Preparation method and application of fibronectin and collagen loaded PLGA nanoparticles

Abstract

The application relates to the field of biomaterials and regenerative medicine, in particular to a preparation method and application of fibronectin and collagen protein-loaded PLGA nanoparticles, wherein the preparation method adopts a complex emulsion solvent evaporation method, a buffer solution containing fibronectin, collagen protein and trehalose is used as an inner water phase, a dichloromethane solution of PLGA is used as an oil phase, and a polyvinyl alcohol and trehalose-containing aqueous solution is used as an outer water phase; and the nanoparticles are prepared through emulsification, high-pressure homogenization, dialysis and freeze-drying. The method has the beneficial effects of stable process and effective protection of protein activity. The obtained nanoparticles have uniform particle sizes (100-200 nm), and can realize synergistic controllable release of double proteins. The product organically combines the physical filling support function of PLGA and the biological repair function of active factors, realizes the integration of "filling-regeneration", can significantly reduce the risk of inflammation, and can be safely applied to soft tissue filling, wrinkle improvement and skin wound repair in the field of medical cosmetology.

Description

Technical Field

[0001] This invention relates to the fields of biomedical materials and regenerative medicine, specifically to a method for preparing polylactic acid-glycolic acid copolymer (PLGA) nanoparticles loaded with fibronectin (FN) and collagen, as well as the product obtained by this method and its application in soft tissue filling and repair. Background Technology

[0002] In the fields of medical aesthetics and wound repair, soft tissue filling and regeneration are core needs. Currently used clinical fillers, such as poly-L-lactic acid (PLLA) and polycaprolactone (PCL), while providing immediate volume-filling effects, still have many limitations. For example, PLLA may produce acidic byproducts during degradation, leading to local microenvironment acidification and triggering inflammatory responses; PCL has a long degradation cycle, and long-term retention may pose long-term risks. More importantly, these traditional fillers primarily act as "physical scaffolds," lacking the ability to actively induce host tissue regeneration and repair, making it difficult to achieve the ideal integrated effect of "filling as repair."

[0003] Fibronectin is a key glycoprotein in the extracellular matrix, promoting cell adhesion, migration, and proliferation, and regulating collagen synthesis, playing a central role in tissue repair. Collagen, especially type III collagen, is a major component of young, healthy dermal tissue, providing regenerative templates and signals for cells. However, directly applying these two bioactive proteins clinically faces significant challenges: they are easily and rapidly degraded by enzymes in vivo, have short half-lives; direct local injection may lead to a burst release of concentration, triggering excessive inflammatory responses or fibrosis; and their activity is easily compromised during preparation and storage.

[0004] PLGA is a biodegradable polymer material with good biocompatibility and a controllable degradation rate. Using PLGA as a carrier to construct nanomedicine delivery systems can protect the loaded active substances and achieve sustained drug release through carrier degradation, thereby maintaining a locally effective drug concentration and reducing toxic side effects. The double emulsion solvent evaporation method (W1 / O / W2) is a commonly used method for preparing PLGA nanoparticles loaded with hydrophilic drugs.

[0005] Therefore, developing a PLGA nanoparticle system that can synergistically load fibronectin and collagen and achieve their controlled sustained release, combining physical filling support with bioactive repair, is of great significance for promoting the development of the field of soft tissue regeneration. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a method for preparing PLGA nanoparticles loaded with fibronectin and collagen. This method is stable, effectively protects protein activity, and achieves co-encapsulation and synergistic sustained release of the two proteins.

[0007] Another object of the present invention is to provide PLGA nanoparticles prepared by the above method. These nanoparticles have uniform particle size, good stability, and possess both immediate physical filling and long-lasting bioactive repair functions.

[0008] Another object of the present invention is to provide the application of the above-mentioned PLGA nanoparticles in the preparation of integrated soft tissue filling and repair materials.

[0009] To achieve the above objectives, the present invention adopts the following technical solution:

[0010] In a first aspect, the present invention provides a method for preparing PLGA nanoparticles loaded with fibronectin and collagen, comprising the following steps:

[0011] (1) Preparation of the inner aqueous phase (W1): Collagen, trehalose and fibronectin were dissolved in phosphate buffer at pH 7.0-7.5.

[0012] (2) Preparation of oil phase (O): PLGA was dissolved in dichloromethane and then emulsifier Span80 was added.

[0013] (3) Preparation of colostrum (W1 / O): The inner aqueous phase is added dropwise to the oil phase under ice-water bath cooling and emulsified by ultrasonic treatment.

