Preparation and application of cartilage-targeted metformin and siRNA self-assembled nano-drug

Abstract

The invention discloses preparation and application of a cartilage targeted metformin and siRNA self-assembled nano-drug, and belongs to the field of medicine, the drug comprises BIS-DPA-Zn, NanoMet and NanoMet / siRNA, and the drug is prepared from the BIS-DPA-Zn, the NanoMet and the NanoMet / siRNA. The nano drug is formed by self-polymerization of DPA-Zn and metformin; the particle size of the nano preparation is 100-120nm, the nano preparation shows positive charges, and the positive charges are reduced after siRNA is carried; the metformin content of the nano-drug is 80-85%, and the nano-drug is applied to drugs for treating osteoarthritis. In addition, Met and siRNA in the prepared medicine can effectively inhibit NF-kappa B, so that cartilage cell autophagy is promoted through an AMPK way, cartilage cell apoptosis is inhibited, and diagnosis and treatment integration and synergistic treatment of osteoarthritis are achieved.

Description

Technical Field

[0001] The invention belongs to the field of medicine, and specifically relates to the preparation and application of a cartilage-targeted metformin and siRNA self-assembly nanomedicine. Background Art

[0002] Osteoarthritis (OA) is a chronic degenerative joint disease that is prevalent in the elderly population. It is a global public health challenge that has a great impact on motor function and overall quality of life. There are currently a variety of methods for treating OA, such as physical therapy, drug intervention, and surgical intervention. Compared with other methods, drug intervention has the advantages of high efficacy, convenient medication, and less trauma, so it has great potential in preventing OA. Traditional oral drugs cannot actively target cartilage lesions. Anti-inflammatory drugs injected simply into the joint cavity will diffuse in the joint cavity and will be rapidly lost with metabolism. In addition, due to the concentration gradient and dense matrix network structure, there will be a certain amount of loss in the process of drugs crossing the dense cartilage matrix to reach chondrocytes, resulting in low bioavailability of drugs in the lesion area. Therefore, it is crucial to develop effective drugs to improve disease progression.

[0003] Autophagy is a metabolic pathway that plays a key role in maintaining cell stability and protecting the intracellular environment. In addition, autophagy also has an important regulatory effect on inflammation and affects the pathological development of inflammatory diseases. In the progression of osteoarthritis, "inflammation" plays a vital role. The activation of the inflammatory NF-κB signaling pathway can lead to leukocyte infiltration and the release of proinflammatory factors, which will stimulate the mTOR signaling cascade, thereby hindering the level of autophagy, and is the "upstream" of inhibiting autophagy. Inflammation and autophagy interact with each other and promote the development of the disease synergistically. In view of this, it is imperative to explore feasible interventions to disrupt the NF-κB inhibition and autophagic degradation process to maintain an appropriate level of chondrocyte autophagy and reduce inflammation during the progression of OA. In addition, there are no reports on methods for nanoparticles to synergistically inhibit inflammation and activate autophagy to alleviate the progression of osteoarthritis.

[0004] Metformin (Met) is currently the drug of choice for the treatment of type 2 diabetes. Studies have shown that metformin is well tolerated and beneficial for various age-related diseases. Metformin can affect cellular processes, including cell metabolism, inflammation, and autophagy. In a mouse arthritis model, Met activated AMPK / SIRT1-mediated autophagy, thereby reducing cartilage degradation. Met can effectively alleviate the progression of arthritis by targeting various types of cells, including adipocytes, synovial macrophages, and chondrocytes. Met can promote the production of collagen-II and lectins in nucleus pulposus cells, indicating that it has a positive effect on the extracellular matrix. In addition, metformin also has the ability to inhibit NF-kB and regulate cytokine networks, which can be used as a potential therapeutic target. However, due to the small molecular weight of metformin, intravenous or intra-articular administration leads to its rapid clearance from the blood, so its oral bioavailability is low and unstable (50-60%), and the biological half-life is relatively short, which is 0.9-2.6 hours. In addition, its hydrophilicity also poses a challenge to penetrating the lipid bilayer of the cell membrane by passive diffusion, further reducing the bioavailability of the drug. Therefore, extending the half-life and improving the bioavailability of the drug are key prerequisites for optimizing the efficacy of Met.

