Osteoarthritis targeted traditional Chinese medicine nano preparation and preparation method thereof

Abstract

The invention belongs to the technical field of traditional Chinese medicine preparations, and particularly relates to an osteoarthritis targeted traditional Chinese medicine nano preparation and a preparation method thereof.The traditional Chinese medicine nano preparation is prepared from, by weight, 15-25 parts of traditional Chinese medicine extract, 4-8 parts of functional peptide, 3 parts of nano zinc oxide, 12-15 parts of coupling agent, 10 parts of modifier, 30-48 parts of phospholipid and 14 parts of cholesterol; the traditional Chinese medicine extract obtained through toxicity attenuation and purification contains rich terpenoids, flavones, saponins and other micromolecular anti-inflammatory immune regulation active substances, multi-target treatment of arthritis is provided, the designed functional polypeptide has inflammation specific response and cell penetrating capacity, focus enrichment of treatment components is achieved, and the treatment effect is good. The toxicity to normal tissues is reduced while the curative effect is improved.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine preparation technology, specifically relating to a traditional Chinese medicine nano-preparation targeting osteoarthritis and its preparation method. Background Technology

[0002] Osteoarthritis is a chronic degenerative joint disease characterized by articular cartilage wear, synovial inflammation, and subchondral bone remodeling. Its pathogenesis involves a complex interplay of multiple factors, including abnormal release of inflammatory mediators, excessive activation of matrix metalloproteinases, chondrocyte metabolic imbalance, and oxidative stress. Current clinical treatment strategies primarily rely on nonsteroidal anti-inflammatory drugs (NSAIDs) and intra-articular corticosteroids. While these can provide short-term symptom relief, they are unlikely to reverse the pathological progression, and long-term use carries risks such as gastrointestinal damage, osteoporosis, and accelerated cartilage degeneration. In recent years, Traditional Chinese Medicine (TCM) has demonstrated its advantages in multi-pathway and multi-target integrated regulation in the treatment of osteoarthritis. TCM compound preparations can exert synergistic effects through anti-inflammatory, immunomodulatory, and cartilage repair-promoting mechanisms. However, traditional TCM preparations suffer from complex chemical compositions, low oral bioavailability, lack of targeted therapy, and systemic exposure leading to toxic side effects, severely hindering their clinical translation and application.

[0003] To address these challenges, nanodelivery systems offer a new direction for the modernization of traditional Chinese medicine (TCM) research. Encapsulation using carriers such as liposomes and polymer micelles can improve drug solubility and stability, and enhance accumulation at lesion sites through passive targeting effects. However, existing simple drug loading strategies for TCM nanoformulations struggle to achieve responsive drug release within the inflammatory microenvironment, easily leading to premature leakage or insufficient release. Furthermore, the low degree of surface functionalization of carriers and the lack of active targeting capabilities make precise delivery to diseased cells highly expressing specific receptors difficult. Additionally, the significant differences in physicochemical properties when multiple TCM components are co-loaded make it challenging to simultaneously ensure the encapsulation efficiency and stability of each active ingredient.

[0004] Therefore, there is an urgent need for a traditional Chinese medicine preparation that can achieve long-term circulation, targeted enrichment, and responsive drug release to osteoarthritis lesions, in order to solve the problems of poor targeting of traditional Chinese medicine components and uncontrollable drug release, and to provide new research ideas for the precise delivery of traditional Chinese medicine compound preparations and the development of modern formulations. Summary of the Invention

[0005] To address the above issues, this invention provides a targeted traditional Chinese medicine nanoparticle formulation for osteoarthritis and its preparation method. Using traditional Chinese medicine extracts as the main active ingredient, the nanoparticle formulation is constructed through innovative design of functional peptides with electrostatic binding segments, enzyme-responsive segments, and cell-penetrating segments. The EPR effect is utilized to target osteoarthritis. The positive surface charge of the electrostatic binding segment forms electrostatic adsorption with abnormally electronegative substances present in the arthritic microenvironment. The MMP matrix metalloproteinase, highly expressed at the inflammatory site, responsively cleaves the enzyme-responsive segment, exposing the cell-penetrating peptide, thus improving the cell entry efficiency of the active ingredient from the traditional Chinese medicine. This provides a new research approach for the precise, efficient, and safe treatment of osteoarthritis.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: This invention provides a traditional Chinese medicine nano-preparation targeting osteoarthritis, the traditional Chinese medicine nano-preparation comprising the following raw materials in parts by weight: 15-25 parts of traditional Chinese medicine extract, 4-8 parts of functional peptide, 3 parts of nano zinc oxide, 12-15 parts of coupling agent, 10 parts of modifier, 30-48 parts of phospholipid and 14 parts of cholesterol.

[0007] Furthermore, the amino acid sequence of the functional peptide from the N-terminus to the C-terminus is H2N-HHHEVGPLGVRGKRRFQFC-CONH2.

[0008] Furthermore, the functional peptide is prepared by solid-phase synthesis.

[0009] Furthermore, the coupling agent is DSPE-PEG2000-Mal (maleimide-modified polyethylene glycol distearatephosphatidylethanolamine).

[0010] Furthermore, the phospholipid is selected from hydrogenated soybean lecithin or sphingomyelin.

[0011] Furthermore, the modifier is selected from any one of hyaluronic acid, carboxymethyl chitosan, and wolfberry polysaccharide.