[0014] (4) Preparation of external aqueous phase (W2): Polyvinyl alcohol and trehalose are dissolved in ultrapure water.

[0015] (5) Preparation of double emulsion (W1 / O / W2): The primary emulsion is added to the external aqueous phase, stirred and premixed, and then subjected to high pressure homogenization.

[0016] (6) Solvent evaporation and solidification: The re-emulsion is placed in a dialysis bag and dialyzed against deionized water or buffer solution to remove organic solvents and free components. After dialysis, the pH of the system is adjusted and the system is sterilized by filtration.

[0017] (7) Freeze-drying: After pre-freezing the purified nanoparticle suspension, freeze-drying is performed to obtain solid nanoparticle powder.

[0018] As a preferred technical solution of the present invention, the collagen in step (1) is recombinant human type III collagen.

[0019] As a preferred technical solution of the present invention, the pressure of the high-pressure homogenization in step (5) is 1300 bar, and the cycle is 5-6 times.

[0020] Secondly, the present invention provides PLGA nanoparticles loaded with fibronectin and collagen prepared by the above method. The nanoparticles have an average particle size of 100-200 nm, a polydispersity index (PDI) of less than 0.3, an encapsulation efficiency of fibronectin ≥75%, and an in vitro release period of 7-14 days.

[0021] Thirdly, this invention provides the application of the PLGA nanoparticles in the preparation of materials for soft tissue filling and repair. Specifically, they can be used in medical aesthetics for wrinkle removal, shaping, and skin texture improvement, or for the repair of soft tissue defects caused by skin trauma, burns, ulcers, and surgery.

[0022] Compared with the prior art, the beneficial effects of the present invention are:

[0023] 1. Functional Integration: This invention creatively integrates the physical support function of the biodegradable polymer material PLGA with the biological functions of fibronectin (initiating repair signals) and collagen (providing regeneration templates) into a single nanocarrier, achieving simultaneous "instant filling" and "active regeneration".

[0024] 2. High safety and controllability: Through PLGA nano-encapsulation and sustained-release technology, easily inactivated and potentially irritating active proteins are transformed into stable and sustained-release "physiological signals," effectively avoiding the risks of inflammation, allergies, and fibrosis caused by sudden concentration release, making the regeneration process gentler and more controllable.

[0025] 3. Excellent activity protection: The preparation process employs low-temperature operation and the addition of a lyophilization protectant (trehalose) to maximize the protection of the bioactivity of fibronectin and collagen. The nanocarrier also improves the stability of the proteins and their delivery efficiency to target cells.

[0026] 4. Stable and reliable preparation process: The double emulsion-solvent evaporation method is a mature process. By optimizing the composition of the internal aqueous phase, emulsification and homogenization parameters, the uniformity of nanoparticle size, high encapsulation efficiency and good batch-to-batch stability are ensured, which has industrialization potential.

[0027] 5. Broad application prospects: The resulting product can be used as an injectable filler or a topical permeation enhancer, and can be widely applied in the fields of medical aesthetics and clinical wound repair, with significant market value and clinical translational significance. Attached Figure Description

[0028] Figure 1 This is a particle size distribution diagram of the nanoparticles prepared in Example 1 of the present invention.

[0029] Figure 2 Transmission electron microscope (TEM) image of the nanoparticles prepared in Example 1 of this invention.

[0030] Figure 3 This is a comparison chart showing the improvement in skin wrinkles in three groups of subjects in Application Example 1 of the present invention. Detailed Implementation

[0031] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0032] Example 1: Preparation of PLGA nanoparticles loaded with fibronectin and recombinant human type III collagen

[0033] (1) Preparation of the inner aqueous phase (W1): Accurately weigh 10 mg of recombinant human type III collagen and 15 mg of trehalose, dissolve them in 0.88 mL of pH 7.2 PBS buffer, and vortex until dissolved. Then add 0.120 mL of fibronectin solution with a concentration of 2 mg / mL, mix gently to obtain the inner aqueous phase (W1).

[0034] (2) Preparation of oil phase (O): Weigh 300 mg PLGA (LA:GA=50:50, Mw≈10,000 Da) into a container, add 10 mL of dichloromethane, and sonicate to dissolve it completely. Then add 50 mg of emulsifier Span80, vortex mix to obtain oil phase (O).

[0035] (3) Preparation of colostrum (W1 / O): Place the container containing the oil phase in an ice-water bath. Under magnetic stirring, slowly add the inner aqueous phase (W1) to the oil phase (O) using a microsyringe. After the addition is complete, emulsify the mixture using an ultrasonic cell disruptor (700W power, 10 seconds operation, 5 seconds interval) for a total time of 10 minutes to obtain a uniform W1 / O colostrum.

