CN111407727A - Application of mammal target of rapamycin (mTOR) blocker in preparation of medicines for treating calcified tendinosis - Google Patents

Abstract

The mammalian target of rapamycin (mTOR) blocker is an application of CHP-P L GA-RAPA nanoparticle in preparing a medicament for treating calcified tendinosis, the nanoparticle can be specifically combined with pathological tendon, and the ectopic ossification process in connective tissues such as tendon and ligament of a mouse injured by the tendon can be prevented by regulating the differentiation of tendon stem progenitor cells through rapamycin, and in the application, the CHP-P L GA-RAPA nanoparticle shows excellent pathological collagen affinity, controlled release capacity and bioactivity.

Description

Mammalian target of rapamycin (mTOR) blocker in preparation for the treatment of calcific muscle Application of tendinopathy drugs

technical field

The invention belongs to the technical field of tendon-related diseases treatment drugs, in particular to the application of a mammalian target of rapamycin (mTOR) blocker in the preparation of a drug for treating calcific tendinopathy.

Background technique

About 30% of clinical consultations regarding disorders of the musculoskeletal system are due to tendon injuries. Heterotopic ossification of the tendon is a subtype of tendinopathy that destroys the structure of the tendon tissue itself, exacerbating symptoms and severely impairing the tendon's mobility.

Currently, there is no specific treatment for tendon calcification, and traditional treatments are still the mainstay, including rest, physical therapy, oral analgesics, local non-steroidal anti-inflammatory drug injection, and surgical treatment after conservative treatment fails. However, the above conservative treatment is not effective, and the operation has a large complication rate and the effect is unstable. The main challenges are insufficient understanding of the key mechanisms of ossification and lack of appropriate systems for precise drug delivery to pathological tendons.

SUMMARY OF THE INVENTION

The purpose of the present invention is to solve the problems existing in the prior art, and propose to use a mammalian target of rapamycin (mTOR) blocker as a medicine for the preparation of calcific tendinopathy, which has a remarkable curative effect.

The invention discloses the application of a mammalian target of rapamycin (mTOR) blocker in preparing a medicine for treating calcific tendinopathy.

Wherein, the mTOR blocker is collagen hybrid peptide-poly(lactic-glycolic acid)-rapamycin nanoparticle, and the corresponding English abbreviation of the nanoparticle is CHP-PLGA-RAPA nanoparticle.

Wherein, the collagen hybrid peptide is a commercially available product, and its specific structure is [(Gly-Pro-Hyp) ₆ -Gly-Gly-Gly] ₂ -Lys.

Wherein, the preparation process of CHP-PLGA-RAPA nanoparticles includes the following steps:

(1) Preparation of PLGA-RAPA nanoparticles: prepared by emulsion-solvent evaporation method with emulsifier molecules as stabilizers;

(2) Preparation of CHP-PLGA-RAPA nanoparticles: prepared by covalently conjugating collagen hybrid peptides to PLGA-RAPA nanoparticles through a cross-linking agent.

Wherein, the emulsifier is bovine serum albumin (BSA) solution; the cross-linking agent is glutaraldehyde.

Among them, calcific tendinopathy is heterotopic ossification of tendon.

The mTOR blocking agent of the present invention is CHP-PLGA-RAPA nanoparticle, which can specifically bind to pathological tendons, mediate the differentiation effect of tendon stem progenitor cells through rapamycin, and can prevent tendon and tendon damage in mice with tendon injury. The process of heterotopic ossification in connective tissues such as ligaments. And in this application, CHP-PLGA-RAPA nanoparticles exhibited excellent pathological collagen affinity, controlled release ability, and bioactivity.

Description of drawings

Fig. 1 is a characterization diagram of PLGA-RAPA nanoparticles and CHP-PLGA-RAPA nanoparticles, wherein, Fig. 1A is a scanning electron microscope image; Fig. 1B is a transmission electron microscope image;

Figure 2 shows the TEM images of CHP-PLGA-RAPA nanoparticles combined with intact collagen (Intact collagen) and damaged collagen (Injured collagen);

Figure 3. In vivo assessment of the pathological tendon affinity of collagen hybrid peptide-poly(lactic-glycolic acid) nanoparticles in a model of tendon heterotopic ossification;

Figure 4 is the characterization diagram of micro-CT and histological examination of the repaired mouse Achilles tendon at 6 weeks after heterotopic ossification modeling, in which Figure 4A is the Micro-CT imaging diagram; Figure 4B is the histological evaluation diagram, Stained with HE, MASSON and SO, respectively.