[0005] Gene therapy is considered a potential treatment for inflammation. However, successful gene therapy, such as the widely used RNA interference (RNAi), largely relies on efficient transfection. Viral vectors are undoubtedly considered the most effective tools in this field. However, the inherent disadvantages of viruses, such as toxicity, immunogenicity and tumorigenicity, have raised concerns about their safety. Based on the excellent characteristics of non-viral vectors such as convenient design, intelligent responsiveness and low cytotoxicity, they have great prospects in gene delivery. Non-viral vectors including liposomes, polymers and inorganic materials may become good substitutes for viruses. Many existing vectors show efficient transfection ability in vitro, but their performance is severely weakened when they encounter complex physiological environments in vivo. In order to effectively deliver therapeutic nucleic acids into target cells, gene vectors must overcome extracellular and intracellular barriers, such as (i) avoiding being captured by the immune system and reaching the lesion; (ii) preventing lysosomal degradation of therapeutic nucleic acids after being taken up by target cells.

[0006] There is growing interest in the use of functionalized nanocarriers to deliver anti-inflammatory drugs or siRNA, with the goal of enhancing drug circulation in the body and reducing the required drug dose. However, these therapeutic agents still face limitations such as loading efficiency, carrier degradation, and incomplete drug release. To address these issues, a new concept has been proposed, namely "self-assembled nanomedicines", which refer to preparations composed only of pharmacologically active substances without non-therapeutic additives. Summary of the invention

[0007] The present invention proposes a cartilage-targeted metformin and siRNA self-assembled nano-drug synergistic system to inhibit inflammation and activate autophagy, thereby achieving the purpose of relieving inflammation and alleviating the progression of osteoarthritis.

[0008] To achieve the above-mentioned purpose, the present application relates to the preparation and application of a cartilage-targeted metformin and siRNA self-assembled nanomedicine, wherein the drug composition includes BIS-DPA-Zn, NanoMet, and NanoMet / siRNA;

[0009] The nano drug is formed by self-polymerization of DPA-Zn and metformin;

[0010] The particle size of the nanoparticle preparation is 100-120 nm, showing a positive charge, which decreases after carrying siRNA;

[0011] The metformin content of the nano drug is 80-85%.

[0012] Furthermore, the preparation method of the cartilage-targeted metformin and siRNA self-assembled nanomedicine comprises:

[0013] Step S1: preparing BIS-DPA-Zn;

[0014] Step S2: Preparation of NanoMet: Dissolve BIS-DPA-Zn in dimethyl sulfoxide until completely dissolved; Dissolve Met hydrochloride in ultrapure water; Slowly add the BIS-DPA-Zn solution to the Met aqueous solution and stir; Ultrafiltration using a 3000K molecular weight cutoff membrane to obtain nanometal; Take a portion of the sample for freeze drying and quantification;

[0015] Step S3: Preparation of NanoMet / siRNA: Disperse siRNA in enzyme-free water, then add NanoMet to the siRNA solution, centrifuge, and collect the precipitate to successfully prepare.

[0016] Furthermore, in step S1, BIS-DPA-Zn includes compound 1, and the preparation method of compound 1 specifically includes:

[0017] Step S101, add methanol and BIS into a flask, and stir until dispersed;

[0018] Step S102, then dissolve BOC in methanol, add the BOC solution into the BIS solution, slowly add K2CO3 and stir;

[0019] Step S103, after rotary evaporation at a certain temperature for 20 to 40 minutes, adding ethyl acetate, and then extracting with water;

[0020] Step S104, take the organic phase, add Na2SO4, stir to remove excess water, filter, concentrate by rotary evaporation, and then put into a vacuum drying oven to obtain solid particle compound 1.

