CN111529713B - AuNPs/miR-140 compound and preparation method and application thereof - Google Patents

AuNPs/miR-140 compound and preparation method and application thereof Download PDF

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CN111529713B
CN111529713B CN202010392604.0A CN202010392604A CN111529713B CN 111529713 B CN111529713 B CN 111529713B CN 202010392604 A CN202010392604 A CN 202010392604A CN 111529713 B CN111529713 B CN 111529713B
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CN111529713A (en
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黄桂华
杨雪华
黄小雨
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Shandong University
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M5/00—Devices for bringing media into the body in a subcutaneous, intra-vascular or intramuscular way; Accessories therefor, e.g. filling or cleaning devices, arm-rests
    • A61M5/178—Syringes
    • A61M5/20—Automatic syringes, e.g. with automatically actuated piston rod, with automatic needle injection, filling automatically
    • A61M2005/2006—Having specific accessories

Abstract

The invention provides an AuNPs/miR-140 compound and a preparation method and application thereof, wherein the AuNPs/miR-140 compound is obtained by compounding gold nanoparticle AuNPs and miR-140 through electrostatic adsorption, and the mass ratio of AuNPs to miR-140 is 5-28.8: 1. The AuNPs/miR-140 compound can efficiently deliver miR-140 to chondrocytes, the cell transfection rate is close to 100%, the AuNPs/miR-140 compound has no obvious toxicity to the chondrocytes, the expression of miR-140 in the chondrocytes can be up-regulated, the expression level of COL2A mRNA in the chondrocytes is increased, and the AuNPs/miR-140 compound has a repairing effect and a certain anti-inflammatory capacity on the chondrocytes. The pharmaceutical composition of the AuNPs/miR-140 compound and lornoxicam has obvious effects in cartilage protection, cartilage repair promotion and arthritis disease prevention and treatment.

Description

AuNPs/miR-140 compound and preparation method and application thereof
Technical Field
The invention relates to the field of medicines, and in particular relates to an AuNPs/miR-140 compound and a preparation method and application thereof.
Background
The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.
Osteoarthritis (OA) is a common degenerative disease of the joint cartilage. Many factors such as obesity, age increase, strain, trauma, congenital abnormality of joint can cause degeneration and loss of joint cartilage, which in turn leads to regeneration of bone at joint margin and subchondral bone and causes synovium inflammation. OA can cause pain, deformity, restricted movement, and muscle atrophy in the joints of patients, and has become one of the most common disabling diseases. It is well developed in middle-aged and elderly people, and the incidence rate of people over 65 years old is over 50%, and the incidence rate of OA also tends to rise year by year with the aging progress of the population. OA poses a serious threat to human health and life, and places a huge economic burden on patients and society. Currently, there is no effective way to cure OA, mainly through symptomatic treatment to improve the condition of OA. Clinical treatment means for OA mainly include basic treatment, drug treatment and surgical treatment. The basic treatment mainly refers to education of patients, self-management of patients, exercise, weight reduction during obesity, walking aid use and the like, and the basic treatment is the preferred treatment mode for OA patients with mild early symptoms. The surgical treatment mainly comprises joint replacement, joint osteotomy and arthroscopic surgery. Surgical treatment is also an effective treatment for advanced stage OA patients. Surgical treatment can improve joint function to some extent, but its long-term efficacy and prognosis are controversial. Drug therapy is also the most prominent mode of treating OA. Non-steroidal anti-inflammatory drugs have been the common drugs for treating OA, and are mainly used clinically to relieve pain and slow OA progression in patients. Hyaluronic acid is also widely used in the treatment of OA. Hyaluronic acid is an endogenous substance, and hyaluronic acid in the joint cavity has a lubricating effect, while hyaluronic acid in the joint cavity of a patient with OA is reduced, so that OA can be treated by injecting hyaluronic acid into the joint cavity of the patient, but there is a concern about the curative effect and safety thereof. Besides, the commonly used medicines include glucocorticoid, glucosamine, bisphosphonate and the like. However, none of the above treatments are effective in treating OA, and thus, there is still a need for a better treatment for OA. Gene therapy for OA means that the desired gene is introduced into chondrocytes to change the expression of the corresponding gene, thereby protecting cartilage. Hung et al, 1994, for the first time demonstrated that the delivery of an interleukin 1receptor antagonist Gene into a joint could inhibit intra-articular pathology, which opened the way for Gene Therapy of OA (Hung G L, Galea-Lauri J, Mueller G M, et al, expression of intra-articular responses to intra-articular in-1by transfer of the intra-articular in-1receptor antagonist Gene to synoveium [ J ]. Gene Therapy,1994,1(1): 64-69). Then, in addition to DNA, interfering RNA (sirna), micro RNA (miRNA), and the like, are also used for gene therapy of OA.
The research on the role of miR-140 in the OA process is relatively mature. miR-140 is a miRNA that is specifically expressed in cartilage tissue. Miyaki et al found that cartilage of miR-140 knockout mice degenerated (Miyaki S, Sato T, Inoue A, et al. MicroRNA-140 plants dual rollers in bone tissue degradation and hoscostasis [ J ]].Genes&Level, 2010,24(11):1173-1185.), indicating that the miR-140 gene may have protective effect on articular cartilage. Although mirnas have potential therapeutic effects on osteoarthritis, mirnas still face many challenges in practical applications. The inventor finds that miRNA is difficult to endocytose into cells to play a role because of large molecular weight, strong hydrophilicity and self-negative charge. Moreover, miRNA is unstable, has short half-life and is easily degraded by nuclease in vivo. The miRNA can be efficiently delivered into cells to play a role by using a proper gene delivery vector, and the gene delivery vector can also protect the miRNA from degradation of nuclease. The inventors have found that, at present, viral vectors and non-viral vectors are commonly used as gene delivery vectors. Viral vectors are highly efficient for transfection, but they have safety problems and are difficult to prepare on a large scale (Yang N.an overview of visual and non-visual delivery systems for microRNA [ J ]]International journal of pharmaceutical initiation 2015,5(4): 179.). Non-viral vectors include primarily liposomes, cationic polymers, and the like. Liposomes are lipid vesicles with an aqueous core composed of a phospholipid bilayer. As early as 1987, Felgner et al used cationic lipid DOTMA to prepare cationic liposomes which condensed DNA and thereby delivered it into cells, and lipofection was 5-100 times more efficient than calcium phosphate or DEAE-dextran transfection techniques, depending on the cell line. Liposomes are then widely used for transfection of genes. Currently, there are many commercially available lipofection reagents, such as Lipofectamine RNAiMAX,
Figure BDA0002486388100000011
SilentFectTMAnd siPORTTMAnd the like. However, the liposome has the disadvantages of short blood circulation time while being well applied, and has poor stability. Cationic polymers are a class of positively charged polymers that can efficiently compress negatively charged genes by electrostatic action to deliver the genes into cells. Currently, most of the cationic polymers studied are Polyethyleneimine (PEI), poly-L-lysine (PLL), dendrimer, cyclodextrin, and the like. The high positive charge density allows cationic polymerizationThe compound has high gene transfection efficiency generally, but has high cytotoxicity just because of high positive charge density. For cationic polymers, how to have low cytotoxicity while maintaining high transfection efficiency is still a difficult problem to overcome.
Lornoxicam (Lnxc), a novel non-steroidal anti-inflammatory drug, has analgesic, anti-inflammatory and antipyretic properties. The mechanism of action is to inhibit the production of Prostaglandins (PGs) at the site of inflammation by selectively inhibiting the synthesis of cyclooxygenase-2. Is mainly used for treating postoperative pain, lumbosacral pain, rheumatoid arthritis, osteoarthritis or ankylosing spondylitis. The dosage forms on the market mainly comprise tablets and injections. Lnxc has two advantages over other non-steroidal anti-inflammatory drugs: firstly, Lnxc has stronger anti-inflammatory and analgesic effects, and research shows that Lnxc inhibits PGD in rat polymorphonuclear leukocytes in vitro2The formed effect is about 100 times stronger than that of tenoxicam, about 50 times stronger than that of piroxicam and about 20 times stronger than that of diclofenac sodium; secondly, Lnxc has relatively small side effect, probably because the elimination half-life period is relatively short (3-5 h) and the clinical dosage is small (4-8 mg). Lnxc is clinically used for OA mainly because of its anti-inflammatory analgesic effect. Although the side effects are relatively small compared to other non-steroidal anti-inflammatory drugs, the inventors found that Lnxc can still cause gastrointestinal side effects including gastrointestinal pain, dyspepsia, nausea, vomiting, etc., after oral administration. In addition, non-steroidal anti-inflammatory drugs administered according to FDA recommendations also increase the risk of cardiovascular diseases such as myocardial infarction and stroke.
Disclosure of Invention
Therefore, the invention aims to provide an AuNPs/miR-140 compound and a preparation method thereof, application of the AuNPs/miR-140 compound in preparation of a medicament for preventing and treating osteoarthritis diseases and/or in preparation of a medicament for promoting cartilage repair and/or in preparation of a medicament for protecting cartilage by alone or in combination with a non-steroidal anti-inflammatory drug lornoxicam, and a method for treating osteoarthritis diseases, promoting cartilage repair and/or protecting cartilage by using the AuNPs/miR-140 compound in treatment of osteoarthritis diseases and in combination with the non-steroidal anti-inflammatory drug lornoxicam.
The AuNPs/miR-140 compound disclosed by the invention has good pharmaceutical activity, has high stability, safety, cell-entering capability and gene regulation effect, can be used for remarkably up-regulating the expression of miR-140 in chondrocytes and increasing the expression level of COL2AmRNA in the chondrocytes, can be used for remarkably protecting cartilage, promoting cartilage repair and inhibiting the development of inflammation in an OA process, and has a certain anti-inflammatory effect. The AuNPs/miR-140 compound can overcome the defects that miR-140 has large molecular weight, strong hydrophilicity and is difficult to enter cells to play a role due to self negative charge, can also overcome the defects that miR-140 is unstable and has short half-life period and is easy to degrade by nuclease in vivo, well delivers miR-140 into chondrocytes, has the test cell transfection rate of up to 100 percent and has better safety, has no obvious toxicity to chondrocytes in a larger dose concentration range, and the verified concentration span can reach 3-200nM, while under the same concentration condition, other gene vectors, such as the most commonly used cationic polymer vectors with extremely high transfection efficiency, such as Polyethyleneimine (PEI) cationic vector (PEI/miR-140 complex), exhibit extremely high cytotoxicity at drug doses exceeding 50 nM. In the embodiment of the invention, the inventor finds that the synovial inflammation and the cartilage injury of OA are mutually promoted processes, the synovial inflammation can aggravate the injury of the cartilage, and the fragments generated by the cartilage injury can also be used as foreign matters to further cause the synovial inflammation, the AuNPs/miR-140 of the invention can repair the cartilage injury and simultaneously inhibit the further development of the inflammation, and the lornoxicam can prevent the further injury of the cartilage by eliminating the synovial inflammation besides having a certain analgesic effect. The lornoxicam and AuNPs/miR-140 are combined to play a role in supplementing each other. The invention adopts the combination of AuNPs/miR-140 compound and lornoxicam, can obviously improve joint swelling, repair and improve the damage of femoral ankle and tibial plateau, prevent cartilage degeneration, improve the treatment effect to the extent equivalent to that of normal tissues, has obvious prevention and treatment effects, greatly shortens the treatment time by combining the application, and can realize the obvious improvement and repair effects after being applied for 4 weeks by combination and reach the extent close to the normal tissues after 6 weeks.
Specifically, the technical scheme of the invention is as follows:
in a first aspect of the invention, the AuNPs/miR-140 compound is obtained by compounding gold nanoparticles AuNPs and miR-140, wherein the mass ratio of the AuNPs to the miR-140 is 5-28.8: 1.
In embodiments of the invention, AuNPs can protect miR-140 from nuclease degradation, which indicates that miR-140 is not only loosely bound to the AuNPs surface simply by electrostatic interaction, but also that AuNPs encapsulated by PEI are tightly compressed inside (it is understood that miR-140 is tightly bound and encapsulated inside the AuNPs particles), which allows the AuNPs/miR-140 complex to be stable in the environment of nucleases such as rnase a for at least 24 h.
