaded with withaferin A were separately added into a dialysis bag (MWCO = 3500 kDa), and immersed in 50 mL of phosphate buffer solution at pH 7.4 at 37°C. The released drug solution was taken at multiple time intervals, an equal volume of the release medium at the same temperature was added, and its absorbance was measured. The cumulative drug release rate (%) was calculated using the following formula: = M t / M0×100%, where M t is the cumulative amount of drug released at time t, and M0 is the initial drug loading amount. The results showed that within 24 h, the cumulative drug release rates of the micelles loaded with withaferin A and the dual-carrier complexes loaded with withaferin A for withaferin A were 60% and 20% respectively.
[0056] Animal experiment
[0057] 1. Experimental animals
[0058] 60 healthy Sprague-Dawley (SD) rats, half male and half female, with a body weight of (142 ± 20) g and a rat age of 4 - 5 weeks (Experimental Animal Center of Liaoning University of Traditional Chinese Medicine, license number: scxk2010.0001).
[0059] 2. Construction of arthritis model rats (OA-Model) and grouping experiments
[0060] Preparation of a type II collagen-induced SD rat arthritis model. An emulsion with a type II collagen concentration of 2.5 mg / mL was prepared with Freund's complete adjuvant, and 100 μL of the inflammatory agent was injected intradermally into the left hind paw of each rat. An arthritis model rat could be obtained in 10 days.
[0061] The experimental groups were divided into a Nomal group, an OA-Model group, an F127 + PEG-PLC group, and an F127 + PEG-PLC + Withaferin A group. In the Nomal group, 100 μL of 0.9% normal saline was injected intradermally into the left hind paw of each normal rat for 10 consecutive days, and no intervention was performed afterwards. The model rats in the OA-Model group were not given any intervention. In the F127 + PEG-PLC group, an equal weight of F127 hydrogel and PEG-PLC micelles at 1.8 mg / kg body weight was injected intradermally into the left hind paw of each arthritis model rat daily. In the F127 + PEG-PLC + Withaferin A group, an equal weight of the dual-carrier complex loaded with withaferin A at 1.8 mg / kg body weight was injected intradermally into the left hind paw of each arthritis model rat daily.
[0062] 3. Tissue section staining
[0063] The knee cartilage of the mice in each group was taken to prepare tissue sections for HE staining, safranin-fast green staining, alcian blue staining, and immunofluorescence staining.
[0064] The results were as Figure 4 shown. The dual-carrier system effectively delivered Withaferin A to the cartilage injury site, increasing the local concentration of the drug in the target area, thereby reducing the degree of cartilage injury. As a natural product, Withaferin A has been shown to have anti-inflammatory and antioxidant properties, which help reduce cartilage inflammation and may promote the regeneration and repair process of chondrocytes. The study also observed that after using the dual-carrier to deliver Withaferin A, the expression of cartilage markers such as COL2A1, SOX9, and Aggrecan increased significantly. These markers are important molecules necessary for chondrocytes to synthesize and maintain the cartilage matrix. COL2A1 encodes collagen II, which is a cartilage-specific collagen; SOX9 is a key transcription factor that regulates chondrocyte differentiation and matrix synthesis; Aggrecan is an important sulfated glycosaminoglycan that is crucial for the mechanical properties of cartilage. F127 and PEG-PLC, as the dual-carrier, combine the advantages of the two carriers, improving the water solubility and stability of Withaferin A, while enhancing its bioavailability in vivo. This system can effectively deliver Withaferin A to the cartilage injury site and continuously release the drug during the treatment process, thereby promoting the regeneration and repair of chondrocytes and accelerating the recovery process of cartilage injury. Using F127 and PEG-PLC as the carrier for dual-carrier delivery of Withaferin A showed significant effects in treating cartilage injury, manifested as a significant reduction in the degree of cartilage injury and an increase in the expression of cartilage markers COL2A1, SOX9, and Aggrecan, which demonstrated the effectiveness and potential of the dual-carrier in improving drug delivery efficiency and promoting cartilage repair.
[0065] As described above, it is only the preferred specific embodiment of the present application, but the protection scope of the present application is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed by the present application should be covered within the protection scope of the present application.
Claims
1. Use of the dual-carrier complex loaded with withanolide A in the preparation of a medicament for treating osteoarthritis; characterized in that: The dual-carrier complex loaded with withanolide A is prepared by the following method, and the steps are as follows: Weigh 400 mg of F127 powder and dissolve it in 1 mL of ddH2O, and fully dissolve it overnight at 4 °C to obtain an F127 hydrogel solution, and the concentration of F127 in the F127 hydrogel solution is 40% w / v; Dissolve 5 mg of withanolide A in 1 mL of DMSO, add it to a 1.5 mL chloroform solution containing 40 mg of PEG-PCL, mix well, and slowly add it dropwise to 20 mL of ultrapure water under ultrasonic action to form an emulsion; Rotate and evaporate to remove chloroform, add it to a dialysis bag with a molecular cut-off of 14,000 Da and dialyze it in pure water to remove DMSO, ultrafiltrate and concentrate, and filter to obtain a micelle solution loaded with withanolide A, and the concentration of the micelle solution loaded with withanolide A is 0.34 mg / mL; and Mix 1 mL of the F127 hydrogel solution and 0.2 mL of the micelle solution loaded with withanolide A to form a gel; Rotate and evaporate to remove chloroform, add it to a dialysis bag with a molecular cut-off of 14,000 Da and dialyze it in pure water for more than 24 h to remove DMSO, ultrafiltrate and concentrate, and filter to obtain the dual-carrier complex loaded with withanolide A.