[0021] Furthermore, in step S1, BIS-DPA-Zn includes compound 2, and the preparation method of compound 2 specifically includes:

[0022] Step S111, adding formaldehyde solution, HCl solution and isopropanol to DPA, and stirring until clear;

[0023] Step S112, dissolving compound 1 in isopropanol and adding the mixture to the reactant, followed by reflux under nitrogen protection;

[0024] Step S113, adding dichloromethane, water and K2CO3 to neutralize, extracting, passing through a silica gel column and then rotary evaporating to concentrate, collecting the brown liquid obtained after passing through the column, to obtain compound 2.

[0025] Furthermore, in step S1, BIS-DPA-Zn includes compound 3, and the preparation method of compound 3 specifically includes:

[0026] Step S121, taking compound 2 and adding it into dichloromethane, slowly adding trifluoroacetic acid solution, and stirring;

[0027] Step S122, adding K2CO3 solution for neutralization, concentrating by rotary evaporation, and separating by silica gel column to obtain compound 3.

[0028] Furthermore, the preparation of BIS-DPA-Zn in step S1 specifically includes: dissolving compound 3 in dimethyl sulfoxide, and then adding Zn(NO3)2·6H2O to obtain BIS-DPA-Zn.

[0029] A cartilage-targeted metformin and siRNA self-assembled nanomedicine is prepared according to the method.

[0030] The cartilage-targeted metformin and siRNA self-assembled nanomedicine is used in preparing medicine for treating osteoarthritis.

[0031] Compared with the prior art, the advantages and effects of this application are as follows:

[0032] 1. The NanoMet prepared in the present application has a clear spherical nanostructure and a relatively high drug content (1.98% for siRNA and 84.21% for Met). The drug prepared in the present application is composed of pharmacologically active substances and does not contain non-therapeutic additives.

[0033] 2. The Met and siRNA in this application can also effectively inhibit NF-κB, thereby promoting chondrocyte autophagy through the AMPK pathway, inhibiting chondrocyte apoptosis, and realizing integrated diagnosis and treatment and synergistic treatment of osteoarthritis.

[0034] The above description is only an overview of the technical solution of the present application. In order to more clearly understand the technical means of the present application so that it can be implemented in accordance with the contents of the specification, and to make the above and other purposes, features and advantages of the present application more obvious and easy to understand, the following is a detailed description of the preferred embodiments of the present application in conjunction with the accompanying drawings as follows.

[0035] Based on the detailed description of the specific embodiments of the present application in combination with the accompanying drawings below, those skilled in the art will become more aware of the above and other objects, advantages and features of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings required for use in the embodiments or the prior art descriptions are briefly introduced below. Obviously, the drawings described below are some embodiments of the present application. For ordinary technicians in this field, other drawings can also be obtained based on these drawings without creative work. In all drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, each element or part is not necessarily drawn according to the actual scale.

[0037] in:

[0038] Figure 1 TEM image of NanoMet.

[0039] Figure 2 This is the DLS image of NanoMet.

[0040] Figure 3 is the Zeta potential of NanoMet and NanoMet / siRNA.

[0041] Figure 4 The XPS spectrum of NanoMet and the fitting diagram of each element peak.

[0042] Figure 5 Aqueous solutions of NanoMet prepared at different ratios.

[0043] Figure 6 UV absorption spectra of DPA-Zn, Met and NanoMet.

[0044] Figure 7 Electrophoretic delay analysis of siRNA (10 pmol) binding to NanoMet.

[0045] Figure 8 This is the chemical synthesis route of BIS-DPA-Zn. Specific embodiments

[0046] To make the purpose, technical scheme and advantages of the embodiment of the present application clearer, the technical scheme in the embodiment of the present application will be clearly and completely described below in conjunction with the accompanying drawings in the embodiment of the present application. Obviously, the described embodiment is a part of the embodiment of the present application, rather than all of the embodiments. In the following description, specific details such as specific configuration and components are provided only to help fully understand the embodiments of the present application. Therefore, it should be clear to those skilled in the art that various changes and modifications can be made to the embodiments described herein without departing from the scope and spirit of the present application. In addition, for clarity and brevity, the description of known functions and structures is omitted in the embodiment.