In the embodiment of the invention, the inventor finds that the compression condition of miR-140 is closely related to the mass ratio of AuNPs and miR-140. Therefore, the invention considers the mass ratio of AuNPs and miR-140, and the result shows that when the mass ratio is not less than 1.8:1, miR-140 is basically and completely compressed by AuNPs, and the drug loading is complete; in a further embodiment, the present inventors continued to examine the effect of the mass ratio and found that the particle size of the composite gradually decreased as the mass ratio increased from 1.8:1 to 28.8:1, and that the particle size of the composite was (27.04 ± 2.59) nm, the particle size was the smallest, and PDI was 0.34 ± 0.03, and the dispersibility was good when the mass ratio of the composite reached 28.8: 1; and when the mass ratio is increased from 1.8:1 to 28.8:1, the potential of the compound is gradually increased and finally stabilizes at about 30 mV. However, when the mass ratio is 1.8:1 or slightly greater than 1.8:1, such as 1.8:1, the average particle size of AuNPs and miR-140 increases to 82nm after combination, and further increases to 3.6:1, and the average particle size of AuNPs and miR-140 increases to 79nm after combination, which indicates that AuNPs may form relatively large aggregates, and the dispersibility and stability are affected. In addition, the size of the particle size of the compound can affect the dispersibility and stability of the compound, and the compound obtained when the mass ratio of the AuNPs to the miR-140 is 5-28.8:1 has a more proper particle size, particularly when the mass ratio of the AuNPs to the miR-140 is 14.4:1, particularly 28.8:1, the particle size of the AuNPs/miR-140 compound is more ideal, and the cell transfection effect is optimal, for example, in some embodiments of the invention, when the ratio is 28.8:1, the chondrocyte transfection rate of the AuNPs/miR-140 compound is close to 100%. Thus, in an embodiment of the invention, the mass ratio of miR-140 to AuNPs is 5-28.8:1, preferably 7.2-28.8:1, more preferably 14.4:1, and most preferably 28.8: 1.
In an embodiment of the invention, the AuNPs/miR-140 complex has a particle size of 24-85nm, preferably 24-40nm, more preferably 24-30nm (average particle size is about 27 nm); PDI is 0.3-0.6, and Zeta potential is 20-30 mV. In a more preferred embodiment, when the mass ratio of AuNPs to miR-140 is 28.8:1, the AuNPs/miR-140 complex has a small particle size (27.04 + -2.59) nm and a suitable zeta potential of 30mV, and has good dispersibility, namely PDI of 0.34 + -0.03.
In an embodiment of the invention, the AuNPs are prepared from HAuCl4Obtained by compounding with Polyethyleneimine (PEI), and has the structure: the inner core is a regular sphere of elemental Au coated PEI, with a particle size of 17-27nm, preferably greater than 20nm (average particle size of about 22nm), a PDI of 0.23-0.3 (average of about 0.25), and a Zeta potential of 31-34mV (average of about 32 mV).
In a second aspect of the invention, the invention provides a method for preparing the AuNPs/miR-140 complex in the first aspect, which comprises the steps of mixing miR-140 with an AuNPs solution uniformly and then standing for incubation. When mixing, the mass ratio of the miR-140 to AuNPs is 1.8-28.8:1, preferably 7.8-28.8:1, and more preferably 28.8: 1.
In a more preferred embodiment of the invention, the miR-140 is linked to AuNPs by electrostatic adsorption, and the preparation method comprises the following steps: and mixing the miR-140 solution and the AuNPs solution, immediately swirling for 30s, uniformly mixing, and then standing and incubating for 30min to obtain the AuNPs/miR-140 compound.
In an embodiment of the present invention, the preparation of the AuNPs solution comprises: adding HAuCl into the mixture under the condition of stirring in water bath4Adding a PEI aqueous solution into the solution, and reacting to obtain the product. In an embodiment of the invention, HAuCl is added to HAuCl4Adding PEI aqueous solution into the solution and adding HAuCl into the PEI aqueous solution4The AuNPs obtained by the two solution modes have obvious difference in structure, the difference is reflected in the embodiment of the invention, the gold core structure completely coated by PEI is easier to obtain, and the AuNPs are easier to disperse well and prevent aggregation.
In an embodiment of the invention, the volume of the PEI aqueous solution is between 400. mu.L and 6.4mL, preferably 400. mu.L, at a concentration of 3 mg/mL.
In an embodiment of the invention, the aqueous PEI solution is mixed with HAuCl4The volume ratio of the solution is not less than 1:1, preferably 1 to 16:1, and for example, may be 1:1, 2:1, 4:1, 8:1, 16:1, etc., but preferably, the volume ratio is 1: 1.
In an embodiment of the invention, the water bath temperature is 40-80 ℃, preferably 60 ℃.
In an embodiment of the invention, the reaction time is from 30min to 3h, preferably 2 h.
Under the conditions of the invention, the obtained AuNPs are regular spheres with an Au simple substance coated with PEI outside, the particle diameter of the regular spheres is 17-27nm, preferably more than 20nm, the PDI is 0.23-0.3, the Zeta potential is 31-34mV, the regular spheres have good stability, good dispersion and difficult aggregation, and are easier to coat completely, and the generation of Au (III) -PEI complex (planar quadrangle) is reduced as much as possible. Greatly improving the stability of AuNPs.
In addition, PEI is coated outside AuNPs prepared by the method, positive potential is presented outside the AuNPs, the particle size and stability of the PEI are more beneficial to the AuNPs to compress miR-140 through electrostatic interaction, and the AuNPs/miR-140 compound prepared by the method is more stable and easy to enter cells.
In a preferred embodiment of the present invention, the preparation method of AuNPs comprises: 20mL of triple distilled water was added to a round-bottomed flask, and HAuCl was added thereto at a concentration of 10mg/mL while stirring in a water bath at 60 ℃4Adding 400 mu L of PEI aqueous solution with the concentration of 3mg/mL, and reacting for 2h to obtain AuNPs. In the embodiment of the present invention, the inventors also tried to prepare AuNPs at normal temperature and reacted for 24 hours, but the obtained structure was Au (iii) -PEI complex, and no Au simple substance was presentAnd generating, wherein the inner core of the aluminum alloy material is a regular spherical AuNPs structure with a simple Au substance coated with PEI. And in further experiments, the complex obtained by further compounding the Au (III) -PEI complex and the miR-140 cannot realize the beneficial effects of the invention, such as low chondrocyte transfection rate, limited repair force on cartilage and development effect on inflammation in OA process, and the like.
In a third aspect of the invention, the invention provides a pharmaceutical formulation comprising the AuNPs/miR-140 complex described in the first aspect above and at least one pharmaceutically acceptable carrier or adjuvant. Alternatively, the pharmaceutical preparation comprises the AuNPs/miR-140 complex described in the first aspect above, at least one non-steroidal anti-inflammatory drug, and at least one pharmaceutically acceptable carrier or adjuvant.
The pharmaceutical preparation comprises the AuNPs/miR-140 compound and one or more pharmaceutic adjuvants or carriers. The invention also provides a method comprising producing a pharmaceutical composition or pharmaceutical formulation comprising mixing an AuNPs/miR-140 complex according to the disclosure herein with a pharmaceutical excipient. The formulations are prepared by any suitable method, generally by uniformly mixing the active compound with liquid and/or finely divided solid excipients in the desired ratio, and then, if desired, shaping the resulting mixture into the desired shape.
In a more preferred embodiment of the invention, the nsaid is lornoxicam.
In a more preferred embodiment of the invention, the pharmaceutical preparation is an injection, a freeze-dried powder injection, a targeted controlled release preparation or a targeted sustained release reagent. The carrier required by the targeted drug can be selected from liposome (such as liposome modified by bisphosphonate derivatives and the like), nanoparticle (such as nanoparticle modified by polylactide glycolide and the like), micelle and the like, and can also be selected or designed according to targeted therapeutic molecule basis and targeted drug design science, which can be realized by the technicians in the field.
The targeting, in embodiments of the present invention, refers to the ability to target the active ingredients of the present invention to the joint, and may also be referred to as joint targeting, or specific cell targeting, such as targeting chondrocytes.
When the medicinal preparation is an injection or a freeze-dried powder injection, the preparation can be prepared by the following method: the complexes of the invention are dissolved in a suitable liquid vehicle, the solution is filter sterilized and then filled into suitable vials or ampoules and sealed. Or adding or not adding lyophilized protectant as required, lyophilizing, and sterilizing to obtain lyophilized powder for injection. In order to improve the dispersibility and stability of the preparation or further adjust the pH or osmotic pressure of the preparation, common pharmaceutical excipients comprise diluents, cosolvents, excipients, osmotic pressure regulators, pH regulators and the like. These methods are merely examples of the many suitable methods for preparing dosage forms known in the art. The compounds of the present invention may of course also be formulated into pharmaceutical compositions or formulations using techniques well known to those skilled in the art. Suitable pharmaceutical adjuvants are known in the art, see for example the 2005 edition handbook of pharmaceutical adjuvants (fourth edition original edition), authors (en) r.c. ro (raymon dcrowe) (usa) p.j. susky (paul jsheskey).
In a fourth aspect of the invention, the invention provides a delivery system comprising an AuNPs/miR-140 complex as described in the first aspect above.
Alternatively, the delivery system comprises an AuNPs/miR-140 complex as described in the first aspect above and at least one non-steroidal anti-inflammatory drug.
In a more preferred embodiment of the invention, the nsaid is lornoxicam.
The drug delivery system can simultaneously deliver the lornoxicam and the AuNPs/miR-140 complex to the joint cavity and optionally prevent the lornoxicam and the AuNPs/miR-140 complex from leaking from the joint cavity, so that the protection and repair effects of the combined drug of the lornoxicam and the AuNPs/miR-140 complex on cartilage and the treatment effect of arthritis can be better played. And the drug delivery system does not affect the stability of the AuNPs/miR-140 complex, or can realize stable coexistence of the lornoxicam and the AuNPs/miR-140 complex, or the whole drug delivery system carrying the lornoxicam and the AuNPs/miR-140 complex is stable, and the lornoxicam and the AuNPs/miR-140 complex are delivered to the joint cavity through the drug delivery system, so that the controllable release of the drug can be realized.
The realization of the drug delivery system depends on drug delivery, drug release devices or carriers, and can carry the drugs to the action part for precise treatment. The drug delivery, release device or carrier can be the same as those mentioned in the present invention, and can be designed according to the design method in the field according to the activity of the complex or drug of the present invention.
In a fifth aspect of the invention, the invention provides a pharmaceutical kit comprising the AuNPs/miR-140 complex described in the first aspect above and at least one non-steroidal anti-inflammatory drug. In a more preferred embodiment of the invention, the nsaid is lornoxicam.
In the embodiment of the invention, in the kit, the AuNPs/miR-140 compound and the AuNPs/miR-140 compound are injection or injection powder. Preferably, the kit further comprises an injection device, a mixing device, a suitable solvent, instructions for use, and the like. Effective use of the kit can be achieved by those skilled in the art according to the instructions for use.
It should be specially noted that the injection or powder injection is preferably selected in the invention, because the lornoxicam is injected into the joint cavity to treat or improve arthritis, protect and repair cartilage tissue, avoid side effect of whole body especially gastrointestinal tract, and increase the concentration of the drug at the action part, thereby achieving better treatment effect.
In a sixth aspect of the present invention, the present invention provides an application of the AuNPs/miR-140 complex described in the first aspect, or the pharmaceutical preparation described in the third aspect, or the drug delivery system described in the fourth aspect, in preparation of a drug for preventing and treating osteoarthritis diseases, or in preparation of a drug for promoting cartilage repair, or in preparation of a drug for protecting cartilage.
In some embodiments of the invention, the concentration of miR-140 in the AuNPs/miR-140 complex is 3-200 nM. The AuNPs/miR-140 compound has no obvious toxic effect on cartilage cells under the concentration in the range, particularly when the concentration is higher than 50nM in the range and even when the concentration is as high as 200nM, and has good safety.
And, in a seventh aspect of the invention, the invention provides the use of an AuNPs/miR-140 complex as described in the first aspect above, or a pharmaceutical formulation as described in the third aspect above, or a delivery system as described in the fourth aspect above, in the preparation of a medicament or agent for upregulating miR-140 expression and/or increasing the level of expression of COL2A mRNA in chondrocytes.
In some embodiments of the invention, in the preparation of a medicament or reagent for up-regulating miR-140 expression and/or increasing expression levels of COL2A mRNA in chondrocytes, when the concentration of miR-140 in the AuNPs/miR-140 complex is 3 to 200nM, particularly above 50nM in this range, the AuNPs/miR-140 complex in the first aspect above, or the pharmaceutical formulation or delivery system described in the third aspect above, still has no significant cytotoxicity to chondrocytes.
In an eighth aspect of the invention, the invention also provides application of lornoxicam and the AuNPs/miR-140 compound of the first aspect in preparation of a medicament for preventing and treating osteoarthritis diseases, or in preparation of a medicament for promoting cartilage repair or a product for protecting cartilage.
Such combinations may include simultaneous, sequential, intermittent, etc. administration, the product optionally being capable of controlling the time, manner or rate of release of the agents. The compounds are used sequentially or at intervals, in particular to the AuNPs/miR-140 compound which is used after lornoxicam is used.