[0012] Furthermore, the herbal extract comprises the following raw materials: safflower, frankincense, myrrh, aconite, and clematis, in a mass ratio of 5-10:15-20:20:10:5. The preparation method of the herbal extract is as follows: S1: Safflower is extracted using CO2 supercritical extraction technology to avoid high-temperature extraction from damaging the activity of heat-sensitive components such as hydroxysaffron yellow pigment A. The extract is collected, concentrated under reduced pressure to a solid content of 60%, and spray-dried to obtain safflower extract. S2: After mixing frankincense, myrrh and Clematis armandii, add 70% ethanol by volume for reflux extraction and filtration to obtain residue and initial extract. Preliminary extraction of active substances such as terpenoids, flavonoids and glycosides with anti-inflammatory and immunomodulatory effects is performed. Add 70% ethanol to the residue for a second extraction and filtration to obtain a second extract. Combine the initial extract and the second extract, concentrate, elute and purify, and freeze dry to obtain a co-extract. S3: After soaking Aconitum carmichaelii in licorice water at pH 8.5 and draining, it is steamed under high pressure and dried to hydrolyze the toxic aconitine in Aconitum carmichaelii, converting it into a less toxic monoester or amino alcohol alkaloid, thus obtaining the steamed product. The steamed product is then extracted with 50% ethanol and filtered to obtain an alcohol extract. The alcohol extract is concentrated and dried to control the residual aconitine content to ≤0.01%, thus obtaining the processed product. The safflower extract, co-extract, and processed product are mixed to obtain the traditional Chinese medicine extract.

[0013] This invention also provides a method for preparing a traditional Chinese medicine nanoparticle formulation targeting osteoarthritis, the specific steps of which are as follows: Step 1: Weigh 10 parts of the modifier and dissolve it. Add EDC (1-ethyl-(3-dimethylaminopropyl)carbodiimide) and NHS (N-hydroxysuccinimide) to activate it. The carboxyl group in the modifier reacts with EDC to generate an unstable O-acylisourea intermediate, which then reacts with NHS to generate an active ester, resulting in an esterification solution. Add 4-8 parts of the functional peptide to the solution. The active ester reacts with the amino group at the N-terminus of the functional peptide to form a stable amide bond, resulting in a crude product. Dialyze the crude product to remove EDC and NHS, and obtain the modified peptide. Step 2: Mix 3 parts of nano zinc oxide, 30-48 parts of phospholipids and 14 parts of cholesterol, add a mixed solvent (chloroform and ethanol in a volume ratio of 2:1), and stir in a 40°C water bath until completely dissolved. Under the hydrophilic-hydrophobic interaction, the phospholipids spontaneously form a lipid bilayer with the hydrophilic end on the outside and the hydrophobic end on the inside. Then add 12-15 parts of a coupling agent and continue stirring to allow the coupling agent, which has a similar molecular structure to the phospholipids, to be embedded into the bilayer structure for subsequent coupling with the modified peptide, thus obtaining a lipid solution. Step 3: Remove the organic solvent from the lipid solution by rotary evaporation under reduced pressure and vacuum drying to obtain a lipid film. Weigh 15-25 parts of the Chinese herbal extract, disperse it in preheated phosphate buffer, and add it to the lipid film. Hydrate in a 60℃ water bath for 1-3 hours to completely detach and disperse the lipid film. The hydration process causes the lipid bilayer to close spontaneously. The hydrophobic regions formed by the hydrophobic ends encapsulate the lipid-soluble components in the Chinese herbal extract. Cholesterol is used to regulate the fluidity of the phospholipid bilayer structure, resist phospholipid oxidation, and stabilize the structure. The hydrophilic ends inside form a hydrophilic core to embed the water-soluble components of the Chinese herbal extract, thus obtaining a liposome suspension. Step 4: The liposome suspension is subjected to ultrasound treatment with a probe under ice bath until it becomes translucent, in order to control the uniformity of liposome particle size in the liposome suspension. Ultrasound causes larger or multi-compartment liposomes to form smaller, predominantly single-compartment nanoliposomes, which is beneficial for tissue penetration, long circulation and EPR effect, thus obtaining liposomes. Step 5: Add the modified peptide to the liposomes and stir in the dark for 4-8 hours. The coupling agent in the liposome structure contains maleimide groups on its surface, which can react with the cysteine ​​(C) thiol group at the C-terminus of the modified peptide to form a stable thioether bond, thus obtaining a traditional Chinese medicine nano-preparation targeting osteoarthritis.

[0014] Furthermore, the aforementioned traditional Chinese medicine nano-preparations can be used to prepare nano-injections, topical transdermal gels, or microneedle patches.

[0015] The beneficial effects achieved by this invention are as follows: The osteoarthritis-targeting traditional Chinese medicine nano-formulation prepared in this invention utilizes the multi-component characteristics of traditional Chinese medicine to achieve multi-target treatment of osteoarthritis. The extract obtained from the traditional Chinese medicine after detoxification processing and biphasic extraction and purification is rich in small molecule components such as hydroxysaffron yellow A, β-boswellic acid, myrrh alkanes, alkaloids, and quercetin-3-O-glucoside. These components exert anti-inflammatory, immunosuppressive, cartilage matrix synthesis-promoting, and macrophage polarization-regulating effects through multiple pharmacological mechanisms. Among these, hydroxysaffron yellow A reduces inflammation by inhibiting the COX-2 / PGE2 pathway. Symptom mediator release; β-boswellic acid and myrrh alkane regulate NF-κB signaling, reduce the expression of pro-inflammatory factors such as IL-1β and TNF-α, inhibit matrix metalloproteinase activity, and protect cartilage joints; processed and detoxified aconitine derivatives block sodium ion channels; Clematis chinensis flavonoids inhibit chondrocyte apoptosis and delay the progression of osteoarthritis; multi-active Chinese medicine components form a multi-pathway intervention network; liposome encapsulation can effectively retain the activity and stability of Chinese medicine components, and modified nano-zinc oxide enhances drug sustained release and targeting, and regulates liposome particle size. In the functional peptide sequence, HHHEV is the electrostatic binding region. Its surface charge is electrically neutral in the bloodstream, reducing adsorption to charged substances such as erythrocytes and prolonging blood circulation time. Upon reaching the weakly acidic microenvironment of osteoarthritis, it undergoes charge reversal, becoming positively charged. This allows it to electrostatically adsorb negatively charged inflammatory factors, nucleic acid fragments, and polysaccharides, remaining at the inflammatory site. GPLGVRGK is an MMP enzyme-responsive peptide. Under the action of abnormally expressed MMP enzymes at the inflammatory site, the functional peptide is cleaved, exposing the drug-loaded liposomes of the surface-modified cell-penetrating peptide RRFQFC. This enables selective and targeted treatment of abnormal chondrocytes and macrophages at the osteoarthritis lesion site. Furthermore, esterification is used to covalently link hyaluronic acid, chitosan, or traditional Chinese medicine polysaccharides to the surface of the functional peptide. This improves the biocompatibility of traditional Chinese medicine nanoparticles, prolongs blood circulation time, protects the stability of the peptide chain and liposome encapsulation, and improves drug utilization. The DSPE-PEG2000-Mal embedded on the liposomes provides a binding site for cysteine ​​in the functional peptide, thereby improving the stability of traditional Chinese medicine nano-preparations.