[0047] It should be understood that the references to "one embodiment" or "this embodiment" throughout the specification mean that the specific features, structures, or characteristics associated with the embodiment are included in at least one embodiment of the present application. Therefore, the references to "one embodiment" or "this embodiment" appearing throughout the specification do not necessarily refer to the same embodiment. In addition, these specific features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0048] In addition, the present application may repeat reference numerals and / or letters in different examples. This repetition is for the purpose of simplicity and clarity, and does not in itself indicate the relationship between the various embodiments and / or settings discussed.

[0049] The term "and / or" in this article is only a description of the association relationship of associated objects, indicating that there can be three relationships. For example, A and / or B can mean: A exists alone, B exists alone, and A and B exist at the same time. The term " / and" in this article describes another type of association object relationship, indicating that there can be two relationships. For example, A / and B can mean: A exists alone, and A and B exist alone. In addition, the character " / " in this article generally indicates that the associated objects before and after are in an "or" relationship.

[0050] The term "at least one" in this article is merely a description of the association relationship of associated objects, indicating that there may be three relationships. For example, at least one of A and B can mean: A exists alone, A and B exist at the same time, and B exists alone.

[0051] It should also be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprises" or any other variations thereof are intended to cover non-exclusive inclusions.

[0052] Example 1

[0053] This example introduces a cartilage-targeted metformin and siRNA self-assembled nanomedicine, the drug comprising BIS-DPA-Zn, NanoMet, and NanoMet / siRNA;

[0054] The carrier-free nanoassembly of Met and siRNA (named NanoMet / siRNA) was formed through Zn(II)-dipicolylamine (DPA-Zn)-promoted interactions, including electrostatic and π-π stacking interactions.

[0055] The two additional guanosine groups in Met can interact with metal ions (including Zn(II), an indispensable metal ion in cells) through ionic interactions or complexation, thereby self-assembling into NanoMet. In addition, the DPA-Zn complex can interact with the phosphate of siRNA, making it an effective gene carrier.

[0056] Furthermore, DPA-Zn is a positively charged organometallic complex that has been reported to bind to fluorescent dyes such as Cy5.5 and Cy7, highlighting its potential in imaging cartilage degeneration and OA by binding to negatively charged glycosaminoglycans (GAGs).

[0057] Please refer to Figure 1 As shown, the prepared NanoMet has a clear spherical nanostructure and a relatively high drug content (1.98% for siRNA and 84.21% for Met); please refer to Figure 2 As shown, the average hydrated particle size of the prepared NanoMet is about 100 nm.

[0058] Met and p65 siRNA can also effectively inhibit NF-κB, thereby promoting chondrocyte autophagy through the AMPK pathway and inhibiting chondrocyte apoptosis, thus achieving integrated diagnosis and treatment of OA and synergistic treatment.

[0059] Example 2

[0060] This embodiment introduces a cartilage-targeted metformin and siRNA self-assembled nanomedicine, which is further introduced based on the above embodiment.

[0061] Please refer to Figure 3 As shown, Figure 3 The Zeta potential diagram of NanoMet and NanoMet / siRNA. The potentials of the prepared NanoMet and NanoMet / siRNA are approximately 15.2±0.4 mV and 6.5±1.2 mV.

[0062] For further information, please refer to Figure 4 As shown, Figure 4 This is the XPS spectrum of NanoMet, the XPS spectrum of the prepared NanoMet and the fitting of each element peak, and the elements include Zn3P, O1s, N1s, and C1s.

[0063] For further information, please refer to Figure 5 As shown, Figure 5 The nanoparticles are formed by different ratios of DPA-Zn and Met. The suspension stability of the aqueous solution of NanoMet prepared at different ratios is different. When the DPA-Zn ratio is 1:1, the aqueous solution of NanoMet is transparent and clear; when the DPA-Zn ratio is 1:20, the aqueous solution of NanoMet is light yellow; when the DPA-Zn ratio is 1:50, the aqueous solution of NanoMet is light yellow and turbid.