Conventional research and development ideas usually consist in using two drugs simultaneously, such as loading the two drugs together in a certain system or constructing the two drugs into a releasable spliced drug form to explore the combined effect of the drugs. However, in embodiments of the present invention, the inventors have found that when lornoxicam and the AuNPs/miR-140 complex described in the first aspect above are used simultaneously, there are serious stability problems that make it difficult to achieve effective therapeutic results, such as, in certain embodiments, the applicant carries the lornoxicam and the AuNPs/miR-140 compound in a gel system for simultaneous administration, and the result shows that although the small molecular drug lornoxicam can be released firstly, and then the miR-140 compound can be released, however, the lornoxicam in the system can affect the stability of the AuNPs/miR-140 complex, the same experimental result can be found when the lornoxicam is mixed with the AuNPs/miR-140 complex, therefore, in a preferred embodiment of the invention, the combination is prepared by using the AuNPs/miR-140 complex after using lornoxicam. For example, when joint cavity injection is adopted for combined administration, lornoxicam is firstly injected, and then AuNPs/miR-140 compound is injected. The time of administration, number of administrations, or interval of administration can be determined under the direction of a clinician, e.g., by first administering lornoxicam by intra-articular injection, and can be administered one or more times per day or more, e.g., once per week, continuously or discontinuously, e.g., after continuous administration for multiple days or more, by intra-articular injection of the AuNPs/miR-140 complex, and can be administered one or more times per day or more, e.g., once per week, continuously or discontinuously. In an embodiment of the invention, a significant improvement and repair effect is achieved by 6 weeks of combined administration, to the extent of approaching normal tissue.
In an embodiment of the invention, the product is a medicament or a medicament-carrying wearable medical device. The drug can realize the controllable release of the lornoxicam and AuNPs/miR-140 compound, such as the control of drug release time, drug release mode, drug release speed and the like. Wearable medical instrument of medicine carrying such as wearable automatic injection system can include the medicine carrying room (the medicine carrying room can be installed in advance as fixed device and fix on injection system, also can install in the junction of reserving when using as an organic whole with the medicine that carries), safety controller, autoinjector, the medicine carrying room can set up a plurality ofly, can be used to deposit lornoxicam and AuNPs/miR-140 complex respectively, and the medicine of depositing can be injection or freeze-dried powder injection, and the medicine room accessible is rotatory to communicate with the autoinjector, when the medicine of depositing is freeze-dried powder injection, still include the mixing chamber among the wearable medical instrument of medicine carrying, can be convenient for inject with the autoinjector intercommunication after this position redissolution with freeze-dried powder injection. The auto-injector may set parameters such as the injection dose, the number of injections, the injection interval, etc.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention. Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:
FIG. 1 is a graph showing the effect of PEI volume on AuNPs production in example 1.
FIG. 2 is a graph showing the effect of reaction temperature on AuNPs production in example 1.
FIG. 3 is a graph showing the effect of reaction time on AuNPs production in example 1.
FIG. 4 is a morphological diagram of AuNPs in example 1 (A: appearance property diagram; B: microscopic morphology diagram under TEM, scale of 50nm indicated by black horizontal line).
FIG. 5 is a graph showing the particle size distribution of AuNPs in example 1.
FIG. 6 is a graph showing the potential distribution of AuNPs in example 1.
FIG. 7 is the agarose gel electrophoresis picture of AuNPs/miR-140 complex with different mass ratios in example 2.
FIG. 8 is the particle size distribution of AuNPs/miR-140 complexes of different mass ratios in example 2.
FIG. 9 is the potentials of AuNPs/miR-140 complexes of different mass ratios in example 2.
FIG. 10 shows the results of the heparin extraction experiment of AuNPs/miR-140 complex in example 2.
FIG. 11 shows the stability of AuNPs/miR-140 complex in RNase A in example 2.
FIG. 12 shows the stability of free miR-140 in RNase A in example 2.
Fig. 13 shows the experimental results of MTT in example 3.
FIG. 14 shows fluorescence microscopy observations of cell transfection in example 3 (white double arrows indicate red fluorescence around the nucleus and white single arrows indicate red fluorescence in the nucleus).
FIG. 15 shows the flow cytometry assay results for cell transfection in example 3 (A: histogram; B: number of miR-140 positive cells) (. about.p < 0.01 compared to control group; p < 0.01 compared to free miR-140 group).
FIG. 16 shows the relative expression levels of miR-140 in example 3 (p < 0.01 compared to control group; p < 0.01 compared to AuNPs/miR-140 group).
FIG. 17 shows the relative expression levels of COL2A mRNA in example 3 (p < 0.01 compared to control group; p < 0.01 compared to AuNPs/miR-140 group).
Fig. 18 shows appearance properties of Lnxc-loaded F127 temperature-sensitive gel prepared in example 4.
FIG. 19 shows appearance properties of temperature-sensitive gels co-loaded with AuNPs/miR-140 and Lnxc prepared in example 4.
FIG. 20 shows the appearance of AuNPs/miR-140-loaded temperature-sensitive gel prepared in example 4.
Fig. 21 shows appearance properties of a mixture of AuNPs/miR-140 solution and Lnxc solution prepared in example 4.
Figure 22 shows the change in body weight of experimental rats in example 4 (p < 0.01 compared to normal control group).
FIG. 23 shows the change in knee width of experimental rats in example 4 (compared with OA model group, # p < 0.01).
FIG. 24 is a photograph showing the appearance of the femoral condyles of the experimental rats in example 4 (A to F are a normal control group, an OA model group, a free miR-140 group, an Lnxc-sol group, an AuNPs/miR-140 group, and a combination treatment group, in this order).
FIG. 25 shows photographs of appearance observation of tibial plateau of experimental rats in example 4 (A to F are a normal control group, an OA model group, a free miR-140 group, an Lnxc-sol group, an AuNPs/miR-140 group, and a combination treatment group, in this order).
FIG. 26 shows HE staining photographs of femoral condyles of experimental rats in example 4 (A to F are a normal control group, an OA model group, a free miR-140 group, an Lnxc-sol group, an AuNPs/miR-140 group and a combination treatment group in this order).
FIG. 27 shows photographs of HE staining of tibial plateau of experimental rats in example 4 (A to F are a normal control group, an OA model group, a free miR-140 group, an Lnxc-sol group, an AuNPs/miR-140 group and a combination treatment group in sequence).
FIG. 28 shows HE staining photographs of synovial membranes of experimental rats in example 4 (A to F are a normal control group, an OA model group, a free miR-140 group, an Lnxc-sol group, an AuNPs/miR-140 group, and a combination treatment group, in this order).
FIG. 29 shows the results of O-fast green staining of the hamstrings of the experimental rats in example 4 (A to F are a normal control group, an OA model group, a free miR-140 group, an Lnxc-sol group, an AuNPs/miR-140 group, and a combination treatment group, in this order).
FIG. 30 shows the results of safranin O-fast green staining of tibial plateau of experimental rats in example 4 (A to F are normal control group, OA model group, free miR-140 group, Lnxc-sol group, AuNPs/miR-140 group, combination treatment group, in that order).
FIG. 31 shows the Mankin score for cartilage of experimental rats in example 4 (p < 0.01 compared to the OA model group; p < 0.01 compared to Lnxc-sol group and AuNPs/miR-140 group).
FIG. 32 shows the histopathological scores of synovium in example 4 (p < 0.01 compared to the OA model group; p < 0.01 compared to the AuNPs/miR-140 group).
FIG. 33 shows the preparation of AuNPs/miR-140 complexes of the invention.
Detailed Description
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out according to conventional conditions or according to conditions recommended by the manufacturers. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The reagents or starting materials used in the present invention can be purchased from conventional sources, and unless otherwise specified, the reagents or starting materials used in the present invention can be used in a conventional manner in the art or in accordance with the product specifications. In addition, any methods and materials similar or equivalent to those described herein can be used in the methods of the present invention. The preferred embodiments and materials described herein are intended to be exemplary only. And, the following examples illustrate the preparation method of the present invention and experimental design and test results of technical effect evaluation directly related to the present invention, the inventors of the present invention have conducted a lot of experiments and examinations to realize the present invention, and some matters will be referred to in the summary of the invention, which is the result verified by the inventors of the present invention, it should be understood that the contents that can be written in the application document are limited, but the limited examples should not be construed as limiting the scope of the present invention.
Reagent and apparatus: chloroauric acid (shanghai national group of pharmaceuticals); branched polyethyleneimine (sigma, usa); miRNA-140 (Shanghai Jima pharmaceutical technology, Inc.); agarose (shanghai alading reagents ltd); type i nucleic acid stain (shanghai source, leaf biotechnology limited); m55 × RNA loading buffer (Beijing, polymeric American Biotechnology Co., Ltd.); low molecular weight heparin sodium injection (zipsign); tris (hydroxymethyl) aminomethane (Shanghai-derived leaf Biotech Co., Ltd.); na (Na)2EDTA.2H2Other reagents such as O, glacial acetic acid, hydrochloric acid, nitric acid and the like are all commercially available analytical reagents. TU-1810 model ultraviolet-visible spectrophotometer (Beijing Pujingyo general instruments, Inc.); a Nano-ZS90 Malvern laser particle size distribution and potential analyzer (Malvern, uk); JEM-1200EX type transmission electron microscope (Japan Electron Ltd.); a gel imager; a constant temperature water bath oscillator; a precision analytical balance of the AL104-IC type (mettler-toledo instruments ltd, switzerland); ETS-D4 model electromagnetic stirrer (IKA, Germany); genius type vortex mixer (IKA, germany); quantitative sample injector (Eppendorf, Germany).
Preparation of main solution: chloroauric acid solution: 1g of HAuCl4 4H2Dissolving O (orange needle crystal) with triple distilled water, transferring to a 100mL volumetric flask, and adding triple distilled water to constant volume to scale to obtain 10mg/mL HAuCl4And (3) solution. PEI solution: 0.1500g of PEI was weighed precisely, dissolved in triple distilled water, transferred to a 100mL volumetric flask, and thenAnd (5) using triple distilled water to fix the volume to a scale, thus obtaining the PEI aqueous solution of 3 mg/mL. 10 × TAE electrophoresis buffer: 4.84g Tris, and 0.744g Na were weighed precisely2EDTA·2H2And O, dissolving the product in triple distilled water, transferring the solution to a 100mL volumetric flask, adding 1.14mL of glacial acetic acid into the volumetric flask, and fixing the volume to a scale by using the triple distilled water to obtain the 10 XTAE electrophoresis buffer solution. miR-140 concentrated solution: centrifuging 1OD miR-140 freeze-dried powder for 1min at 4000rpm, dissolving the miR-140 freeze-dried powder by 125 mu L DEPC water to obtain a miR-140 concentrated stock solution of 264 mu g/mL (20 mu M), subpackaging and storing at-20 ℃.
Example 1Preparation of AuNPs
20mL of triple distilled water was added to a round-bottomed flask, and HAuCl was added thereto at a concentration of 10mg/mL while stirring in a water bath at 60 ℃4Adding 800 mul of PEI aqueous solution with the concentration of 3mg/mL into 400 mul of the solution, and reacting for 1h to obtain AuNPs.
Effect of PEI volume on AuNPs preparation: 20mL of triple distilled water was added to a round-bottomed flask, and HAuCl was added thereto at a concentration of 10mg/mL while stirring in a water bath at 60 ℃4And adding 100 mu L, 200 mu L, 400 mu L, 800 mu L, 1.6mL, 3.2mL and 6.4mL of PEI aqueous solution with the concentration of 3mg/mL into the solution, reacting for 1h, diluting the obtained solution by 2 times, scanning the wavelength at the position of 300-700nm, and determining the optimal PEI volume according to the characteristic curve of ultraviolet-visible absorption of different solutions.
The reaction temperature and time were fixed, the volume of PEI was varied, and the influence of the volume of PEI on the UV-visible absorption spectrum of AuNPs was examined, the results are shown in FIG. 1. Due to the Surface Plasmon Resonance (SPR) effect, the synthesized AuNPs have ultraviolet-visible absorption peaks at certain wavelengths (510-550 nm), and the positions, the intensities and the half-peak widths of the UV-Vis absorption peaks can roughly represent the particle sizes, the particle size distributions and the generated AuNPs. Generally, the smaller the wavelength of the absorption peak, the smaller the particle size of the synthesized AuNPs; the narrower the half-width of the absorption peak is, the more uniform the particle size of AuNPs is; the greater the intensity of the absorption peak, the greater the amount of AuNPs produced.
Based on the above theory, it can be seen from fig. 1 that, when the volume of PEI is constant within the range of 400 μ L to 6.4mL, the generated AuNPs have a plasmon resonance absorption peak at about 520nm, while when the volume of PEI is 100 μ L and 200 μ L, there is no plasmon resonance absorption peak characteristic of AuNPs, which indicates that AuNPs can be successfully synthesized when the volume of PEI is within the range of 400 μ L to 6.4 mL. On the other hand, when the volume of PEI is 400. mu.L, the intensity of the absorption peak is the largest, indicating that the amount of AuNPs generated under the condition is the largest, so that the optimum preparation condition of AuNPs is selected as the PEI volume of 400. mu.L.
The reason why the synthesis of gold nanoparticles was not successful when the volume of PEI was 100. mu.L and 200. mu.L is presumed to be that this amount of PEI was not effective for reducing the chloroauric acid solution and was not effective for coating the surface of the gold nanoparticles to prevent the agglomeration of the gold nanoparticles.