[0016] The present invention constructs a traditional Chinese medicine nano-formulation with long-acting circulation, osteoarthritis targeting, and microenvironment-responsive drug release, which realizes the co-loading of multiple components of traditional Chinese medicine and the safety of drug administration, thereby improving the therapeutic effect of traditional Chinese medicine on osteoarthritis. Attached Figure Description

[0017] Figure 1 This is a schematic diagram of the structure of a traditional Chinese medicine nano-preparation targeting osteoarthritis according to the present invention; Figure 2 The molecular structure and preferred conformation of functional peptides; Figure 3The particle size detection results are for the traditional Chinese medicine nano-preparations prepared in Examples 1-4 and Comparative Examples 1-2; Figure 4 The potential detection results are for the traditional Chinese medicine nano-preparations prepared in Examples 1-4 and Comparative Examples 1-2. Figure 5 The scanning electron microscope characterization results are for the traditional Chinese medicine nano-preparation prepared in Example 1.

[0018] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used together with the embodiments of the invention to explain the invention and do not constitute a limitation thereof. Detailed Implementation

[0019] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to this invention. The preferred embodiments and materials described herein are for illustrative purposes only and do not limit the scope of this application.

[0021] The schematic diagram of the osteoarthritis-targeting traditional Chinese medicine nano-formulation structure is shown in the following embodiments. Figure 1 Unless otherwise specified, all methods are conventional. Unless otherwise specified, all materials used in the following examples are new materials purchased from the market. Specifically, the Chinese medicinal herbs safflower, frankincense, myrrh, aconite, and *Clematis chinensis* are all standard processed medicinal slices according to the *Chinese Pharmacopoeia*; the functional peptides used are prepared by solid-phase synthesis, in N-terminal amidated lyophilized powder form, with a purity of 97.2%; the DSPE-PEG2000-Mal used has a purity ≥95%; the hydrogenated soybean lecithin used has a purity ≥98%; the cholesterol used is of plant origin, with a purity ≥98%; the sphingomyelin used is of synthetic origin, with a purity ≥98%; the hyaluronic acid used has a molecular weight of 10-30 kDa and is pharmaceutical injection grade; the carboxymethyl chitosan used has a carboxylation degree ≥80% and a viscosity ≤100 mPa·s; the *Lycium barbarum* polysaccharide used has a purity ≥50%; and the EDC used is in hydrochloride form.

[0022] Example 1: This invention provides a traditional Chinese medicine nano-preparation targeting osteoarthritis, the traditional Chinese medicine nano-preparation comprising the following raw materials in parts by weight: 15 parts of traditional Chinese medicine extract, 8 parts of functional peptide, 3 parts of nano zinc oxide, 12 parts of DSPE-PEG2000-Mal, 10 parts of hyaluronic acid, 30 parts of hydrogenated soybean lecithin and 14 parts of cholesterol.

[0023] The herbal extract comprises the following raw materials in parts by weight: 500 parts safflower, 1500 parts frankincense, 2000 parts myrrh, 1000 parts aconite, and 500 parts clematis. The preparation method is as follows: S1: Weigh 500 parts of safflower and perform CO2 supercritical extraction at a pressure of 25 MPa, a temperature of 45℃, and an extraction time of 3 h to avoid high-temperature extraction from damaging the activity of heat-sensitive components such as hydroxysafflower yellow pigment A. Collect the extract, concentrate the extract under reduced pressure to a solid content of 60%, and spray dry to obtain safflower extract. S2: Mix 1500 parts of frankincense, 2000 parts of myrrh, and 500 parts of Clematis chinensis, add 40,000 parts of 70% ethanol, heat and reflux at 78°C for 2 hours for a first extraction, filter to obtain residue and initial extract. Preliminary extraction of active substances such as terpenes, flavonoids, and glycosides with anti-inflammatory and immunomodulatory effects. Add 40,000 parts of 70% ethanol to the residue for a second extraction for 2 hours, filter to obtain a second extract. Combine the initial extract and the second extract, concentrate to 1 / 10 of the combined weight, elute and purify with macroporous resin (eluting agent is 50% ethanol), freeze dry to obtain a co-extract. S3: Soak 1000 parts of Aconitum carmichaelii in licorice water at pH 8.5 for 24 hours, drain, then steam at 120℃ for 2 hours and dry to hydrolyze the toxic aconitine in Aconitum carmichaelii into a less toxic monoester or amino alcohol alkaloid, obtaining a steamed product. Add 10000 parts of 50% ethanol to the steamed product for alcohol extraction to obtain an alcohol extract. Concentrate the alcohol extract to 1 / 10 of its original weight, remove residual aconitine through a cation exchange resin, and then dry. The residual aconitine content is 0.008%, obtaining a processed product. Mix the safflower extract, co-extract, and processed product to obtain a traditional Chinese medicine extract.