[0064] For further information, please refer to Figure 6 As shown, the UV absorption spectra of the prepared NanoMet, Met and Dpa-Zn;

[0065] For further information, please refer to Figure 7 As shown, Figure 7 This is the electrophoresis delay analysis of the binding of siRNA (10 pmol) to NanoMet. The results show that the higher the mass content of NanoMet, the stronger its binding ability to siRNA.

[0066] Example 3

[0067] This embodiment introduces a method for preparing a cartilage-targeted metformin and siRNA self-assembled nanomedicine, which is further described based on the above embodiment. The preparation method includes:

[0068] Step S1: preparing BIS-DPA-Zn;

[0069] Step S2: Preparation of NanoMet: First, 5 mg of BIS-DPA-Zn was dissolved in 100 μL of dimethyl sulfoxide until completely dissolved. Then, 100 mg of Met hydrochloride was dissolved in 40 mL of ultrapure water. The BIS-DPA-Zn solution was slowly added to the Met aqueous solution and stirred for 4 hours. Ultrafiltration was performed using a 3000K molecular weight cutoff membrane to obtain the nanometal. A portion of the sample was freeze-dried for quantification.

[0070] Step S3: Preparation of NanoMet / siRNA: Disperse siRNA in enzyme-free water, then add NanoMet to the siRNA solution, centrifuge for 10 minutes, and collect the precipitate to successfully prepare.

[0071] The final drug is a round nanoformulation formed by self-polymerization of DPA-Zn and metformin, with a particle size of about 110 nm and a positive charge. The positive charge is reduced after carrying siRNA. Drug release measurements show that the metformin content of the nanoformulation is about 83%.

[0072] Example 4

[0073] This example introduces a method for preparing a cartilage-targeted metformin and siRNA self-assembled nanomedicine. Please refer to Figure 8 As shown, based on the above embodiment, the preparation of BIS-DPA-Zn in step S1 includes compound 1, compound 2, and compound 3. The preparation method of compound 1 in BIS-DPA-Zn specifically includes:

[0074] Step S101, add 40 ml of methanol and 2.74 g of Bis into a 250 ml flask, and stir until dispersed;

[0075] Step S102, then take 4.84 ml of BOC and dissolve it in 5 ml of methanol, add the BOC solution into the BIS solution, slowly add 8.3 g of K2CO3 and stir for 4 hours;

[0076] Step S103, after rotary evaporation at 60° C. for 30 minutes, 50 ml of ethyl acetate was added, and then 100 ml of water was used for extraction;

[0077] Step S104: Take the organic phase, add 20-30 g Na2SO4, stir to remove excess water, filter, and concentrate by rotary evaporation to obtain 4 g, and then put it into a vacuum drying oven to obtain solid particle compound 1.

[0078] Furthermore, the preparation method of compound 2 in step S1 specifically includes:

[0079] Step S111, add 1.013 ml of formaldehyde solution, 1 ml of 1M HCl solution and 30 ml of isopropanol to 2.49 g of DPA, and stir until clear;

[0080] Step S112, dissolving 1.48 g of compound 1 in 5 ml of isopropanol and adding the mixture to the reactant, and then refluxing at 80° C. for 2-3 d under nitrogen protection;

[0081] Step S113, adding 50 ml of dichloromethane, 100 ml of water and K2CO3 for neutralization, extracting, passing through a silica gel column and then rotary evaporating and concentrating, collecting the brown liquid obtained after passing through the column to obtain compound 2.

[0082] Furthermore, the preparation method of compound 3 in step S1 specifically includes:

[0083] Step S121, take 1 g of compound 2 and add it into 10 ml of dichloromethane, slowly add 30 ml of 10% trifluoroacetic acid (TFA) solution, and stir for 4 hours;

[0084] Step S122, adding K2CO3 solution for neutralization, concentrating by rotary evaporation, and separating by silica gel column to obtain compound 3;

[0085] The preparation of BIS-DPA-Zn in step S1 specifically includes: dissolving compound 3 in dimethyl sulfoxide, and then adding Zn(NO3)2·6H2O to obtain BIS-DPA-Zn.