Effect of reaction temperature on AuNPs production: adding 20mL of triple distilled water into a round-bottom flask, and adding HAuCl with the concentration of 10mg/mL into the round-bottom flask under the conditions of water bath stirring at 40 ℃, 60 ℃ and 80 ℃ respectively4Adding 400 mu L of PEI aqueous solution with the concentration of 3mg/mL, reacting for 1h, finally diluting the obtained solution by 2 times, and then scanning the wavelength at the position of 300-700nm, and determining the optimal reaction temperature according to the ultraviolet-visible absorption characteristic curves of different solutions.
The volume of PEI was fixed and the reaction time was not changed, the reaction temperature was changed, and the effect of temperature on the UV-visible absorption spectrum of AuNPs was examined, the results are shown in FIG. 2. As can be seen from fig. 2, the volume of the fixed PEI and the reaction time are not changed, when the reaction temperature is changed within a range of 40 to 80 ℃, the position and half-peak width of the absorption peak do not change much, the intensity of the absorption peak increases first and then decreases, and the intensity of the absorption peak is the greatest at a reaction temperature of 60 ℃, indicating that the number of AuNPs generated is the greatest at a reaction temperature of 60 ℃, so the reaction temperature of 60 ℃ is selected as the optimal preparation condition for the AuNPs.
Effect of reaction time on AuNPs production: 20mL of triple distilled water was added to a round-bottomed flask, and HAuCl was added thereto at a concentration of 10mg/mL while stirring in a water bath at 60 ℃4Adding 400 μ L of PEI aqueous solution with concentration of 3mg/mL, reacting for 30min, 1h, 2h and 3h, and obtaining the solutionAfter dilution by 2 times, wavelength scanning is carried out at the position of 300-700nm, and the optimal reaction time is determined according to the ultraviolet-visible absorption characteristic curves of different solutions.
The volume and temperature of PEI were fixed, the reaction time was varied, and the effect of the reaction time on the UV-visible absorption spectrum of AuNPs was examined, the results are shown in FIG. 3. As can be seen from FIG. 3, the volume of PEI is fixed and the reaction temperature is not changed, when the reaction time is changed within 30min-3h, the position and half-peak width of the absorption peak are not changed much, the intensity of the absorption peak is increased firstly and then is not changed along with the extension of the reaction time, and the intensity of the absorption peak is not changed basically after 2h, which indicates that the reaction is almost completely carried out when the reaction time is 2h, so the reaction time of 2h is selected as the optimal preparation condition of AuNPs.
In summary, the best preparation method of AuNPs comprises the following steps: 20mL of triple distilled water was added to a round-bottomed flask, and HAuCl was added thereto at a concentration of 10mg/mL while stirring in a water bath at 60 ℃4Adding 400 mu L of PEI aqueous solution with the concentration of 3mg/mL, and reacting for 2h to obtain AuNPs.
Characterization of AuNPs
(1) Appearance and microscopic morphology: the appearance of freshly prepared AuNPs solutions was visually observed and photographed. Microscopic morphology of AuNPs was observed using Transmission Electron Microscopy (TEM) as follows: diluting AuNPs solution by a proper multiple, sucking a proper amount of AuNPs solution by a dropper, dripping the AuNPs solution on a copper net special for a transmission electron microscope, sucking excessive liquid by filter paper after 15s, putting the copper net on clean filter paper, drying under an infrared lamp, and then putting the copper net in the transmission electron microscope to observe the microscopic morphology of the AuNPs. The appearance and micro-morphology of AuNPs are shown in fig. 4. As can be seen from fig. 4, the AuNPs solution is a wine red clear solution, and under TEM, the AuNPs are in a regular spherical shape, uniform in size, and uniform in dispersion, and do not clump together.
(2) Particle size and potential distribution: and (3) preparing three batches of AuNPs repeatedly according to the optimal preparation method, putting an AuNPs solution with the volume of about 1mL into an absorption cell, and measuring the average particle size and Zeta potential distribution of the AuNPs solution by using a Malvern laser particle size distribution and potential analyzer. Three batches of AuNPs are prepared according to the optimal preparation conditions, and the particle size distribution and potential distribution results of the AuNPs are respectively shown in the figure 5 and the figure 6; the average particle size and potential measurement results of AuNPs are shown in table 1.
TABLE 1 particle size and potential measurement results of AuNPs
Figure BDA0002486388100000081
Figure BDA0002486388100000091
As is clear from Table 1, the AuNPs have a hydrated particle size of (22.31. + -. 4.41) nm, PDI of 0.25. + -. 0.03 and a potential of (32.23. + -. 1.16) mV, and it is clear from FIGS. 5 and 6 that the synthesized AuNPs have relatively uniform particle sizes and potential distributions. The fact that the potential of the AuNPs is positive indicates that PEI is used as a stabilizer to be coated on the surface of the AuNPs, and the PEI is favorable for the AuNPs to be combined with miRNA through electrostatic interaction, so that the AuNPs can be used as a carrier for delivering miRNA into cells.
Example 2Preparation of gold nanoparticle/miR-140 compound
Connecting miR-140 to AuNPs through electrostatic adsorption, mixing miR-140 solutions with different concentrations with AuNPs solutions, immediately swirling for 30s for uniform mixing, and then standing and incubating for 30min to obtain AuNPs/miR-140 compounds with different mass ratios.
1. Screening of optimal mass ratio of complex (W/W ═ Au/miR-140)
Agarose gel block electrophoresis: the ability of AuNPs to compress miR-140 was examined by agarose gel blocking electrophoresis experiments. Accurately weighing 0.2g of agarose, placing the agarose in a 50mL triangular flask, adding 20mL of 1 XTAE electrophoresis buffer solution into the triangular flask, placing the triangular flask in a microwave oven for heating until the agarose is boiled and dissolved, adding 2 microliter of I type nucleic acid coloring agent into the triangular flask when the agarose is cooled to 50-65 ℃, uniformly mixing, then gently pouring the agarose into a horizontal groove inserted with a comb, pulling out the comb after the agarose is cooled to gel, placing the gel into an electrophoresis tank, adding 100mL of 1 XTAE electrophoresis buffer solution into the electrophoresis tank, finally uniformly mixing 2 microliter of 5 XTAE Loading buffer and 8 microliter of compound with different mass ratios, adding the compound into a sample Loading hole, setting the electrophoresis voltage to be 90V, setting the time to be 10min, and after the electrophoresis is finished, placing the agarose gel into an imager for imaging.
AuNPs of the PEI with the surface modified are positively charged, and the negatively charged miR-140 can be compressed through electrostatic action, so that an AuNPs/miR-140 compound is formed. And an agarose gel electrophoresis experiment is adopted to characterize the miR-140 compression capability of AuNPs, miR-140 with negative charges can migrate from a negative electrode to a positive electrode during electrophoresis, so that a bright strip is displayed on the positive electrode of an electrophoresis chart, and when miR-140 is compressed by AuNPs, the miR-140 migration can be blocked, and the strip can be weakened or no strip can be seen. The migration of miR-140 in agarose gel electrophoresis was examined when the AuNPs/miR-140 complex mass ratio was 0, 0.9:1, 1.8:1, 3.6:1, 7.2:1, 14.4:1, and 28.8:1, and the results are shown in FIG. 7. As is clear from FIG. 7, when the mass ratio is 0, a bright band is visible on the electrophoretogram, when the mass ratio is 0.9:1, the brightness of the band becomes weak, and when the mass ratio is 1.8:1 or more, the miR-140 band is hardly visible. This indicates that miR-140 is not completely compressed by AuNPs when the mass ratio of AuNPs/miR-140 is less than 1.8:1, and miR-140 is substantially completely compressed by AuNPs when the mass ratio is 1.8:1 or more, so in the next experiment, the particle size and the electrical size of the complex having a mass ratio of 1.8:1 or more were examined to determine the optimum mass ratio of the complex.
Particle size and potential distribution: the particle size and potential distribution influence the fate of the nano particles in vivo, and the endocytosis of the nano particles is influenced by the particle size and potential. Therefore, the composite solutions with different mass ratios and the volume of about 1mL are put into an absorption cell, and the mean particle size and the Zeta potential distribution of the composite solutions are measured by a Malvern laser particle size distribution and potential analyzer. The optimum mass ratio of the composite is determined by the measurement results of the particle size and the potential distribution.
The particle size and the potential of the AuNPs/miR-140 complex are important factors influencing the entry of the complex into cells, nanoparticles with small particle size are more easily taken up by the cells, and nanoparticles with positive charges are more easily taken into the cells. Therefore, on the basis of the gel retardation electrophoresis experiment, the particle size and potential of AuNPs/miR-140 complexes with different mass ratios are investigated in the experiment, and the experiment results are shown in FIGS. 8 and 9. In the embodiment, the average particle size of AuNPs is 22nm, and when the AuNPs and miR-140 are combined according to the mass ratio of 1.8:1, the average particle size is increased to 82nm, which indicates that the AuNPs can form relatively large aggregates; however, when the mass ratio of the AuNPs to the miR-140 is continuously increased, the particle size of the compound is reduced, and finally, when the mass ratio is 28.8:1, the particle size of the compound is reduced to 30nm, and only a small increase is caused compared with the particle size of the AuNPs, which shows that the AuNPs and miRNA are combined through electrostatic interaction, and the AuNPs/miRNA nanoclusters with relatively large particle sizes are formed by the original AuNPs which are uniformly distributed. As can be seen from fig. 8, when the mass ratio of the composite was increased from 1.8:1 to 28.8:1, the particle size of the composite gradually decreased, and when the mass ratio of the composite reached 28.8:1, the particle size of the composite was (27.04 ± 2.59) nm, the particle size was the smallest, and PDI was 0.34 ± 0.03, and dispersibility was good. As can be seen from FIG. 9, when the mass ratio of the composite was increased from 1.8:1 to 28.8:1, the potential of the composite gradually increased and finally stabilized at about 30 mV. In conclusion, when the mass ratio is 28.8:1, the composite has the smallest particle diameter and the appropriate zeta potential, so 28.8:1 was further investigated as the optimum mass ratio of the composite.
2. Stability experiment of gold nanoparticle/miR-140 compound
Heparin extraction experiments: for stability experiments of the complex, miR-140 in the complex needs to be extracted. Selecting low molecular weight heparin for extraction, mixing the compound with low molecular weight heparin solution of 0.75%, 1.5%, 3%, 6%, 12%, 18%, and 24% (V/V), mixing, placing in a constant temperature water bath oscillator, oscillating (37 deg.C, 100rpm), extracting for 1h, and performing agarose gel electrophoresis experiment.
In order to verify whether the AuNPs/miR-140 compound can protect the miR-140 from degradation of nuclease, the miR-140 in the compound needs to be extracted, the part selects low-molecular-weight heparin with different concentrations to extract the miR-140 in the compound, and the experimental result is shown in FIG. 10. As can be seen from FIG. 10, the free mi-140 has a distinct band in the electrophoretogram, and the AuNPs/miR-140 has no band in the electrophoretogram, when miR-140 in the complex is extracted by using low molecular weight heparin with different concentrations, the electrophoresed band gradually brightens, and when miR-140 in the low molecular weight heparin with the concentration of 24% is extracted, the brightness of the band is the same as that of the free miR-140, which indicates that miR-140 in the AuNPs/miR-140 complex can be completely extracted by 24% of low molecular weight heparin, so that the optimal extraction concentration of heparin is selected to be 24%.
Stability of the complexes in RNase A experiments: and adding 10 mu L of 0.1mg/mL RNase A solution into 90 mu L of AuNPs/miR-140 compound to ensure that the final concentration of the RNase A solution is 10 mu g/mL. Then incubating for 0.5, 1, 2, 4, 6, 8, 12 and 24 hours under the condition of constant-temperature water bath oscillation at 37 ℃, placing the sample in a refrigerator at-80 ℃ for freezing for 20min to stop reaction, and using the same method operation of naked miR-140 as a control. And finally, extracting miR-140 in the sample by using low molecular weight heparin to perform an agarose gel electrophoresis experiment.
miR-140 is unstable and is easily degraded by RNase in vivo, in order to verify whether AuNPs can protect miR-140 from degradation of nuclease after AuNPs/miR-140 complex is formed, AuNPs/miR-140 and RNase A are mixed and incubated for different times, miR-140 in AuNPs/miR-140 is extracted by heparin and then subjected to agarose gel electrophoresis experiment, and the experimental result is shown in figure 11 and figure 12. As can be seen from FIG. 12, no band was seen in the electrophoretogram after 30min of mixed incubation of free miR-140 and RNase A, indicating that free miR-140 is degraded by RNase A at 30 min. As can be seen from FIG. 11, after the AuNPs/miR-140 complex and RNase A are mixed and incubated for 24h, a band can still be seen in an electrophoretogram, which indicates that the AuNPs/miR-140 complex can stably exist in an RNase A environment for at least 24 h. AuNPs can protect miR-140 from nuclease degradation, which shows that miR-140 is not only loosely bound on the surface of AuNPs simply through electrostatic interaction, but also tightly compressed inside AuNPs coated by PEI. When the compound is used for treating OA in vivo, AuNPs can protect miR-140 from degradation before the compound enters cells, so that miR-140 can better exert a treatment effect.