[0024] This embodiment also provides a method for preparing a traditional Chinese medicine nano-formulation targeting osteoarthritis, the specific steps of which are as follows: Step 1: Weigh 10 parts of hyaluronic acid and dissolve it in 100 parts of 0.5 M pH 6 MES buffer. Add 1 part of EDC and 2 parts of NHS to the solution and shake in the dark for 30 min to activate the esterification solution. Add 8 parts of functional peptide to the solution. The active ester reacts with the amino group at the N-terminus of the functional peptide to form a stable amide bond, resulting in a crude product. Dialyze the crude product using a 3.5 kDa dialysis bag to remove EDC and NHS, obtaining the modified peptide. Step 2: Mix 3 parts of nano zinc oxide, 30 parts of hydrogenated soybean lecithin and 14 parts of cholesterol, add 300 parts of mixed solvent (chloroform and ethanol in a volume ratio of 2:1), and stir in a 40°C water bath until completely dissolved. Under the hydrophilic-hydrophobic interaction, the phospholipid spontaneously forms a lipid bilayer with the hydrophilic end on the outside and the hydrophobic end on the inside. Then add 12 parts of DSPE-PEG2000-Mal and continue stirring until completely dissolved. This allows the coupling agent, which has a similar molecular structure to the phospholipid, to be embedded in the bilayer structure for subsequent coupling with the modified peptide, thus obtaining a lipid solution. Step 3: Remove the organic solvent by rotary evaporation at 40℃ and 30 rpm under reduced pressure, and vacuum dry for 12 h to obtain a uniform lipid film. Disperse 15 parts of the traditional Chinese medicine extract in 300 parts of pH 7.4 phosphate buffer preheated at 60℃, and hydrate by rotary evaporation in a 60℃ water bath for 1 h to completely detach and disperse the lipid film. The hydration process causes the lipid bilayer to close spontaneously, resulting in a liposome suspension. Step 4: Sonicate the liposome suspension under an ice bath with a 300 W power probe for 10 minutes until it becomes translucent, in order to control the uniformity of liposome particle size in the liposome suspension. Sonication causes larger or multi-compartment liposomes to form smaller, predominantly single-compartment nanoliposomes, achieving long-cycle and EPR effects, and thus obtaining liposomes. Step 5: Add the modified peptide to the liposomes and stir in the dark for 4 h. The coupling agent in the liposome structure contains maleimide groups on its surface, which can react with the cysteine ​​thiol group at the C-terminus of the modified peptide to form a stable thioether bond, thus obtaining a traditional Chinese medicine nano-preparation targeting osteoarthritis.

[0025] Example 2: This invention provides a traditional Chinese medicine nano-preparation targeting osteoarthritis, the traditional Chinese medicine nano-preparation comprising the following raw materials in parts by weight: 20 parts of traditional Chinese medicine extract, 6 parts of functional peptide, 3 parts of nano zinc oxide, 14 parts of DSPE-PEG2000-Mal, 10 parts of carboxymethyl chitosan, 40 parts of hydrogenated soybean lecithin and 14 parts of cholesterol.

[0026] The herbal extract comprises the following raw materials in parts by weight: 800 parts safflower, 1700 parts frankincense, 2000 parts myrrh, 1000 parts aconite, and 500 parts clematis root. The preparation method is as follows: S1: Weigh 800 parts of safflower and perform CO2 supercritical extraction at a pressure of 25 MPa, a temperature of 45℃, and an extraction time of 3 h to avoid high-temperature extraction from damaging the activity of heat-sensitive components such as hydroxysafflower yellow pigment A. Collect the extract, concentrate the extract under reduced pressure to a solid content of 60%, and spray dry to obtain safflower extract. S2: Mix 1700 parts of frankincense, 2000 parts of myrrh, and 500 parts of Clematis chinensis, add 42000 parts of 70% ethanol, heat and reflux at 78℃ for 2 h for a first extraction, filter to obtain residue and initial extract. Preliminary extraction of active substances such as terpenes, flavonoids, and glycosides with anti-inflammatory and immunomodulatory effects. Add 42000 parts of 70% ethanol to the residue for a second extraction for 2 h, filter to obtain a second extract. Combine the initial extract and the second extract, concentrate to 1 / 10 of the combined weight, elute and purify with macroporous resin (eluting agent is 50% ethanol), freeze dry to obtain a co-extract. S3: Soak 1000 parts of Aconitum carmichaelii in licorice water at pH 8.5 for 24 hours, drain, then steam at 120℃ for 2 hours and dry to hydrolyze the toxic aconitine in Aconitum carmichaelii into a less toxic monoester or amino alcohol alkaloid, obtaining a steamed product. Add 10000 parts of 50% ethanol to the steamed product for alcohol extraction to obtain an alcohol extract. Concentrate the alcohol extract to 1 / 10 of its original weight, remove residual aconitine through a cation exchange resin, and then dry. The residual aconitine content is 0.006%, obtaining a processed product. Mix the safflower extract, co-extract, and processed product to obtain a traditional Chinese medicine extract.