[0086] The above description is only the preferred embodiment of the present invention, which does not limit the protection scope of the present invention. For those skilled in the art, the present invention can be modified and varied in various ways. Within the spirit and principle of the present invention, any change, modification, replacement, integration and parameter change of these embodiments by conventional substitution or capable of achieving the same function without departing from the principle and spirit of the present invention shall fall within the protection scope of the present invention.

Claims

1. A cartilage-targeted metformin and siRNA self-assembled nanomedicine, characterized in that: Its composition includes BIS-DPA-Zn, NanoMet, and NanoMet / siRNA; The nano drug is formed by self-polymerization of DPA-Zn and metformin; The particle size of the nanoparticle preparation is 100-120 nm, showing a positive charge, which decreases after carrying siRNA; The metformin content of the nano drug is 80-85%.

2. The method for preparing a cartilage-targeted metformin and siRNA self-assembled nanomedicine according to claim 1, characterized in that: The preparation method comprises: Step S1: preparing BIS-DPA-Zn; Step S2: Preparation of NanoMet: Dissolve BIS-DPA-Zn in dimethyl sulfoxide until completely dissolved; Dissolve Met hydrochloride in ultrapure water; Slowly add the BIS-DPA-Zn solution to the Met aqueous solution and stir; Ultrafiltration using a 3000K molecular weight cutoff membrane to obtain nanometal; Take a portion of the sample for freeze drying and quantification; Step S3: Preparation of NanoMet / siRNA: Disperse siRNA in enzyme-free water, then add NanoMet to the siRNA solution, centrifuge, and collect the precipitate to successfully prepare.

3. The method for preparing a cartilage-targeted metformin and siRNA self-assembled nanomedicine according to claim 2, characterized in that: In step S1, BIS-DPA-Zn includes compound 1, and the preparation method of compound 1 specifically includes: Step S101, add methanol and BIS into a flask, and stir until dispersed; Step S102, then dissolve BOC in methanol, add the BOC solution into the BIS solution, slowly add K2CO3 and stir; Step S103, after rotary evaporation at a certain temperature for 20 to 40 minutes, adding ethyl acetate, and then extracting with water; Step S104, take the organic phase, add Na2SO4, stir to remove excess water, filter, concentrate by rotary evaporation, and then put into a vacuum drying oven to obtain solid particle compound 1.

4. The method for preparing a cartilage-targeted metformin and siRNA self-assembled nanomedicine according to claim 2, characterized in that: In step S1, BIS-DPA-Zn includes compound 2, and the preparation method of compound 2 specifically includes: Step S111, adding formaldehyde solution, HCl solution and isopropanol to DPA, and stirring until clear; Step S112, dissolving compound 1 in isopropanol and adding the mixture to the reactant, followed by reflux under nitrogen protection; Step S113, adding dichloromethane, water and K2CO3 to neutralize, extracting, passing through a silica gel column and then rotary evaporating to concentrate, collecting the brown liquid obtained after passing through the column, to obtain compound 2.

5. The method for preparing a cartilage-targeted metformin and siRNA self-assembled nanomedicine according to claim 2, characterized in that: In step S1, BIS-DPA-Zn includes compound 3, and the preparation method of compound 3 specifically includes: Step S121, taking compound 2 and adding it into dichloromethane, slowly adding trifluoroacetic acid solution, and stirring; Step S122, adding K2CO3 solution for neutralization, concentrating by rotary evaporation, and separating by silica gel column to obtain compound 3.

6. The method for preparing a cartilage-targeted metformin and siRNA self-assembled nanomedicine according to claim 2, characterized in that: The preparation of BIS-DPA-Zn in step S1 specifically includes: dissolving compound 3 in dimethyl sulfoxide, and then adding Zn(NO3)2·6H2O to obtain BIS-DPA-Zn.

7. A cartilage-targeted metformin and siRNA self-assembled nanomedicine prepared according to the method of claims 2-6.

8. Use of the cartilage-targeted metformin and siRNA self-assembled nanomedicine according to claim 1 in the preparation of a drug for treating osteoarthritis.

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