Detailed ways

Unless otherwise defined, technical terms used in the following embodiments have the same meanings as commonly understood by those skilled in the art to which the present invention belongs. The test reagents used in the following examples are conventional biochemical reagents unless otherwise specified; the experimental methods are conventional methods unless otherwise specified. The present invention will be described in detail below with reference to the embodiments.

Example 1 Preparation and Characterization of Nanoparticles

(1) Preparation of PLGA-RAPA nanoparticles

Both poly(lactic-glycolic acid) (PLGA, Mw～78kD, Jinan Daigang Biotechnology Co., Ltd., China) and rapamycin (RAPA, S1039, Selleck) were dissolved in dichloromethane at a concentration of 20 mg/ml, respectively And 1mg/ml, get PLGA/RAPA solution. 1 ml of the PLGA/RAPA solution was quickly added to 4 ml of the emulsifier bovine serum albumin (BSA) solution (3%) (Sigma-Aldrich) under magnetic stirring. Probe sonication was applied and adequate emulsification was performed on ice at 10 W for 30 s. Then, stir for at least 3 hours until set. PLGA-RAPA nanoparticles were collected and purified by centrifugation at 12,000 rpm for 10 minutes, 3 times; they were then resuspended in ultrapure water to remove excess emulsifier solution molecules.

(2) Preparation of CHP-PLGA-RAPA nanoparticles

Prior to preparation, the peptides were heated (80°C; 5 minutes) and then cooled on ice for 1 minute to unwind the helical structure of the peptides. A suspension of PLGA-RAPA nanoparticles (1 mg/ml) was mixed with glutaraldehyde (final concentration of 1 mg/ml) for 12 hours at room temperature with gentle shaking. After centrifugation and ultrapure water washing, the collagen hybrid peptide [(Gly-Pro-Hyp) ₆ -Gly-Gly-Gly] ₂ -Lys was added to the above system and further conjugated and left at room temperature for one week. The peptide-modified PLGA-RAPA was collected by centrifugation at 12,000 rpm for 10 minutes, and washed with water for 3 times under sonication to remove free peptide molecules, resulting in CHP-PLGA-RAPA nanoparticles.

The SEM and TEM results of PLGA-RAPA nanoparticles and CHP-PLGA-RAPA nanoparticles are shown in Figure 1.

Example 2 Binding specificity of CHP-PLGA-RAPA nanoparticles

Collagen type 1 (extracted from porcine tendon) (0.5 mg/ml) was denatured by heating (80°C; 5 min) and placed in an ice bath. Digestion with 2% collagenase for 30 minutes in a 37°C incubator damages porcine tendons. Immobilize 10 μl of wounded type 1 collagen or 10 μl of intact type 1 collagen onto the nickel mesh. After 1 minute, remove the excess solution with absorbent paper, then add 10 μl poly(lactic-glycolic acid) or collagen hybrid peptide-poly(lactic-glycolic acid) nanoparticles (10 nM) to incubate the assembled collagen nickel mesh 10 minutes and washed 3 times with double distilled water. Another 10 μl of 2% (w/v) uranyl acetate staining solution was added to the grid to stain the sample, and after 1 minute, the excess solution was blotted off with absorbent paper. The above nickel mesh was left to dry overnight at room temperature. Finally, observe under a projection electron microscope. Subsequently, intact and injured tendons were separately incubated with CHP-PLGA-RAPA nanoparticles (10 μM) for 2 h on a shaker at room temperature. After that, the above tendons were washed three times with PBS and further processed for scanning electron microscopy.

According to scanning electron microscopy and transmission electron microscopy results, CHP-PLGA-RAPA nanoparticles were specifically attached to damaged collagen or tendon, but rarely in intact collagen or intact tendon, as shown in Figure 2.