[0027] This embodiment also provides a method for preparing a traditional Chinese medicine nano-formulation targeting osteoarthritis, the specific steps of which are as follows: Step 1: Weigh 10 parts of carboxymethyl chitosan and dissolve it in 100 parts of 0.5 M pH 6 MES buffer. Add 1 part of EDC and 2 parts of NHS to the solution and shake in the dark for 30 min to activate the solution and obtain an esterification solution. Add 6 parts of functional peptide to the solution. The active ester reacts with the amino group at the N-terminus of the functional peptide to form a stable amide bond and obtain the crude product. Dialyze the crude product using a 3.5 kDa dialysis bag to remove EDC and NHS and obtain the modified peptide. Step 2: Mix 3 parts of nano zinc oxide, 30 parts of hydrogenated soybean lecithin and 14 parts of cholesterol, add 300 parts of mixed solvent (chloroform and ethanol in a volume ratio of 2:1), and stir in a 40°C water bath until completely dissolved. Under the hydrophilic-hydrophobic interaction, the phospholipid spontaneously forms a lipid bilayer with the hydrophilic end on the outside and the hydrophobic end on the inside. Then add 14 parts of DSPE-PEG2000-Mal and continue stirring until completely dissolved. This allows the coupling agent, which has a similar molecular structure to the phospholipid, to be embedded in the bilayer structure for subsequent coupling with the modified peptide, thus obtaining a lipid solution. Step 3: Remove the organic solvent by rotary evaporation at 40℃ and 30 rpm under reduced pressure, and vacuum dry for 12 h to obtain a uniform lipid film. Disperse 20 parts of the traditional Chinese medicine extract in 300 parts of pH 7.4 phosphate buffer preheated at 60℃, and hydrate by rotary evaporation in a 60℃ water bath for 1 h to completely detach and disperse the lipid film. The hydration process causes the lipid bilayer to close spontaneously, resulting in a liposome suspension. Step 4: Sonicate the liposome suspension under an ice bath with a 400 W power probe for 10 minutes until it becomes translucent, in order to control the uniformity of liposome particle size in the liposome suspension. Sonication causes larger or multi-compartment liposomes to form smaller, predominantly single-compartment nanoliposomes, achieving long-term circulation and EPR effect, and thus obtaining liposomes. Step 5: Add the modified peptide to the liposomes and stir in the dark for 6 h. The coupling agent in the liposome structure contains maleimide groups on its surface, which can react with the cysteine ​​thiol group at the C-terminus of the modified peptide to form a stable thioether bond, thus obtaining a traditional Chinese medicine nano-preparation targeting osteoarthritis.

[0028] Example 3: The present invention provides a traditional Chinese medicine nano-preparation for osteoarthritis, the traditional Chinese medicine nano-preparation comprising the following raw materials in parts by weight: 25 parts of traditional Chinese medicine extract, 4 parts of functional peptide, 3 parts of nano zinc oxide, 15 parts of DSPE-PEG2000-Mal, 10 parts of wolfberry polysaccharide, 48 parts of hydrogenated soybean lecithin and 14 parts of cholesterol.

[0029] The herbal extract comprises the following raw materials in parts by weight: 1000 parts safflower, 2000 parts frankincense, 2000 parts myrrh, 1000 parts aconite, and 500 parts clematis root. The preparation method is as follows: S1: Weigh 1000 parts of safflower and perform CO2 supercritical extraction at a pressure of 25 MPa, a temperature of 45℃, and an extraction time of 3 h to avoid high-temperature extraction from damaging the activity of heat-sensitive components such as hydroxysafflower yellow pigment A. Collect the extract, concentrate the extract under reduced pressure to a solid content of 60%, and spray dry to obtain safflower extract. S2: Mix 2000 parts of frankincense, 2000 parts of myrrh, and 500 parts of Clematis chinensis, add 45000 parts of 70% ethanol, heat and reflux at 78°C for 2 hours for a first extraction, filter to obtain residue and initial extract. Preliminary extraction of active substances such as terpenes, flavonoids, and glycosides with anti-inflammatory and immunomodulatory effects. Add 45000 parts of 70% ethanol to the residue for a second extraction for 2 hours, filter to obtain secondary extract. Combine the initial extract and secondary extract, concentrate to 1 / 10 of the combined weight, elute and purify with macroporous resin (eluting agent is 50% ethanol), freeze dry to obtain co-extract; S3: Soak 1000 parts of Aconitum carmichaelii in licorice water at pH 8.5 for 24 hours, drain, then steam at 120℃ for 2 hours and dry to hydrolyze the toxic aconitine in Aconitum carmichaelii into a less toxic monoester or amino alcohol alkaloid, obtaining a steamed product. Add 10000 parts of 50% ethanol to the steamed product for alcohol extraction to obtain an alcohol extract. Concentrate the alcohol extract to 1 / 10 of its original weight, remove residual aconitine through a cation exchange resin, and then dry. The residual aconitine content is 0.009%, obtaining a processed product. Mix the safflower extract, co-extract, and processed product to obtain a traditional Chinese medicine extract.