[0036] (4) Preparation of external aqueous phase (W2): Weigh 1.0g polyvinyl alcohol (PVA) and 2.5g trehalose, dissolve them in 50mL of ultrapure water, stir magnetically and heat appropriately until completely dissolved, cool to room temperature to obtain external aqueous phase (W2).

[0037] (5) Preparation of double emulsion (W1 / O / W2): The pre-emulsion obtained in step (3) was slowly poured into the external aqueous phase (W2) at a stirring speed of 400 rpm, and stirring was continued for 10 minutes for pre-emulsification. Then the mixed emulsion was transferred to a high-pressure homogenizer and homogenized 5 times at a pressure of 1300 bar to obtain a milky white, uniform and delicate W1 / O / W2 double emulsion.

[0038] (6) Solvent evaporation and solidification: The re-emulsion was transferred into a dialysis bag with a molecular weight cutoff of 8-14 kDa and placed in a beaker containing a large amount of PBS (pH 7.4) at 4°C for dialysis for 24 hours, during which the dialysis solution was changed 3-4 times to completely remove dichloromethane and free small molecules. After dialysis, the pH of the system was adjusted to 7.2 with dilute NaOH solution and filtered through a 0.45 μm microporous membrane.

[0039] (7) Freeze-drying: The filtered nanoparticle suspension was dispensed into freeze-drying bottles and pre-frozen in an ultra-low temperature freezer at -80℃ for more than 4 hours. Then the freeze-drying bottles were transferred to a freeze dryer and freeze-dried for 24-48 hours at a condenser temperature of -80℃ and a vacuum degree of 0.1mbar to obtain a white loose powder product, which was then sealed and stored at room temperature.

[0040] Experimental Example 1: Characterization of Nanoparticles

[0041] The lyophilized powder obtained in Example 1 was reconstituted with deionized water and then characterized.

[0042] (1) Particle size determination: Dynamic light scattering particle size analyzer was used. The results showed that the average hydrated particle size of the nanoparticles was 116.85 nm and the polydispersity index (PDI) was 0.24. This indicates that the particle size is uniform and the system has good stability.

[0043] (2) Morphological observation: A small amount of the reconstituted solution was dropped onto a copper grid, negatively stained, and observed under a transmission electron microscope. For example... Figure 2 As shown, the nanoparticles are regular spherical in shape with smooth surfaces, and their particle size distribution is consistent with the dynamic light scattering results.

[0044] (3) Encapsulation efficiency and drug loading determination: The content of free and total fibronectin in the system was determined by high performance liquid chromatography, and the encapsulation efficiency was calculated by the difference method. The total protein content was determined by the BCA protein quantification kit, and the collagen encapsulation efficiency was calculated. The results showed that the encapsulation efficiency of fibronectin was 79.2%, and the drug loading was 2.3%; the encapsulation efficiency of collagen was approximately 75.5%.

[0045] Application Example 1: Evaluation of the use of nanoparticle reconstituted solutions for improving skin wrinkles

[0046] (1) Sample preparation: Take 50 mg of the lyophilized nanoparticle powder prepared in Example 1, add 5 mL of sterile physiological saline under sterile conditions, and gently vortex to fully reconstitute it, so as to prepare a 10 mg / mL nanoparticle essence. Use an equal volume of sterile physiological saline as a placebo control.

[0047] (2) Experimental grouping and protocol: Sixty healthy female volunteers with mild to moderate static wrinkles around the eyes were recruited and randomly divided into three groups (n=20). Experimental group: After applying the essence of this invention, the essence was introduced into the skin using a medical ultrasound infusion device (frequency 1MHz, intensity 0.5W / cm²) for 5 minutes; Essence control group: Only an equal amount of essence was applied and massaged for absorption; Placebo control group: An equal amount of saline solution was applied and ultrasound infusion was performed. All groups were treated 3 times a week for 4 weeks.

[0048] (3) Evaluation of results: Before treatment, 30 minutes after each treatment, and at the end of the 4-week treatment course, the wrinkle depth in the same area was quantitatively measured using a three-dimensional skin imaging system, and the percentage of improvement was calculated. The results are shown in Table 1 and... Figure 3 As shown.