This example identifies the preferred conditions for the preparation of AuNPs from PEI, i.e., PEI volume of 400. mu.L, reaction temperature of 60 ℃ and reaction time of 2 h. The prepared AuNPs solution is clear wine red in appearance, is spherical particles on a micro scale, and is uniform in size. The AuNPs particle size measured by a nanometer particle size potential analyzer is (22.31 +/-4.41) nm, PDI is (0.25 +/-0.03), and zeta potential is (32.23 +/-1.16) mV. When the mass ratio of AuNPs/miR-140 complex is 28.8:1, miR-140 can not only be completely compressed by AuNPs, but also has the smallest particle size (27.04 +/-2.59 nm) and proper potential (30.4 +/-1.93 mv). The results of heparin extraction experiments show that 24% heparin can extract miR-140 in the AuNPs/miR-140 complex. The stability experiment result shows that the AuNPs/miR-140 compound can protect the miR-140 from degradation of nuclease within 24h, and has better dispersity and stability.
Example 3In vitro evaluation of AuNPs/miR-140 complexes
The AuNPs/miR-140 compound has to have small cytotoxicity and high cell transfection capacity to play a role in gene regulation of miR-140 so as to play a role in treating OA. In this example, chondrocytes are cultured in vitro, and then the cytotoxicity, cell transfection capacity and in vitro gene regulation effect of the AuNPs/miR-140 complex are examined through MTT (methyl thiazolyl tetrazolium) experiment, cell transfection experiment and qPCR (quantitative polymerase chain reaction) experiment.
Reagent and instrument
cy3-miR-140 (Shanghai Jima pharmaceutical technology, Inc.); tetramethylazoazolium salt (MTT, Sigma, USA); DMSO for cell culture (Beijing Soilebao Tech Co., Ltd.); 4% paraformaldehyde (beijing solibao technologies ltd); DAPI (beijing solibao science and technology ltd); GAPDH, COL2 primer, U6, miR-140 primer (Shanghai Jima technology pharmaceuticals, Inc.); TRNzol Universal reagent (Tiangen Biochemical technology Co., Ltd.); HiFiScript CdnaSynthesis Kit (kang is a century); ilex purpurea Hassk fetal bovine serum (Hangzhou Biotechnology Co., Ltd., Zhejiang Tian); cell culture medium (DMEM/F12, Thermoscientific Corp.); (ii) trypsin; the isopropanol, the absolute ethanol and the chloroform are all commercially available analytical pure.
Inverting the fluorescence microscope; a flow cytometer; a CO2 incubator; microplate reader (Biotek corporation, usa); (Genius type vortex mixer (IKA, Germany); Nanodrop; reverse transcription incubator; quantitative fluorescence instrument; AL104-IC type precision analytical balance, Mettler-Torledo instruments, Inc.); quantitative sample injector (Eppendorf, Germany).
Cell: primary chondrocytes were purchased from Sainbow biotech, Inc. and cultured in DMEM/F12 medium containing 10% fetal bovine serum at 37 deg.C, 5% CO2Culturing in an incubator.
Preparation of the principal solution
MTT solution: 50mg of MTT was precisely weighed, dissolved in 10mL of PBS, filtered and sterilized with a 0.22 μm filter, and then, the mixture was placed in a dark place, stored for a short period at 4 ℃ and stored for a long period at-20 ℃.
Phosphate Buffered Saline (PBS) for cell experiments: precisely weighing 4.5g NaCl and 0.531g Na2HPO4And 0.072g KH2PO4, dissolving with triple distilled water, transferring to a 500mL volumetric flask, and fixing the volume to the scale to obtain PBS for cell experiments.
1. Cytotoxicity experiments: the safety of the complex was verified by cytotoxicity experiments. The cells were cultured at 1X 104Density of one well was seeded in 96-well plates at 37 ℃ with 5% CO2And after culturing for 24h under the condition of saturation humidity, diluting the AuNPs/miR-140 complex and the PEI/miR-140 complex to different concentrations (3.125nM, 6.25nM, 12.5nM, 25nM, 50nM and 200nM) by using a culture medium, respectively adding the diluted AuNPs/miR-140 complex and the PEI/miR-140 complex into corresponding holes, incubating with the cells for 24h, adding 20 mu L of MTT solution (5mg/mL) into each hole, and continuously incubating in the incubator for 4 h. After incubation was complete, the supernatant was discarded and DMSO (200 μ L/well) was added and the formazan crystals were fully dissolved. Finally, the absorbance value (A) at 570nm of each well was measured by a microplate reader. Cell viability was calculated as follows.
Cell survival rate (%) ═ aSample (I)-ABlank space)/(AControl-ABlank space) X 100; wherein A isSample (I)The absorbance after the medicine adding treatment is obtained; a. theControlAbsorbance of control wells (cells added, no drug added); a. theBlank spaceAbsorbance of blank wells (no cells, no drug).
2. Cell transfection assay
(1) And (3) observation by a fluorescence microscope: cells were plated at 1.5X 105Hole/holeThe cell density of (2) was inoculated in a 12-well plate, and after 24 hours of culture in an incubator, cell transfection experiments were performed. Absorbing original culture medium, adopting DMEM/F12 culture medium to dilute free cy3-miR-140, AuNPs/cy3-miR-140 and PEI/cy3-miR-140, and then respectively adding the diluted free cy3-miR-140, AuNPs/cy3-miR-140 and PEI/cy3-miR-140 into corresponding holes, wherein the concentration of miR-140 in each hole is 50nM, and the volume is 1 mL. After incubation at 37 ℃ for 4h, carefully wash with PBS for 2 times, add 4% paraformaldehyde 500. mu.L, fix at room temperature for 20min, remove the fixative and wash twice with PBS, add 500. mu.L of 1. mu.g/mL DAPI solution, incubate at room temperature for 15min, remove the DAPI solution, wash twice with PBS, immediately place under inverted fluorescence microscope for observation.
(2) Flow cytometry measurement: cells were plated at 1.5X 105The density per well was seeded in 12-well plates and after 24h incubation in an incubator, cell transfection experiments were performed. Absorbing original culture medium, adopting DMEM/F12 culture medium to dilute free cy3-miR-140, AuNPs/cy3-miR-140 and PEI/cy3-miR-140, and then respectively adding the diluted free cy3-miR-140, AuNPs/cy3-miR-140 and PEI/cy3-miR-140 into corresponding holes, wherein the concentration of miR-140 in each hole is 50nM, and the volume is 1 mL. After incubation for 4h at 37 ℃, wash 2 times with PBS and digest with EDTA-containing trypsin, after which the cell suspension is transferred to a flow tube, centrifuged at 1000rpm for 5min and the supernatant discarded, washed twice with PBS, and finally resuspended chondrocytes in the flow tube with PBS and assayed with a flow cytometer.
3. qPCR assay for miR-140 and COL2A mRNA relative expression: cartilage cells were cultured at 3X 105The density of each well was inoculated into 6-well plates at 37 ℃ with 5% CO2And culturing for 24 hours under the condition of saturation humidity, absorbing the original culture medium, diluting the AuNPs/miR-140 compound, the PEI/miR-140 compound, the free miR-140 and the AuNPs/NC to the concentration of 50nM by using a DMEM/F12 serum-free culture medium, adding the diluted AuNPs/miR-140 compound, the PEI/miR-140 compound, the free miR-140 and the AuNPs/NC into a 6-hole plate, and after 4 hours, replacing the serum-free culture medium with a serum-containing culture medium to continue culturing for 48 hours. And extracting total RNA in different wells after 48h, then carrying out reverse transcription of microRNA and total RNA, finally carrying out a real-time fluorescence quantitative PCR experiment, determining the change of miR-140 expression level by taking U6 as an internal reference, and determining the change of COL2A mRNA expression level by taking GAPDH as an internal reference.
(1) Extraction of total RNA:<1>the cells of each group were washed gently with PBS 2 times, 1mL of TRNzol Universal reagent was added to each well to lyse the cells,fully blowing the chondrocytes at the bottom of the hole by a pipette tip, and placing the homogenate sample at room temperature for 5min to completely separate the nucleic acid-protein complex.<2>The homogenate was transferred to an EP tube, 200. mu.L of chloroform was added to each 1mL of TRNzol reagent, mixed by manual rapid inversion for 2min, and left at room temperature for 3 min.<3>Centrifuge at 12000rpm for 15min at 4 ℃. The sample will be divided into three layers: the pink organic layer, the middle layer and the upper colorless aqueous phase, with the RNA mainly in the aqueous phase, are transferred to a new EP tube.<4>Adding isopropanol with the same volume into the obtained water phase solution, mixing, and standing at room temperature for 10 min.<5>Centrifugation was carried out at 12000rpm for 10min at 4 ℃ to remove the supernatant. RNA precipitation before centrifugation is often not visible, and after centrifugation a gelatinous precipitate forms on the tube side and base.<6>1mL of RNase-free 75% ethanol was added and centrifuged at 12000rpm at 4 ℃ for 5 min. The supernatant was discarded.<7>Drying at room temperature, adding RNase-Free dd H2O30. mu.L dissolved the extracted RNA.<8>The concentration and purity of RNA was determined using Nanodrop. (2) Reverse transcription step of microRNA-140: a microRNA reverse transcription reaction system is prepared according to the table 2, the total volume is 20 mu L, and the amount of reverse transcribed RNA is 600 ng. The reaction conditions for cDNA synthesis were: incubate at 42 ℃ for 15 minutes and 85 ℃ for 5 minutes. According to the reaction condition, the reverse transcription of microRNA-140 and U6 is carried out.
TABLE 2 reverse transcription System for MicroRNA
Figure BDA0002486388100000111
Figure BDA0002486388100000121
(3) Reverse transcription of total RNA: a total RNA reverse transcription reaction system was prepared in accordance with Table 3, and the total volume was 20. mu.L, and the amount of reverse-transcribed RNA was 600 ng. The reaction conditions for cDNA synthesis were: incubate at 42 ℃ for 15 minutes and 85 ℃ for 5 minutes. According to this reaction condition, reverse transcription of total RNA was performed.
TABLE 3 reverse transcription System for Total RNA
Figure BDA0002486388100000122
(4) The real-time fluorescent quantitative PCR reaction step: the reverse transcribed cDNA was diluted 10-fold and prepared into the fluorescent quantitative PCR reaction system of Table 4, and the fluorescent quantitative PCR experiment was performed according to the PCR reaction procedure of Table 5. After the reaction is finished, using
Figure BDA0002486388100000125
The 96SW1.1 software calculates the relative expression of each group of RNA.
TABLE 4 fluorescent quantitative PCR reaction System
Figure BDA0002486388100000123
TABLE 5 fluorescent quantitative PCR reaction procedure
Figure BDA0002486388100000124
5. Statistical analysis: the experimental results are expressed as mean ± standard deviation (mean ± SD). Student's t test was performed on the experimental data using SPSS 10.0 software, with P <0.05 indicating significant differences.
6. Results and analysis
(1) Results of cytotoxicity experiments: branched polyethyleneimine (molecular weight 25000) is recognized as the "gold standard" for gene transfection because of its high transfection efficiency, so this example was performed with PEI and miR-140 as a "10: 1 as a positive control, and researching the cytotoxicity, cell transfection capability and in-vitro gene regulation and control capability of the AuNPs/miR-140 compound.
The MTT method is widely used to detect the viability of cells. The detection principle is that succinate dehydrogenase in the mitochondria of the living cells can reduce MTT into water-insoluble blue-violet formazan crystals, and after the formazan crystals are dissolved by dimethyl sulfoxide (DMSO), the absorbance of the formazan crystals is measured by an enzyme labeling instrument, so that the relative activity of the cells is calculated. Therefore, in order to determine whether the AuNPs/miR-140 complex is toxic to chondrocytes, an MTT (maximum temperature test) experiment of the complex is carried out, and the result is shown in FIG. 13. As can be seen from FIG. 13, when the miR-140 concentration is between 3.125-50nM, the cell survival rates of the AuNPs/miR-140 and PEI/miR-140 groups are both above 90%, which indicates that neither AuNPs/miR-140 nor PEI/miR-140 has obvious toxicity to chondrocytes in the concentration range of 3.125-50 nM. However, when the miR-140 concentration is increased to 200nM, the cell survival rate of the AuNPs/miR-140 group is still over 90%, and the cell survival rate of the PEI/miR-140 group is reduced to 78%, which shows that the PEI/miR-140 has higher cytotoxicity to chondrocytes when the concentration is 200 nM. The results show that compared with the complex formed by cationic polymer carriers such as PEI and the like, the AuNPs/miR-140 complex improves cytotoxicity and improves use safety.