[0030] This embodiment also provides a method for preparing a traditional Chinese medicine nano-formulation targeting osteoarthritis, the specific steps of which are as follows: Step 1: Weigh 10 parts of Lycium barbarum polysaccharide and dissolve it in 100 parts of 0.5 M pH 6 MES buffer. Add 1 part of EDC and 2 parts of NHS to the solution and shake in the dark for 30 min to activate the solution and obtain an esterification solution. Add 4 parts of functional peptide to the solution. The active ester reacts with the amino group at the N-terminus of the functional peptide to form a stable amide bond and obtain the crude product. Dialyze the crude product using a 1000 Da dialysis bag to remove EDC and NHS and obtain the modified peptide. Step 2: Mix 3 parts of nano zinc oxide, 30 parts of hydrogenated soybean lecithin and 14 parts of cholesterol, add 300 parts of mixed solvent (chloroform and ethanol in a volume ratio of 2:1), and stir in a 40°C water bath until completely dissolved. Under the hydrophilic-hydrophobic interaction, the phospholipid spontaneously forms a lipid bilayer with the hydrophilic end on the outside and the hydrophobic end on the inside. Then add 15 parts of DSPE-PEG2000-Mal and continue stirring until completely dissolved. This allows the coupling agent, which has a similar molecular structure to the phospholipid, to be embedded in the bilayer structure for subsequent coupling with the modified peptide, thus obtaining a lipid solution. Step 3: Remove the organic solvent by rotary evaporation at 40℃ and 30 rpm under reduced pressure, and vacuum dry for 12 h to obtain a uniform lipid film. Disperse 25 parts of the traditional Chinese medicine extract in 300 parts of pH 7.4 phosphate buffer preheated at 60℃, and hydrate by rotary evaporation in a 60℃ water bath for 1 h to completely detach and disperse the lipid film. The hydration process causes the lipid bilayer to close spontaneously, resulting in a liposome suspension. Step 4: Sonicate the liposome suspension under an ice bath with a 500 W power probe for 10 minutes until it becomes translucent, in order to control the uniformity of liposome particle size in the liposome suspension. Sonication causes larger or multi-compartment liposomes to form smaller, predominantly single-compartment nanoliposomes, achieving long-term circulation and EPR effect, and thus obtaining liposomes. Step 5: Add the modified peptide to the liposomes and stir in the dark for 8 h. The coupling agent in the liposome structure contains maleimide groups on its surface, which can react with the cysteine ​​thiol group at the C-terminus of the modified peptide to form a stable thioether bond, thus obtaining a traditional Chinese medicine nano-preparation targeting osteoarthritis.

[0031] Example 4: The present invention provides a traditional Chinese medicine nano-preparation for osteoarthritis, the traditional Chinese medicine nano-preparation comprising the following raw materials in parts by weight: 20 parts of traditional Chinese medicine extract, 8 parts of functional peptide, 3 parts of nano zinc oxide, 14 parts of DSPE-PEG2000-Mal, 10 parts of hyaluronic acid, 42 parts of sphingomyelin and 14 parts of cholesterol.

[0032] The herbal extract comprises the following raw materials in parts by weight: 800 parts safflower, 2000 parts frankincense, 2000 parts myrrh, 1000 parts aconite, and 500 parts clematis root. The preparation method is as follows: S1: Weigh 800 parts of safflower and perform CO2 supercritical extraction at a pressure of 25 MPa, a temperature of 45℃, and an extraction time of 3 h to avoid high-temperature extraction from damaging the activity of heat-sensitive components such as hydroxysafflower yellow pigment A. Collect the extract, concentrate the extract under reduced pressure to a solid content of 60%, and spray dry to obtain safflower extract. S2: Mix 2000 parts of frankincense, 2000 parts of myrrh, and 500 parts of Clematis chinensis, add 45000 parts of 70% ethanol, heat and reflux at 78°C for 2 hours for a first extraction, filter to obtain residue and initial extract. Preliminary extraction of active substances such as terpenes, flavonoids, and glycosides with anti-inflammatory and immunomodulatory effects. Add 45000 parts of 70% ethanol to the residue for a second extraction for 2 hours, filter to obtain secondary extract. Combine the initial extract and secondary extract, concentrate to 1 / 10 of the combined weight, elute and purify with macroporous resin (eluting agent is 50% ethanol), freeze dry to obtain co-extract; S3: Soak 1000 parts of Aconitum carmichaelii in licorice water at pH 8.5 for 24 hours, drain, then steam at 120℃ for 2 hours and dry to hydrolyze the toxic aconitine in Aconitum carmichaelii into a less toxic monoester or amino alcohol alkaloid, obtaining a steamed product. Add 10000 parts of 50% ethanol to the steamed product for alcohol extraction to obtain an alcohol extract. Concentrate the alcohol extract to 1 / 10 of its original weight, remove residual aconitine through a cation exchange resin, and then dry. The residual aconitine content is 0.008%, obtaining a processed product. Mix the safflower extract, co-extract, and processed product to obtain a traditional Chinese medicine extract.

[0033] This embodiment also provides a method for preparing a traditional Chinese medicine nano-formulation targeting osteoarthritis, the specific steps of which are as follows: Step 1: Weigh 10 parts of hyaluronic acid and dissolve it in 100 parts of 0.5 M pH 6 MES buffer. Add 1 part of EDC and 2 parts of NHS to the solution and shake in the dark for 30 min to activate the solution and obtain an esterification solution. Add 8 parts of functional peptide to the solution. The active ester reacts with the amino group at the N-terminus of the functional peptide to form a stable amide bond and obtain the crude product. Dialyze the crude product using a 1000 Da dialysis bag to remove EDC and NHS and obtain the modified peptide. Step 2: Mix 3 parts of nano zinc oxide, 30 parts of hydrogenated soybean lecithin and 14 parts of cholesterol, add 300 parts of mixed solvent (chloroform and ethanol in a volume ratio of 2:1), and stir in a 40°C water bath until completely dissolved. Under the hydrophilic-hydrophobic interaction, the phospholipid spontaneously forms a lipid bilayer with the hydrophilic end on the outside and the hydrophobic end on the inside. Then add 14 parts of DSPE-PEG2000-Mal and continue stirring until completely dissolved. This allows the coupling agent, which has a similar molecular structure to the phospholipid, to be embedded in the bilayer structure for subsequent coupling with the modified peptide, thus obtaining a lipid solution. Step 3: Remove the organic solvent by rotary evaporation at 40℃ and 30 rpm under reduced pressure, and vacuum dry for 12 h to obtain a uniform lipid film. Disperse 20 parts of the traditional Chinese medicine extract in 300 parts of pH 7.4 phosphate buffer preheated at 60℃, and hydrate by rotary evaporation in a 60℃ water bath for 1 h to completely detach and disperse the lipid film. The hydration process causes the lipid bilayer to close spontaneously, resulting in a liposome suspension. Step 4: Sonicate the liposome suspension under an ice bath with a 300 W power probe for 20 minutes until it becomes translucent, in order to control the uniformity of liposome particle size in the liposome suspension. Sonication causes larger or multi-compartment liposomes to form smaller, predominantly single-compartment nanoliposomes, achieving long-term circulation and EPR effect, and thus obtaining liposomes. Step 5: Add the modified peptide to the liposomes and stir in the dark for 6 h. The coupling agent in the liposome structure contains maleimide groups on its surface, which can react with the cysteine ​​thiol group at the C-terminus of the modified peptide to form a stable thioether bond, thus obtaining a traditional Chinese medicine nano-preparation targeting osteoarthritis.