Example 3 Animal Experiments - In Vivo Fluorescence Imaging and Analysis

To establish a tendon heterotopic ossification model, type 1 collagenase (12.5 U/leg, Gibco, 17100017) was injected into the midpoint of the right Achilles tendon of 8-week-old mice, in PLGA-RAPA nanoparticles or CHP-PLGA-RAPA nanoparticles Cy5 (Lumiprobe, 23020) was used to replace RAPA during the synthesis of PLGA-Cy5 or CHP-PLGA-Cy5, respectively. One week after tendon heterotopic ossification modeling, mice were injected with PLGA-Cy5 or CHP-PLGA-Cy5. Mice were anesthetized with isoflurane and analyzed by a fluorescence imaging system (IVIS Spectrum CT, PerkinElmer) at the indicated time points.

Fig. 3 is the in vivo fluorescence imaging of mice after the above nanoparticles were injected into the injured Achilles tendon, and the total radiation efficiency of PLGA-Cy5 or CHP-PLGA-Cy5 at different time points. The results showed that compared with PLGA-Cy5, CHP-PLGA-Cy5 significantly enhanced the retention of Cy5 in tendon tissue at various time points, indicating that the former has a stronger affinity for damaged tendons and is a more effective drug delivery vehicle.

Example 4 Animal experiment-CT imaging and histological examination

To establish a tendon heterotopic ossification model, type 1 collagenase (12.5 U/leg, Gibco, 17100017) was injected into the midpoint of the right Achilles tendon of 8-week-old mice, and the same operation was performed 3 days later to stabilize the effect. 10 WT mice (mTOR ^floxed ) and 10 mTOR-TKO mice (Scx-cre; mTOR ^floxed ) were sacrificed 6 weeks after modeling and legs were removed for micro-CT imaging and subsequent histological examination.

In the in vivo drug experiment, 60 mice were equally divided into four groups and injected subcutaneously once a week: control group (sterile PBS, 5 μl), RAPA group (rapamycin, 70 μM, 5 μl), PLGA-RAPA group (70 μM, 5 μl) and CHP-PLGA-RAPA group (70 μM, 5 μl). Six weeks after treatment, 10 mice per group were sacrificed. The legs were taken for micro-CT and histological examination, as shown in Figure 4. Figure 4A is the Micro-CT image of the repaired mouse Achilles tendon at 6 weeks after heterotopic ossification modeling. Micro-CT imaging show that poly(lactic-glycolic acid)-rapamycin nanoparticles and collagen hybrid peptide-poly(lactic-glycolic acid)-rapamycin nanoparticles, especially the latter, significantly reduce ectopic The occurrence of ossification; Figure 4B is histological assessment, including HE, MASSON and SO staining, only little or no heterotopic bone tissue was observed in the latter two groups, confirming the significant improvement in the sustained-release drug group.

Matters not addressed in the present invention are known in the art.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the scope of the present invention. within the scope of protection.

Claims (7)

1. Application of a mammalian target of rapamycin (mTOR) blocker in the preparation of a medicament for treating calcific tendinopathy. 2 . The application according to claim 1 , wherein the mTOR blocking agent is collagen hybrid peptide-poly(lactic-glycolic acid)-rapamycin nanoparticles (CHP-PLGA-RAPA nanoparticles). 3 . The application according to claim 2, wherein the collagen hybrid peptide has a structure of [(Gly-Pro-Hyp) ₆ -Gly-Gly-Gly] ₂ -Lys. 4. application according to claim 2, is characterized in that, CHP-PLGA-RAPA nano particle preparation process comprises the steps: (1) Preparation of PLGA-RAPA nanoparticles: prepared by emulsion-solvent evaporation method with emulsifier molecules as stabilizers; (2) Preparation of CHP-PLGA-RAPA nanoparticles: prepared by covalently conjugating collagen hybrid peptides to PLGA-RAPA nanoparticles through a cross-linking agent. 5. The application according to claim 4, wherein the emulsifier is a bovine serum albumin (BSA) solution. 6. The application according to claim 4, wherein the cross-linking agent is glutaraldehyde. 7. The application according to claim 1-6, wherein the calcific tendinopathy is tendon heterotopic ossification.

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