[0049] Table 1 Comparison of the percentage improvement in wrinkle depth among the three groups of subjects (mean ± SD, %)

[0050] Group 0min (baseline) 30 minutes after treatment 4 weeks after treatment Experimental group (serum + ultrasonic infusion) 0 58.7±6.2 76.3±7.5 Serum control group (serum only) 0 22.4±5.1 45.6±6.8 Placebo control group (saline solution + ultrasound infusion) 0 5.2±2.3 8.1±3.0

[0051] Statistical analysis showed that the experimental group exhibited significantly better improvement at both 30 minutes and 4 weeks compared to the other two groups (P<0.01). The results indicate that the nanoparticle essence of this invention, combined with physical penetration enhancement technology, enables rapid penetration and long-lasting release of active ingredients, producing significant immediate and lasting wrinkle-reducing effects with good safety profile.

[0052] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for preparing PLGA nanoparticles loaded with fibronectin and collagen, characterized in that: Includes the following steps: Step (1): Preparation of the inner aqueous phase: Collagen, trehalose and fibronectin were dispersed in PBS buffer at pH 7.0-7.5 and stirred to dissolve, thus obtaining the inner aqueous phase (W1). Step (2): Oil phase preparation: PLGA was dissolved in dichloromethane, and after dissolution, Span80 emulsifier was added and stirred evenly to obtain the oil phase (O). Step (3): Preparation of colostrum: The inner aqueous phase (W1) is added dropwise to the oil phase (O), and ultrasonic emulsification is performed at low temperature to form W1 / O colostrum; Step (4): Preparation of external aqueous phase: Polyvinyl alcohol and trehalose were dissolved in ultrapure water and stirred to obtain external aqueous phase (W2). Step (5): Preparation of double emulsion: The W1 / O primary emulsion is added to the external aqueous phase (W2), stirred and mixed, and then subjected to high pressure homogenization to form W1 / O / W2 double emulsion; Step (6): Solvent evaporation and solidification: The re-emulsion is dialyzed to remove organic solvents and free components, then the pH is adjusted and filtered; Step (7): Freeze-drying: The dialyzed and filtered system was freeze-dried to obtain solid PLGA nanoparticles co-loaded with fibronectin and collagen.

2. The method for preparing PLGA nanoparticles loaded with fibronectin and collagen according to claim 1, characterized in that: In step (1), the collagen is recombinant human type III collagen; in the internal aqueous phase (W1), the mass-volume concentration of recombinant human type III collagen is 0.1%-1%, the mass-volume concentration of trehalose is 1%-3%, and the mass-volume concentration of fibronectin is 0.1%-1%.

3. The method for preparing PLGA nanoparticles loaded with fibronectin and collagen according to claim 2, characterized in that: In step (1), the internal aqueous phase (W1) contains 0.3% by mass volume of recombinant human type III collagen, 1.5% by mass volume of trehalose, and 0.24% by mass volume of fibronectin.

4. The method for preparing PLGA nanoparticles loaded with fibronectin and collagen according to claim 1, characterized in that: In step (2), the molar ratio of lactic acid to glycolic acid in the PLGA is 50:50, and the molecular weight is 7000-17000 Da; the amount of emulsifier Span80 added is 10%-30% of the mass of PLGA.

5. The method for preparing PLGA nanoparticles loaded with fibronectin and collagen according to claim 1, characterized in that: In step (3), the power of ultrasonic emulsification is 600-800W, the time is 5-15 minutes, and the low temperature environment is an ice-water bath.

6. The method for preparing PLGA nanoparticles loaded with fibronectin and collagen according to claim 1, characterized in that: In step (4), the mass volume concentration of polyvinyl alcohol in the external aqueous phase (W2) is 1%-3% and the mass volume concentration of trehalose is 3%-7%.

7. The method for preparing PLGA nanoparticles loaded with fibronectin and collagen according to claim 1, characterized in that: In step (5), the pressure of the high-pressure homogenization process is 1000-1500 bar, and the number of cycles is 3-8.

8. The PLGA nanoparticles loaded with fibronectin and collagen prepared by the preparation method according to any one of claims 1-7, characterized in that: The nanoparticles have an average particle size of 100-200 nm, and the encapsulation efficiency of fibronectin is not less than 75%. In in vitro release experiments, fibronectin can be continuously released for 7-14 days.

9. The application of the PLGA nanoparticles loaded with fibronectin and collagen according to claim 8, characterized in that: The PLGA nanoparticles loaded with fibronectin and collagen are used in the preparation of materials for soft tissue filling and / or repair.

10. The application of the PLGA nanoparticles loaded with fibronectin and collagen according to claim 9, characterized in that: The material is used in the field of medical aesthetics for wrinkle smoothing, contour reshaping, skin texture improvement, or repair of depressed scars; or for skin wound healing and soft tissue defect repair.

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