[0034] Example 5: This example provides a topical transdermal gel based on traditional Chinese medicine nano-preparations, comprising the following raw materials in parts by weight: 38.8 parts of traditional Chinese medicine nano-preparations prepared according to the raw material composition ratio of Example 1, 0.8 parts of borneol, 0.4 parts of azone, and 10 parts of poloxamer 407. The specific preparation method is as follows: 38.8 parts of traditional Chinese medicine nano-preparations and 10 parts of poloxamer 407 are mixed and stirred at 100 rpm until completely dissolved. 0.8 parts of borneol and 0.4 parts of azone are added, and stirring is continued for 20 min to disperse. The mixture is then allowed to stand at 4°C for 24 h to form a gel, thus obtaining the topical transdermal gel.

[0035] Example 6: This example provides a microneedle patch based on traditional Chinese medicine nano-preparations, comprising the following raw materials by weight: 35 parts of traditional Chinese medicine nano-preparations, 12 parts of hyaluronic acid, 3 parts of polyvinylpyrrolidone (PVP K30), and 0.8 parts of borneol were prepared according to the raw material composition ratio of Example 1. The specific preparation method is as follows: A polydimethylsiloxane (PDMS) mold was prepared by laser engraving, with a needle height of 300 μm and a needle tip diameter of 50 μm. 12 parts of hyaluronic acid, 3 parts of PVP K30, and 0.8 parts of borneol were added to 35 parts of traditional Chinese medicine nano-preparations and stirred to dissolve, obtaining a drug solution. The drug solution was dropped into the PDMS mold, and after vacuum degassing, it was centrifuged at 3000 rpm for 10 min, dried at room temperature for 24 h, and then demolded to obtain the microneedle patch.

[0036] The difference between Comparative Example 1 and Example 1 is that the functional peptide is not modified with hyaluronic acid, while the rest is the same as in Example 1. The difference between Comparative Example 2 and Example 1 is that hyaluronic acid and functional peptides were not added, while the rest were the same as in Example 1. Comparative Example 3 was a commercially available safflower and Panax notoginseng patch preparation.

[0037] Functional peptide molecular conformation investigation After importing the functional peptide molecular structure into molecular dynamics simulation software, setting the C-terminus to amidation, and fitting the preferred conformation, the results are shown in [Figure number missing]. Figure 2 .

[0038] Microscopic morphology of traditional Chinese medicine nano-preparations Particle size determination: 1 mL of the traditional Chinese medicine nano-preparations prepared in Examples 1-4 and Comparative Examples 1-2 were taken respectively, and the particle size was determined using a nanoparticle size analyzer. The results are shown in the figure. Figure 3 ; Potential testing: 10 mL of the traditional Chinese medicine nanoparticles prepared in Examples 1-4 and Comparative Examples 1-2 were taken respectively, divided into two equal portions, and the pH was adjusted to neutral and weakly acidic pH 6.0 with 0.1 M NaOH solution and 1 M HCl solution, respectively, to simulate the physiological environment and the weakly acidic environment of osteoarthritis. The potential was then tested in a nanoparticle size analyzer. The results are shown in […]. Figure 4 ; Morphological examination: 1 mg of the traditional Chinese medicine nano-preparation prepared in Example 1 was weighed and added to 1 mL of physiological saline. The solution was dissolved by sonication to obtain a 1 mg / mL solution. 10 μL of this solution was dropped onto a copper mesh and allowed to precipitate for 1 min. After absorbing the surface liquid with filter paper, the copper mesh was dried under an electric heating lamp. Baking was stopped when the surface liquid was almost gone. 5 μL of a 2% phosphotungstic acid solution was added, and the mesh was allowed to precipitate for 1 min. The surface liquid was then absorbed with filter paper and dried in a cool place. TEM (transmission electron microscopy) was performed at 100 kV. The results are as follows: Figure 5 As shown.

[0039] Transdermal efficiency study The transdermal efficiency of the topical transdermal gel prepared in Example 5 and the safflower and Panax notoginseng patch preparation in Comparative Example 3 was investigated using the Franz diffusion cell method. The cumulative transdermal amount and transdermal rate constant over 24 hours were investigated using the content of hydroxysafflower yellow A as the detection index. The results are shown in Table 1.

[0040] Anti-inflammatory activity investigation RAW264.7 inflammatory cell models were constructed using LPS (lipopolysaccharide) induction. Cells were cultured in cell culture flasks and seeded into 12-well plates. Control group (no drug administration), Example 5 group (drug administration concentration of 10 μg / mL based on the content of traditional Chinese medicine extract), and diclofenac group (10 μg / mL) were set up. The levels of TNF-α and IL-6 in cells before and after drug administration were measured in each group. The inhibition rate was calculated as (cytokine concentration in control group - cytokine concentration in drug administration group) / cytokine concentration in control group × 100%. The results are shown in Table 2.

[0041] Table 1 Transdermal Efficiency Evaluation

[0042] Table 2 Anti-inflammatory activity

[0043] Figure 2 The results of molecular simulation of the functional peptide's superior conformation show that the designed functional peptide has a certain helical folding conformation and can spontaneously assemble into a molecular structure with greater steric hindrance. This helps to prevent it from being adsorbed and destroyed by normal tissues, cells or molecules during blood circulation and can stabilize the liposome membrane loaded with the active ingredients of traditional Chinese medicine.