(2) Results of cell transfection experiments
Fluorescence microscope observation results: AuNPs deliver negatively charged miR-140 into chondrocytes is a prerequisite for miR-140 to function. In order to detect whether AuNPs can deliver miR-140 into chondrocytes, miR-140 is firstly marked with cy3 capable of emitting red fluorescence, then the distribution of red fluorescence in chondrocytes is observed after free cy3-miR-140, PEI/cy3-miR-140 and AuNPs/cy3-miR-140 complex are transfected into chondrocytes through a fluorescence microscope, and transfection reagents can generally transfect into cells within about 4h, so that the fluorescence distribution in the cells after transfection is measured within 4h, and the result is shown in FIG. 14. In fig. 14, blue represents nuclei, red represents miR-140 labeled with cy3, white double arrows represent red fluorescence around nuclei, and white single arrows represent red fluorescence in nuclei. As can be seen from the figure, no red fluorescence was observed in the cells in the free cy-3miR-140 group, indicating that the negatively charged naked miR-140 cannot be endocytosed by the cells. In the PEI/cy3-miR-140 group and the AuNPs/cy3-miR-140 group, stronger red fluorescence is observed in cells, which indicates that PEI and AuNPs can successfully deliver miR-140 to cells; and the red fluorescence inside the cell is mostly distributed around the nucleus and a small part is distributed in the nucleus, indicating that miR-140 is mostly delivered into the cytoplasm and a very small part is delivered into the nucleus. However, the red fluorescence in the PEI/cy3-miR-140 group cells is brighter than that in the AuNPs/cy3-miR-140 group cells, which indicates that AuNPs can well deliver miR-140 to chondrocytes, but the delivery capacity of the AuNPs is possibly weaker than that of PEI.
Flow cytometry detection results:
in order to quantitatively detect the capacity of AuNPs to deliver miR-140 cells, after miR-140 is labeled with cy3 capable of emitting red fluorescence, the fluorescence intensity of cy3 in chondrocytes is detected by a flow cytometer after free cy3-miR-140, PEI/cy3-miR-140 and AuNPs/cy3-miR-140 complex transfect the chondrocytes for 4 h. The results are shown in FIG. 15. As can be seen from FIG. 15(B), the percentage of miR-140 positive cells in the free miR-140 group to the total number of cells is about 4.5%, while the percentage of positive cells in the AuNPs/miR-140 group and PEI/miR-140 group is close to 100%, which indicates that the free miR-140 can be hardly taken up by chondrocytes, and both AuNPs and PEI can efficiently deliver miR-140 into chondrocytes, and the cell transfection rate is close to 100%, which is consistent with the result observed by a fluorescence microscope. Although the cell transfection rates of the two are close to 100%, it can be seen from fig. 15(a) that the fluorescence intensity in the PEI/miR-140 group cells is still stronger than that in the AuNPs/miR-140 group cells, which indicates that the AuNPs have a weaker ability to deliver miR-140 into cells than PEI, but the AuNPs have lower cytotoxicity than PEI, and the comprehensive comparison shows that the AuNPs can be used as a gene delivery carrier with low toxicity and high efficiency.
(3) qPCR determination of miR-140 and COL2A mRNA expression
qPCR determination of miR-140 expression results: after the miR-140 is delivered into the chondrocytes by the carrier, the miR-140 expression level in the cells is increased, the change of the miR-140 expression level of the chondrocytes after treatment of free miR-140, AuNPs/NC and PEI/miR-140 is detected by using a real-time fluorescence quantitative PCR experiment, and the experimental result is shown in figure 16. As can be seen from FIG. 16, after the chondrocytes are treated by free miR-140 and AuNPs/NC for 48 hours, the expression level of miR-140 in the chondrocytes is not changed greatly compared with that of a control group, and after the chondrocytes are treated by AuNPs/miR-140 and PEI/miR-140, the expression level of miR-140 in the chondrocytes is increased by 220 times and 1676 times respectively, which indicates that the AuNPs can effectively deliver miR-140 to the chondrocytes, although the delivery capacity of the AuNPs is weaker than that of PEI in the positive control group, the high toxicity of PEI limits further application of PEI, and the application is also confirmed in the previous cytotoxicity experiment.
Results of qPCR assay for expression of COL2A mRNA: the change of COL2A mRNA expression level after the chondrocytes are treated by free miR-140, AuNPs/NC and PEI/miR-140 is detected by a real-time fluorescent quantitative PCR experiment, and the experimental result is shown in FIG. 17. As can be seen from FIG. 17, after the chondrocytes are treated with the free miR-140 and AuNPs/NC for 48h, the expression level of COL2A mRNA in the chondrocytes is not changed greatly compared with that of a control group, and after the chondrocytes are treated with the PEI/miR-140 and the AuNPs/miR-140, the expression level of COL2A mRNA in the chondrocytes is increased by about 1.61 times and 1.37 times respectively, which shows that miR-140 transfected into the cells can increase the expression level of COL2A mRNA. Type II collagen is an important component of cartilage extracellular matrix, and when cartilage is degenerated, the extracellular matrix is damaged, and the content of type II collagen is reduced. After the expression level of COL2A mRNA in the chondrocytes is increased, the collagen II synthesized by the chondrocytes can be increased, thereby promoting cartilage repair. In conclusion, after AuNPs/miR-140 transfects chondrocytes, the expression level of COL2A mRNA can be increased, which has positive significance for maintaining normal function of cartilage.
This example uses PEI as a positive control to evaluate the cytotoxicity, cell transfection ability, and in vivo gene regulation ability of AuNPs. From the results, AuNPs have less cytotoxicity than PEI as a gene delivery vehicle, but their cell transfection ability is weaker than PEI. However, compared with the experimental results of other documents, the AuNPs/miR-140 complex prepared by the experiment has excellent miR-140 cell delivery capability, and the AuNPs and miR-140 have excellent suitability. Pan et al tested that the transfection rates of hMSC cells (human mesenchymal stem cells) of AuNPs/Cy3-miR-29b and positive control lipo/miR-29b are only 54 +/-0.71% and 65.12 +/-1.85% respectively, and the transfection rates of MC3T3-E1 cells are 88 +/-1.42% and 80.57 +/-1.77% respectively; a nanocapsule is prepared by Liuzhongong and the like and is used for delivering miRNA into cells, and the result shows that the transfection efficiencies of U87 (human glioma cells) and MCF-7 (human breast cancer cells) of the nanocapsule are about 90%, and the transfection efficiencies of positive control lipofectamine are about 40% and about 20% respectively; the transfection rate of the chondrocytes of the AuNPs/miR-140 compound prepared by the experiment is close to 100%, the AuNPs/miR-140 compound has better delivery capacity, has no cytotoxicity to the chondrocytes, and has obvious advantages over the prior art.
In the embodiment, the safety, the cell-entering capacity and the gene regulation effect of the AuNPs/miR-140 compound are evaluated through an MTT experiment, a cell transfection experiment and a qPCR experiment. MTT experimental results show that the positive control group PEI/miR-140 generates obvious toxicity to chondrocytes at the concentration of 200nM, and the AuNPs/miR-140 group does not generate toxicity to chondrocytes at the concentration range of 3.125nM-200nM, which shows that the AuNPs/miR-140 compound improves cytotoxicity and improves use safety. Cell transfection experiments show that the chondrocyte transfection rates of AuNPs/miR-140 and PEI/miR-140 are close to 100%, AuNPs and PEI can efficiently deliver miRNA-140 into chondrocytes, but the fluorescence intensity in chondrocytes of AuNPs/miR-140 group is weaker than that in chondrocytes of PEI/miR-140 group, which shows that the AuNPs delivery miR-140 celling capacity is possibly lower than that of PEI, but PEI/miR-140 has higher cytotoxicity than AuNPs/miR-140, and in a comprehensive way, the AuNPs/miR-140 complex is safer and more efficient. The qPCR experiment shows that the AuNPs/miR-140 compound can up-regulate the expression of miR-140 in chondrocytes, and can also increase the expression level of COL2A mRNA in the chondrocytes, so that the AuNPs/miR-140 compound has positive significance for repairing the chondrocytes.
Example 4Combination of Lnxc and AuNPs/miR-140 complex
1. Temperature-sensitive gel loaded with AuNPs/miR-140 and Lnxc together
The AuNPs/miR-140 compound and Lnxc are co-loaded in the temperature-sensitive gel, the administration system can simultaneously deliver the miR-140 and the Lnxc into a joint cavity, the temperature-sensitive gel can prevent the miR-140 and the Lnxc from leaking from the joint cavity, the AuNPs/miR-140 plays a role in repairing cartilage injury, and the Lnxc plays a role in eliminating inflammation, so that OA is treated.
Lnxc-loaded F127 temperature-sensitive gel: precisely weighing 2g F127, 0.23g F68, 0.04g Lnxc and 0.06g of tris (hydroxymethyl) aminomethane (adjusting the pH of the solution to dissolve lornoxicam; the Lnxc structure contains keto-enol structure which is weakly acidic drug, the pKa is 4.7, and the solubility exponentially increases with the increase of the pH) in a small beaker, adding 10mL of water, sealing the beaker with a preservative film, stirring the beaker on a magnetic stirrer for 30min, then placing the beaker in a refrigerator at 4 ℃ for 24h, and fully swelling F127 and F68 in the water to obtain the temperature-sensitive Lnxc-loaded F127 gel, wherein the morphology of the temperature-sensitive Lnxc-loaded F127 gel is as shown in FIG. 18, and as shown in FIG. 18, the temperature-sensitive gel is yellowish at room temperature, is in a solution state (a), and is in a gel state (b) at 36 ℃.
Temperature-sensitive gel loaded with AuNPs/miR-140 and Lnxc together: precisely weighing 2g F127, 0.23g F68, 0.04g Lnxc and 0.06g of tris (hydroxymethyl) aminomethane in a small beaker, adding 10mL of AuNPs/miR-140 aqueous solution, sealing with a preservative film, stirring for 30min on a magnetic stirrer, then placing the beaker in a refrigerator at 4 ℃ for 24h to fully swell F127 and F68 in water, and obtaining the temperature-sensitive gel loaded with AuNPs/miR-140 and Lnxc. As shown in FIG. 19, it is clear from FIG. 19 that the temperature-sensitive gel loaded with both AuNPs/miR-140 and Lnxc is black green and has a black precipitate. The AuNPs/miR-140 solution is wine red, the Lnxc-loaded temperature-sensitive gel is yellow, the AuNPs/miR-140 solution and the Lnxc-loaded temperature-sensitive gel are mixed to be orange, the reason for analyzing the phenomenon is that the AuNPs are aggregated, and the AuNPs are aggregated by the temperature-sensitive gel or the Lnxc solution and black precipitates are separated out.
In order to search the reason for the aggregation of AuNPs, the AuNPs/miR-140-loaded temperature-sensitive gel and a mixture of AuNPs/miR-140 solution and Lnxc solution are also prepared in the embodiment.
AuNPs/miR-140-loaded temperature-sensitive gel: 2g F127 and 0.23g F68 are precisely weighed in a small beaker, 10mL of AuNPs/miR-140 aqueous solution is added, the beaker is sealed by a preservative film, the mixture is stirred for 30min on a magnetic stirrer, and then the beaker is placed in a refrigerator at 4 ℃ for 24h, so that F127 and F68 are fully swelled in water, and the temperature-sensitive gel loaded with AuNPs/miR-140 is obtained. The results are shown in fig. 20, and it is clear from fig. 20 that the prepared AuNPs/miR-140-loaded temperature-sensitive gel is purple, and the phenomenon of AuNPs aggregation appears, which shows that AuNPs can be aggregated by F127 and F68. Presumably, the reason for this was that anions were dissociated from the aqueous solutions of F127 and F68, resulting in aggregation of the cation-modified AuNPs.
Mixture of AuNPs/miR-140 solution and Lnxc solution: and precisely weighing 0.04g of Lnxc and 0.09g of tris (hydroxymethyl) aminomethane in a small beaker, and adding 10mL of AuNPs/miR-140 aqueous solution to obtain a mixture of the AuNPs/miR-140 solution and the Lnxc solution, wherein the result is shown in figure 21, and the mixture is earthy yellow and precipitates, which indicates that the Lnxc solution causes the AuNPs to aggregate. Lnxc has low solubility in water, but the Lnxc solubility is increased after a certain amount of tris is added. It is presumed that, as the pH value increases, in the alkaline solution, weakly acidic Lnxc dissociates anions to increase the solubility in water, and the anions aggregate the cation-modified AuNPs.
2. Combination drug
This example evaluates the efficacy of Lnxc and AuNPs/miR-140 complexes in combination for treating OA, particularly for protecting and promoting cartilage repair, primarily by in vivo pharmacodynamic experiments in rats. Firstly, a rat OA model is established by adopting a joint cavity injection papain method, and the successful establishment of the rat OA model is proved by measuring the width of a knee joint. Then, the rats were divided into A, B, C, D, E, F six groups, a was a normal control group (not modeled), B was an OA model group, C was a free miR-140 group, D was a drug (Lnxc-sol, lornoxicam injection) group, E was an AuNPs/miR-140 complex group, and F was a combination treatment group. A. B, injecting normal saline into joint cavities respectively; injecting free miR-140 into group C; group D is injected with Lnxc-sol, group E is injected with AuNPs/miR-140 compound, and the groups are injected once per week for six weeks; in group F, Lnxc-sol is injected into the joint cavity once a week for three weeks; the joint cavity was injected with the AuNPs/miR-140 complex once a week for three consecutive weeks starting at week four. Finally, knee joint cartilage repair and inflammation improvement of each group of rats were evaluated by using knee width and body weight of the rats, appearance observation results of femoral condyles and tibial plateaus, HE staining of femoral condyles and tibial plateaus, safranin O-fast green staining condition and ManKin score, HE staining condition of synovium and histopathological score as evaluation indexes.
Experimental materials:papain (sigma, usa); cysteine (Shanghai-derived leaf Biotech Co., Ltd.); safranin O fast green dye liquor (beijing solibao); hematoxylinRed dyeing liquor (Beijing solibao); reagents such as formaldehyde, nitric acid, ethanol, xylene and the like are all commercially available analytical reagents. AL104-IC type precision analytical balance, mettler-toledo instruments ltd); model RM2235 paraffin wax microtomes (leica, germany); model EG 1150 paraffin embedding machine (leica, germany); an ASP200s type dehydrator (Leica, Germany); HI 1220 type sheet spreading and baking machine (leica, germany); autostainer type dyeing machine (leica, germany); TE2000 type fluorescence microscope (NIKON corporation, japan); a balance. Wistar rat (300g +/-20 g, provided by New drug evaluation center of Shandong province pharmaceutical academy of sciences)
Experimental methods
Preparation of main solution:
1% papain and 0.03M cysteine mixed solution: 50mg of papain and 18.174mg of cysteine were precisely weighed, dissolved in 5mL of physiological saline, and sterilized by filtration through a 0.22 μm microporous membrane. Lornoxicam injection (Lnxc-sol): precisely weighing 40mg of lornoxicam and 90mg of tris (hydroxymethyl) aminomethane, dissolving in 10mL of distilled water, and filtering and sterilizing through a 0.22 mu m microporous filter membrane to obtain 4mg/mL Lnxc-sol. Establishment of rat knee joint OA model: the method of establishing The rat knee joint model was determined by reference to The relevant literature (Huang, M.H., et al., "The early evaluation of induced osteo arthritis in rates with 99 Tcm-permethate scales." Nuclear media communications 17.6(1996): 529-. Firstly, anesthetizing a rat by adopting an ether inhalation method, and the method comprises the following steps: after placing the rat in the desiccator, 2mL of diethyl ether was poured into the desiccator and the lid of the desiccator was closed. Then, after the knee joint of the anesthetized rat is slightly bent and disinfected by iodophor, a syringe is punctured into the joint cavity through the patellar ligament tendon, and 60 mu L of papain and cysteine mixed solution is injected into the knee joint cavity of the rat. Injecting 60 mu L of papain and cysteine mixed solution into the cavity of the left knee joint of the rat respectively on the first, third and fifth days, injecting three times in total, and completing the establishment of the OA model of the knee joint of the rat two weeks after the first injection. Measuring the knee width of the rat in the whole molding process, preliminarily judging the molding condition according to the knee joint swelling degree (knee width) of the rat, and removing animals with unsatisfactory molding.
Design of experimental protocol
Grouping: let group a be a normal control group (no model), and then the modeled rats were randomly divided into 5 groups (B, C, D, E, F) of 6 rats each. Group A: normal control group, group B: OA model group, group C: free miR-140 group and D group: drug groups, i.e., Lnxc-sol group, E group: the AuNPs/miR-140 compound group and the F group are as follows: is a drug and gene combined treatment group. The following regimen was followed:
the administration scheme is as follows: group A: injecting 80 μ L normal saline into joint cavity once per week for 6 weeks as normal control group without molding; group B: for OA model group, injecting 80 μ L physiological saline into joint cavity, once per week, and continuously injecting for 6 weeks; group C: for the free miR-140 group, 80 μ L of free miR-140 solution is injected into the joint cavity once a week for 6 weeks; group D: for the drug group, Lnxc-sol group, 80. mu.L of Lnxc-sol (containing Lnxc of 4mg/mL) was injected into the joint cavity once a week for 6 weeks; group E: for the AuNPs/miR-140 compound group, injecting 80 mu L of AuNPs/miR-140 compound solution (containing miR-140 of 27.2 mu g/ml) into the joint cavity, and continuously injecting once per week for 6 weeks; and F group: for drug and gene combination treatment group, 80 μ L of Lnxc-sol (containing Lnxc of 4mg/mL) was injected into joint cavity first, 1 time per week, and 3 weeks were continuously injected; then 80. mu.L of AuNPs/miR-140 complex solution (containing miR-140 of 27.2. mu.g/ml) is injected into the joint cavity, and the injection is performed once a week and continuously for 3 weeks.
The knee joint diameter was measured with a vernier caliper at various time points after dosing, while the weight of the rats was recorded. The effect of the combination treatment of OA by Lnxc and miR-140 is evaluated by the following indexes of detection and observation after the rats are killed by ether over-anesthesia for six weeks after the last administration.
Observation and determination of pharmacodynamic evaluation index
1. Appearance of cartilage: after sacrifice, the left knee femoral condyle, tibial plateau and synovium were isolated and observed for appearance.
2. Histological evaluation of cartilage and synovium
Treatment of tissues such as femoral condyles, tibial plateau, and synovium
The femoral condyles, tibial plateau and synovium were processed as follows:
(1) fixing and decalcifying: the femoral condyle, the tibial plateau and the synovial membrane are soaked in 10% formaldehyde solution (formalin) for at least 48h for fixation, and after being washed by running water, the femoral condyle and the tibial plateau are soaked in decalcifying liquid (10% nitric acid) for decalcification for 5 days, and the decalcification is completed when bone tissues can be easily penetrated by a needle or the tissues have no resistance by the needle. The specimen was soft and elastic. After decalcification was completed, the plate was washed with running water.
(2) Dehydrating and transparent: dehydrating and transparent at room temperature. The dehydration process proceeds from low concentration ethanol to high concentration ethanol step by step. After the specimen is soaked in 80% ethanol overnight, the specimen is sequentially immersed in 95% ethanol I and II for 2 hours respectively and 100% ethanol I and II for 1.5 hours respectively. Xylene I, II are transparent 2 times, each for 40 min.
(3) And (3) paraffin embedding and slicing, namely sequentially immersing the specimen into soft wax I at 50-52 ℃ for 12h, hard wax II at 56-58 ℃ for 1h, and embedding by using embedding wax (the melting point is 58-60 ℃, and beeswax at 10-20 ℃) can be added). The tissue is sliced into slices of 3-5 μm thickness with a microtome. The cut thin slices are stuck on a glass slide and put in an incubator for drying.
(4) Staining, namely performing hematoxylin-eosin (HE) staining and safranin O-fast green staining on the cartilage section, and performing HE staining on the synovial membrane section.
HE staining:hematoxylin-eosin staining (HE staining for short) is one of the commonly used staining methods in paraffin section technology. The hematoxylin staining fluid is alkaline, and mainly makes chromatin in cell nucleus and nucleic acid in cytoplasm bluish: eosin is an acid dye that primarily reddens components in the cytoplasm and extracellular matrix. HE staining of cartilage and synovial membrane sections allows for study of pathological changes in cartilage and synovial tissue, and allows for assessment of the severity of cartilage damage and the size of synovial inflammation in various groups of rats.
The HE staining method comprises the following steps: dewaxing by dimethylbenzene I and dimethylbenzene II for 10min respectively, rehydrating absolute ethyl alcohol and 95% ethyl alcohol to 70% ethyl alcohol step by step, carrying out hematoxylin dyeing for 15min after each step of 2min by adding distilled water for 3min, carrying out color separation for 10s by 1% hydrochloric acid alcohol (prepared by 70% ethyl alcohol), washing for 20min by running water, carrying out eosin dyeing for 10min, dehydrating by 70% ethyl alcohol, 85% ethyl alcohol and 95% ethyl alcohol I and II at each step of 2min finally, carrying out transparency for 5min in the dimethylbenzene I and II respectively, and sealing by neutral gum.
And (3) dyeing with fast green and fast green by using pink O-dye:cartilage is composed of chondrocytes and extracellular matrix, and safranin O is a cationic dye that binds to polyanions and binds to polysaccharides of the cartilage matrix, reflecting the proteoglycan content and distribution in the matrix, and when cartilage is damaged, glycoproteins in cartilage are released, rendering the matrix components less distributed, resulting in less or no staining of safranin O. Cartilage sections were stained with safranin O-fast green to assess cartilage damage.
The method for dyeing the pink O-fast green comprises the following steps: dewaxing to water conventionally. Adding a freshly prepared Weibert dye liquor to dye for 3-5min, and washing with water. And (5) differentiating the acidic differentiation solution for 15 s. Washing with distilled water for 10 min. Dip-dyeing in a solid green dyeing solution for 5 min. The sections were washed rapidly with a weak acid solution for 10-15s to remove residual fast green. Dip-dyeing in Safranin O stain for 5 min. Dehydrating with 95% ethanol for 2-3s, anhydrous ethanol for 2-3s, and anhydrous ethanol for 1 min. Xylene is transparent and the optical resin is sealed.
3. Histopathological scoring of cartilage and synovium: based on HE staining of the femoral condyles and tibial plateau and safranin O-fast green staining, the reference performed histopathological scoring (ManKin scoring) of the femoral condyles and tibial plateau, the ManKin scoring criteria being shown in table 3-1. The synovial tissue was histopathologically scored according to HE staining of the synovial tissue, and the scoring criteria are shown in table 3-2.
4. Statistical analysis: the experimental results are expressed as mean ± standard deviation (mean ± SD). Student's t test was performed on the experimental data using SPSS 10.0 software, with P <0.05 indicating significant differences.
Table 6: mankin scoring criteria for articular cartilage
Figure BDA0002486388100000171
Table 7: criteria for histopathological scoring of synovium
Figure BDA0002486388100000172
5. Results and analysis
(1) Measurement results of rat body weight and knee width: body weight and knee width were recorded for rats during molding and at various time points after dosing, and the results are shown in fig. 22 and 23. As can be seen from FIG. 22, the weight of the rats in each group was increased after molding (2 weeks) compared to before molding, but the weight of the rats in the normal control group was larger than that in other molding groups (P < 0.01), indicating that the molding may affect the appetite and physical condition of the rats; the body weight of rats in each group is increased from the first administration to before death (3 weeks to 8 weeks), except that the body weight of the rats in the normal control group at week 4 is slightly lower than that of the free miR-140 group and Lnxc-sol group, the body weight of the rats in the normal control group at other time points (3 weeks, 5 weeks, 6 weeks, 7 weeks and 8 weeks) is larger than that of the rats in other experimental groups, which indicates that the appetite and the physical condition of the rats in the treatment group are still not as same as those in the normal control group, but the difference between the body weights of the rats in each group has no statistical significance, indicating that the appetite of the rats is basically normal and the physical condition of the rats in the whole experimental process is good. As can be seen from fig. 23, the average knee width of each group of rats before molding was about 9mm, and the average knee width of each group of rats after molding reached about 10.1mm, which indicates that the knee joints of the rats after molding were swollen, indicating that the molding was successful. The knee widths of rats in the OA model group and the free miR-140 group increase with time after administration, and the knee widths of rats in the Lnxc-sol group, the AuNPs/miR-140 group and the combination treatment group decrease with time. The average knee widths of the OA model group before the sacrifice of the rats and the free miR-140 group of rats are respectively 10.80mm and 10.75mm, and the average knee widths are not statistically different compared with the average knee widths of the free miR-140 group of rats; while the average knee widths of the Lnxc-sol group, the AuNPs/miR-140 group and the combined treatment group are respectively 9.72, 9.81 and 9.58mm, which is smaller than the average knee width of the OA model group (P < 0.01), but the difference of the average knee widths among the Lnxc-sol group, the AuNPs/miR-140 group and the combined treatment group has no statistical significance. These results indicate that the OA of the rat can cause the swelling of the knee joint of the rat, the free miR-140 can not relieve the swelling of the knee joint of the rat, and the OA has no treatment effect, because the free miR-140 is easily degraded by the enzyme in the body and is difficult to enter the cell to play a role; and Lnxc-sol, AuNPs/miR-140 and the combination of the Lnxc-sol and the AuNPs/miR-140 can relieve the swelling of the knee joint of the rat, but the combined treatment group has better effect and is closer to the level of a normal control group.