[0044] Figure 3 The results showed that the preparation methods of traditional Chinese medicine nanoparticles provided in Examples 1-4 can stably prepare nanoparticles with a particle size of about 120 nm, with a small SD value, relatively uniform particle size, and stable process.

[0045] Figure 4 The results showed that, under neutral conditions, the potentials of the traditional Chinese medicine nanoparticles prepared in Examples 1-4 and Comparative Examples 1-2 were negative, and the potentials of the traditional Chinese medicine nanoparticles in Examples 1-4 were larger, which could effectively prevent red blood cell adsorption, prolong blood circulation time, and increase the concentration of the drug reaching the osteoarthritis site. Under weakly acidic conditions, due to the change in the charge of the N-terminus of the functional peptide molecule, it became positively charged. At this time, it could form electrostatic binding with substances such as abnormally expressed electronegative cytokines at the inflammatory site, promote drug retention at the inflammatory site, and help improve drug utilization.

[0046] Figure 5The results showed that the traditional Chinese medicine nano-preparation prepared in Example 1 was a spherical particle with a uniform particle size of about 100 nm, which was consistent with the particle size detection results. This nano-preparation can utilize the EPR effect to achieve drug aggregation at the lesion site.

[0047] Table 1 shows that the topical transdermal gel prepared in Example 5 has a higher transdermal efficiency than the traditional safflower and Panax notoginseng patch. On the one hand, the prepared topical transdermal gel is a nano-scale formulation, which is more conducive to skin penetration. On the other hand, the drug-loaded liposomes optimized by surface modification have both water-soluble and lipid-soluble properties, resulting in higher drug utilization compared to traditional patches with a single structural component.

[0048] Table 2 shows that the topical transdermal gel prepared in Example 5 has better anti-inflammatory activity than conventional nonsteroidal anti-inflammatory preparations. This is mainly due to the detoxification and efficacy enhancement processing tailored to the different properties of traditional Chinese medicines. The traditional Chinese medicine components with anti-inflammatory activity have high extraction efficiency, high content, and high activity, showing great application prospects.

[0049] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

[0050] The present invention and its embodiments have been described above. This description is not restrictive, and the accompanying drawings are only one embodiment of the present invention. The actual application is not limited to this. In conclusion, if those skilled in the art are inspired by this description and design similar methods and embodiments without departing from the spirit of the present invention, they should all fall within the protection scope of the present invention.

Claims

1. A traditional Chinese medicine nano-formulation targeting osteoarthritis, characterized in that, The traditional Chinese medicine nano-preparation comprises the following raw materials in parts by weight: 15-25 parts of traditional Chinese medicine extract, 4-8 parts of functional peptide, 3 parts of nano zinc oxide, 12-15 parts of coupling agent, 10 parts of modifier, 30-48 parts of phospholipid and 14 parts of cholesterol. The sequence of the functional peptide is: H2N-HHHEVGPLGVRGKRRFQFC-CONH2; The herbal extract comprises the following raw materials: safflower, frankincense, myrrh, aconite, and clematis root. The specific preparation method is as follows: S1: Safflower extract is obtained by extraction, concentration and drying. S2: Mix, extract, and filter frankincense, myrrh, and clematis root to obtain an extract. The extract is then concentrated, purified, and freeze-dried to obtain a co-extract. S3: Soak Aconitum carmichaelii, steam, dry, extract with alcohol, and filter to obtain alcohol extract. After concentration and drying, the alcohol extract is mixed with safflower extract and co-extract to obtain traditional Chinese medicine extract.

2. The osteoarthritis-targeting traditional Chinese medicine nano-formulation according to claim 1, characterized in that, The mass ratio of safflower, frankincense, myrrh, aconite, and clematis is 5-10:15-20:20:10:5; In step S1, the extraction method is supercritical CO2 extraction; In step S2, the extraction process uses a 70% volume fraction of ethanol aqueous solution.

3. The osteoarthritis-targeting traditional Chinese medicine nano-formulation according to claim 1, characterized in that, The coupling agent is DSPE-PEG2000-Mal; The phospholipid is selected from hydrogenated soybean lecithin or sphingomyelin; The modifier is selected from any one of hyaluronic acid, carboxymethyl chitosan, and wolfberry polysaccharide.

4. A method for preparing a traditional Chinese medicine nano-formulation targeting osteoarthritis according to any one of claims 1-3, characterized in that, The specific preparation method is as follows: Step 1: Dissolve and esterify the modifier to obtain an esterified solution, add the functional peptide to it, react and dialyze to obtain the modified peptide; Step 2: Mix and dissolve nano zinc oxide, phospholipids and cholesterol, then add a coupling agent to obtain a lipid solution; Step 3: Disperse the Chinese herbal extract in a buffer solution to obtain a dispersion. After drying the lipid solution into a film, add the dispersion to hydrate and obtain a liposome suspension. Step 4: Homogenize the liposome suspension by sonication to obtain liposomes; Step 5: Add the modified peptide to the liposomes to react and obtain a traditional Chinese medicine nano-formulation targeting osteoarthritis.

5. The method for preparing a traditional Chinese medicine nano-formulation targeting osteoarthritis according to claim 4, characterized in that, In step 1, the esterification process uses EDC and NHS; In step 2, the dissolution process uses a chloroform-ethanol mixed solvent with a volume ratio of 2:

1. In step 3, the buffer solution is a pH 7.4 phosphate buffer.

6. The method for preparing a traditional Chinese medicine nano-formulation targeting osteoarthritis according to claim 4, characterized in that, The aforementioned traditional Chinese medicine nano-preparations can be used to prepare nano-injections, topical transdermal gels, or microneedle patches.

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