(2) Appearance observations of femoral condyles and tibial plateau in rats: after 6 weeks of dosing, the rats were sacrificed and the femoral condyles and tibial plateau of each group of rats were dissected out and photographed, see fig. 24 and 25. The effect of treatment after administration to model rats was evaluated from the photographs. As can be seen from FIG. 24, the surface of the femoral condyles of the rats in the normal control group was white, smooth and glossy; while the surfaces of the femoral condyles of rats in the OA model group and the free miR-140 group are yellow, the surface gloss is reduced, deeper fissures can be seen, and even local small erosion can be seen on the surfaces of the femoral condyles of the free miR-140 group. The surfaces of the thighbone ankles of the rats in the Lnxc-sol group, the AuNPs/miR-140 group and the combination treatment group are glossy, and the treatment effect of the combination treatment group is closest to that of a normal control group except that only small cracks are formed on the outer surface of the combination treatment group. As can be seen from FIG. 25, the tibial plateau surfaces of the rats in the normal control group were smooth and glossy and had no cracks, while the tibial plateau surfaces of the rats in the OA model group and the free miR-140 group had much visible cracks with reduced gloss, and the cracks in the tibial plateau surfaces of the rats in the Lnxc-sol, AuNPs/miR-140 group and the combination treatment group were reduced, and the cracks in the surfaces of the combination treatment group were the least, the best improvement effect was obtained, and the most similar to the normal control group.
The appearance observation results show that the cartilage of rats in the OA model group and the free miR-140 group is seriously damaged, the cartilage of the rats in the three treatment groups is not so seriously damaged, the three treatment groups can have certain treatment effect on OA, and the effect of the combined treatment group is superior to that of the other two groups.
(3) Results of histological evaluation of cartilage and synovium
HE staining results of femoral condylar and tibial plateau sections:HE staining results for femoral condylar and tibial plateau sections of each group of rats are shown in fig. 26 and 27. As can be seen from FIG. 26, the surface of the femoral condyles of the rats in the normal control group was smooth and seamless, and the chondrocytes were aligned. Chondrocytes were hardly visible in sections of the OA model group and free miR-140 group, and were absent. L isnxc-sol group has reduced chondrocyte number and disordered arrangement. The AuNPs/miR-140 group has a large number of chondrocytes, but the cells have clustering phenomenon. The rat cartilage cells of the combined treatment group are more in quantity and more orderly arranged, and are closest to the normal control group. As can be seen from fig. 27, the tibial plateau of the rats in the normal control group had a smooth and seamless surface, and the number of chondrocytes was large and aligned. Chondrocytes were hardly seen in the sections of the OA model group, the surface of cartilage in the free miR-140 group was fractured, and only a small number of chondrocytes were seen in the sections, whereas chondrocytes were relatively large and well-aligned in the Lnxc-sol group, AuNPs/miR-140 group and combination treatment group.
The HE staining results of the sections show that the cartilage damage conditions of rats in the Lnxc-sol group, the AuNPs/miR-140 group and the combined treatment group caused by OA are improved, and particularly the improvement effect of the combined treatment group is optimal.
HE staining of synovial sections:HE staining of rat synovial sections is shown in fig. 28. As can be seen from fig. 28, the synovium of the normal control group had no inflammatory cell infiltration. The OA model group and the free miR-140 group have a large amount of inflammatory cell infiltration in the synovium. There was also some inflammatory cell infiltration in synovium of AuNPs/miR-140 group, but less inflammatory cell infiltration than in OA model group. Only a small amount of inflammatory cells infiltrate into the synovium of the Lnxc-sol group and the combined treatment group, which shows that AuNPs/miR-140 and Lnxc both have certain anti-inflammatory effect.
Safranin O-fast green staining of femoral condylar and tibial plateau sections:safranin O stains proteoglycan in the cartilage matrix, while cartilage is composed of 1% chondrocytes and 99% extracellular matrix, so safranin O-fast green staining of cartilage sections can be used to characterize articular cartilage defects. The results of safranin O-fast green staining of femoral condylar and tibial plateau sections of each group of rats are shown in fig. 29 and fig. 30. As can be seen from fig. 29, the normal control group had a large staining range and a deep staining of cartilaginous safranin O in the femoral condyles. The OA model group and the free miR-140 group are subjected to safranin O transfection; and the coloring range of the safranin O in the Lnxc-sol group, the AuNPs/miR-140 group and the combination treatment group is gradually enlarged, and the coloring range of the safranin O in the combination treatment group is the largest and is closest to that in the normal control group. As can be seen from FIG. 30, the normal pairThe cartilaginous safranin O staining of the tibial plateau of the control was extensive and dark. OA model group and safranin O loss in most regions of free miR-140; while the Lnxc-sol group, the AuNPs/miR-140 group and the combination treatment group only partially lost safranin O.
The safranin O-fast green staining result of the section shows that compared with an OA model group and a free miR-140 group, the cartilage matrixes of rats in an Lnxc-sol group, an AuNPs/miR-140 group and a combined treatment group are more, the Lnxc-sol, the AuNPs/miR-140 and the combined application all have certain treatment effect on OA, and particularly the combined application has the best treatment effect.
(4) Histopathological scoring of cartilage and synovium
Mankin scores for femoral condyles and tibial plateau:the results of the Mankin scores for the femoral condyles and tibial plateau are shown in fig. 31. As can be seen from FIG. 31, the Mankin scores of the OA model groups of the femoral condyles and the tibial plateau were 8.13. + -. 0.67 and 7.44. + -. 0.88, respectively, and the Mankin scores of the femoral condyles and the tibial plateau of the free miR-140 group were not significantly different from those of the OA model group, indicating that the free miR-140 could not play a role in repairing cartilage damage, whereas the Mankin scores of the femoral condyles and the tibial plateau of the Lnxc-sol group, the AuNPs/miR-140 group and the combination treatment group were all lower than those of the OA model group (P < 0.01), indicating that the Lnxc-sol, the AuNPs/miR-140 and the combination treatment group were all capable of repairing cartilage damage, and the Mankin score of the combination treatment group was lower than those of the AuNPs/miR-140 and the Lnxc-sol group (P < 0.01), indicating that the cartilage damage of rats could be better repaired by applying the Lnxc-sol and the combination treatment group.
Histopathological scoring of synovium:the results of the histopathological scoring of synovium are shown in figure 32. As can be seen from FIG. 32, the histopathological score of the synovium of the OA model group is 12.33 + -0.49, the score of the free miR-140 group is not significantly different from that of the OA model group, while the histopathological scores of the synovium of the Lnxc-sol group, the AuNPs/miR-140 group and the synovium of the combined treatment group are all lower than that of the OA model group (P < 0.01), indicating that the Lnxc-sol, the AuNPs/miR-140 and the combined application can improve the synovium inflammation of OA to some extent, and the score of the Lnxc-sol group and the combined treatment group is lower than that of the AuNPs/miR-140 (P < 0.01), indicating that the combined application of the Lnxc-sol and the AuNPs/miR-140 to treat OA can achieve eliminationRemoving synovial inflammation and repairing cartilage damage.
In addition to the spontaneous OA model, the OA model of animals is mainly induced, i.e. the animals are induced to produce OA symptoms by certain experimental means. The OA molding method commonly used at present comprises a joint braking method, a joint cavity injection medicine method, an operation method and the like. Papain is a proteolytic enzyme, and injection of papain into the joint cavity of animals can produce models of OA by disrupting the cartilage matrix. The method has the advantages of short molding time, high success rate and similarity to human OA. The rat is a common experimental animal and has the advantages of easy feeding and simple experimental operation. Therefore, the OA model was established in this experiment by injecting papain into the joint cavity of rats.
In the embodiment of the invention, the inventor finds that the Lnxc-sol and AuNPs/miR-140 are combined to play complementary roles in treating OA, so that the OA can be treated and cartilage can be repaired better. The HE staining and safranin O-fast green staining results of the cartilage sections show that the cartilage damage degree of the Lnxc-sol group is smaller than that of the OA model group, and meanwhile, the HE staining results of the synovial membrane sections also show that the synovial membrane inflammation of the AuNPs/miR-140 group is reduced compared with the OA model group, so that the synovial membrane inflammation and the cartilage damage of OA are mutually promoted, the injury of cartilage is aggravated by the synovial membrane inflammation, and fragments generated by the cartilage damage can be used as foreign matters to cause the synovial membrane inflammation. The Lnxc-sol and AuNPs/miR-140 are combined to play a better role in treating OA and protecting and repairing chondrocytes.
And (4) conclusion:the weight measurement result shows that the weight of the rats in the model group is smaller than that of the rats in the normal control group, but the difference between the weights of the rats in each group after the last administration for one week has no statistical significance, which indicates that the model building can influence the appetite and the state of the rats, but the state of the rats in each group after the last administration for one week is better. Measurement of Knee WidthThe average knee width of the rat after the model is made is increased from about 9mm to about 10.1mm, which shows that the model is successfully made; after administration, the knee width of the free miR-140 group rats has no significant difference with that of the OA model group, while the knee widths of the Lnxc-sol group, the AuNPs/miR-140 group and the combined treatment group rats are smaller than those of the OA model group, and the knee width of the combined treatment group is the smallest, which indicates that the Lnxc-sol and the AuNPs/miR-140 can relieve knee joint swelling of the OA rats, and the combined effect of the Lnxc-sol and the AuNPs/miR-140 is better. The appearance observation result of cartilage shows that the cartilage surface of the normal control group rat is smooth and glossy, the cartilage of the free miR-140 group and the OA model group rat is seriously damaged, the cartilage damage condition of the Lnxc-sol group and the AuNPs/miR-140 group is improved, and the cartilage surface of the combined treatment group is almost the same as that of the normal group, so that the treatment effect is best. HE staining of cartilage, safranin O-fast green staining and Mankin scoring result show that the rat cartilage of the normal control group is not damaged, and the cartilage damage degree of the rest groups of rats is sequenced into that an OA model group is approximately equal to a free miR-140 group which is larger than an Lnxc-sol group which is larger than or equal to an AuNPs/miR-140 group which is larger than a combined treatment group; HE staining of synovium and histopathological scoring result show that the synovium of the rats in the normal control group has no inflammation, and the rest groups have the following synovium inflammation ranking: the OA model group is approximately equal to the free miR-140 group, the AuNPs/miR-140 group, the Lnxc-sol group and the combination treatment group. The results show that the injection of the free miR-140 can not treat the OA of the rat, while the Lnxc-sol, the AuNPs/miR-140 and the combination of the Lnxc-sol and the AuNPs/miR-140 can treat the OA, and particularly the combined medicine has the best treatment effect.
Although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (17)

1. A pharmaceutical kit comprising an AuNPs/miR-140 complex and at least one non-steroidal anti-inflammatory drug;
the AuNPs/miR-140 compound is obtained by compounding gold nanoparticle AuNPs and miR-140, wherein the mass ratio of the AuNPs to the miR-140 is 5-28.8: 1.
2. The pharmaceutical kit of claim 1, wherein the AuNPs/miR-140 complex has a particle size of 24-85nm, a PDI of 0.3-0.6, and a Zeta potential of 20-30 mV.
3. The pharmaceutical kit of claim 1, wherein the AuNPs are prepared from HAuCl4The composite material is obtained by compounding with polyethyleneimine, and the structure of the composite material is that an inner core is in a spherical shape with an Au simple substance coated with PEI, the particle size is 17-27nm, the PDI is 0.23-0.3, and the Zeta potential is 31-34 mV.
4. The pharmaceutical kit of claim 1, wherein the non-steroidal anti-inflammatory drug is lornoxicam.
5. The pharmaceutical kit according to claim 1, wherein the AuNPs/miR-140 complex in the kit is an injection.
6. The pharmaceutical kit of claim 5, wherein the injection is an injectable powder.
7. The pharmaceutical kit according to claim 1, wherein the preparation method of the AuNPs/miR-140 compound comprises the steps of mixing miR-140 and AuNPs solution uniformly and then standing for incubation.
8. The pharmaceutical kit according to claim 7, wherein the mass ratio of AuNPs to miR-140 is 5-28.8: 1.
9. The pharmaceutical kit of claim 8, wherein the preparation of the AuNPs solution comprises: adding HAuCl into the mixture under the condition of stirring in water bath4Adding a PEI aqueous solution into the solution, and reacting, namelyAnd (5) obtaining the product.
10. The pharmaceutical kit according to claim 9, wherein the volume of the PEI aqueous solution is 400 μ L-6.4mL with a concentration of 3 mg/mL.
11. The pharmaceutical kit of claim 9, wherein the aqueous PEI solution is mixed with HAuCl4The volume ratio of the solution is not less than 1: 1.
12. The pharmaceutical kit of claim 9, wherein the water bath temperature is 40-80 ℃.
13. The pharmaceutical kit according to claim 9, wherein the reaction time is 30min to 3 h.
14. Use of a pharmaceutical kit according to any one of claims 1 to 13 for the manufacture of a medicament for the prevention and treatment of osteoarthritic diseases or for the manufacture of a medicament for promoting cartilage repair or for the manufacture of a medicament for the protection of cartilage.
15. Use of a pharmaceutical kit of any one of claims 1-13 in the manufacture of a medicament or agent for up-regulating expression of miR-140 in chondrocytes and/or increasing expression levels of COL2A mRNA in chondrocytes.
16. Use of lornoxicam in combination with an AuNPs/miR-140 complex as described in any of claims 1 to 3 for the manufacture of a medicament for the prevention and treatment of osteoarthritic diseases or for the manufacture of a medicament for promoting cartilage repair or for the manufacture of a product for protecting cartilage.
17. The use of claim 16, wherein the product is a pharmaceutical or a wearable medical device.
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