A trehalose nanogel carrier system for delivering recombinant human elastin and preparation method and application thereof

Abstract

The application provides a preparation method of a trehalose nanogel carrier system for delivering recombinant human elastin, and belongs to the technical field of biological medicine and cosmetics. The carrier system is prepared by using trehalose acrylate and acrylamide as monomers, preparing a nanogel core through a reverse microemulsion polymerization method, and sequentially constructing a hyaluronic acid surface modification and a glutathione-responsive recombinant human elastin compound loading. The system has a triple controlled release mechanism: amide catalytic hydrolysis realizes pH response slow release, hyaluronidase enzymatic degradation enhances epidermal permeation, and glutathione triggers disulfide bond rupture to realize intracellular targeted release. The nanogel has an encapsulation efficiency higher than 85%, a 72-hour cumulative release rate of 63% in PBS, and can significantly improve the transdermal absorption rate of recombinant human elastin and the intracellular bioavailability. The carrier system has good stability and high biocompatibility, and can be used for preparing high-efficiency anti-aging skin care products.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine and cosmetics, and in particular to a trehalose nanogel carrier system for delivering recombinant human elastin, its preparation method, and its application. Background Technology

[0002] With the increasing demand for anti-aging skincare, delivery systems for functional active ingredients have become a research hotspot. Recombinant human elastin (rhElastin) is widely used in anti-aging skincare products due to its good biocompatibility and skin elasticity-promoting function. However, the large molecular weight, poor stability, and difficulty in transdermal absorption of rhElastin limit its application efficacy.

[0003] In existing technologies, nanocarrier systems such as liposomes and polymer nanoparticles are used to improve the delivery efficiency of active ingredients, but problems such as low drug loading rate, uncontrollable release, and poor targeting still exist. Therefore, developing a nanocarrier system with efficient drug loading, controllable sustained release, enhanced transdermal absorption, and targeted recognition functions has significant application value.

[0004] This invention develops a novel nanogel carrier system. A polymer network carrier with hydrolyzable ester bonds is constructed by copolymerizing trehalose (meth)acrylate with hydrophilic acrylamide. Combined with the skin-penetration-enhancing effect of hyaluronic acid (HA) and the targeted modification of glutathione (GSH), this system is used to deliver recombinant human elastin (rhElastin) to achieve highly effective anti-aging effects. This carrier system possesses three major advantages: controlled and sustained release, environmental stability, and targeted recognition, providing a completely new solution for the nanodelivery of anti-aging skincare products. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing a trehalose nanogel carrier system for delivering recombinant human elastin, so as to solve the problems of low delivery efficiency, poor stability and difficulty in transdermal absorption of recombinant human elastin in the prior art.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a method for preparing a trehalose nanogel carrier system for delivering recombinant human elastin, comprising the following steps: 1) Dissolve trehalose acrylate, acrylamide, 3-acrylamidopropyltrimethylammonium chloride, bisacryloylcysteine ​​and photoinitiator in buffer solution to form an aqueous phase; 2) The surfactant is dissolved in cyclohexane to form an oil phase; 3) The aqueous phase is added to the oil phase, and ultrasonic emulsification is performed to form a microemulsion; 4) The microemulsion is subjected to a polymerization reaction under ultraviolet light irradiation to obtain the initial nanogel product; 5) The initial nanogel product was subjected to acetone precipitation, dialysis purification and freeze-drying in sequence to obtain trehalose nanogel carrier; 6) After activating hyaluronic acid by EDC / NHS method, it reacts with the amino groups on the surface of the trehalose nanogel carrier to perform HA surface grafting modification, and obtain HA modified nanogel. 7) Recombinant human elastin and glutathione are linked by disulfide bonds to form a complex, which is then loaded into HA-modified nanogels to obtain the carrier system.

[0007] Preferably, the molar ratio of trehalose acrylate, acrylamide, 3-acrylamidopropyltrimethylammonium chloride, and bisacryloylcysteine ​​in step 1) is 0.31~0.51:0.45~0.56:0.081~0.098:0.050~0.061.

[0008] Preferably, the amount of photoinitiator used in step 1) is 2.2~2.4 mg, and the amount of buffer solution used is 1~2 mL.

[0009] Preferably, the surfactant mentioned in step 2) is Span80, and the mass-to-volume ratio of the surfactant to cyclohexane is 0.5~0.6 g: 8~12 mL.

[0010] Preferably, the volume ratio of the oil phase to the water phase in step 3) is 1~2:7~15; the conditions for ultrasonic emulsification are 50~60% amplitude and ultrasonication for 5~10 min.

[0011] Preferably, the wavelength of ultraviolet light irradiation in step 4) is 395 nm, and the polymerization time is 25~30 min.

[0012] The present invention also provides a trehalose nanogel carrier system prepared by the above-described preparation method.

[0013] The present invention also provides an application of the above-mentioned trehalose nanogel carrier system in the preparation of anti-aging skin care products.

[0014] The present invention also provides an application of the above-mentioned trehalose nanogel carrier system in the preparation of medical dressings.

[0015] Beneficial effects

[0016] 1. This invention achieves efficient loading, controlled sustained release, and targeted delivery of rhElastin through a trehalose nanogel carrier system, significantly improving its transdermal absorption efficiency and bioavailability.

[0017] 2. This invention enhances skin permeability through HA surface modification, achieves targeted intracellular release through GSH-responsive disulfide bonds, and enhances protein stability through trehalose structure, thereby synergistically improving the anti-aging effect through multiple mechanisms.

[0018] 3. The nanogel carrier system provided by this invention has good stability, biocompatibility and safety, and can be widely used in skin care products, medical dressings and other fields. Attached Figure Description

[0019] Figure 1 The release curves of rhElastin at different time conditions at pH 7.4 in Example 3 are shown. Figure 2 This is a comparison chart of the permeability of the HA-modified and unmodified groups in Example 3; Figure 3 This is a graph showing the cell safety test results in Example 3; Figure 4 The image shows the results of the physiological stability test in Example 3. Detailed Implementation

[0020] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0021] Example 1: Preparation method of trehalose nanogel carrier system

[0022] The trehalose nanogel carrier system was prepared according to the amounts of each raw material in Table 1 below, and the specific preparation process is as follows: 1) Dissolve trehalose acrylate, acrylamide, 3-acrylamidopropyltrimethylammonium chloride, disulfide-containing crosslinking agent bisacryloylcysteine, and photoinitiator lithium phenyl-2,4,6-trimethylbenzoylphosphinic acid in PBS buffer to form an aqueous phase; 2) Dissolve Span 80 in cyclohexane to form an oil phase; 3) Add the aqueous phase from step 1) to the oil phase prepared in step 2), and ultrasonically emulsify for 5 minutes at 60% amplitude to form a microemulsion; 4) The microemulsion was irradiated with 395 nm ultraviolet light for 30 min to carry out a polymerization reaction, and the initial nanogel product was obtained. 5) The initial nanogel product was subjected to acetone precipitation (150 mL, 2 times), dialysis (pH 5.0 sodium acetate buffer, 2 L × 3 times, 24 h) and lyophilization for 48 h to obtain trehalose nanogel carrier. 6) After activating HA by EDC / NHS method, it reacts with amino groups on the surface of trehalose nanogel carrier (25℃, 12h) to perform HA surface grafting modification. The zeta potential is -15 mV, and HA-modified nanogel is obtained. 7) rhElastin and GSH were linked by disulfide bonds to form a GSH-SS-rhElastin complex, which was then loaded into the HA-modified nanogel by physical encapsulation (50 μg / mg nanogel) to obtain the trehalose nanogel carrier system. The encapsulation efficiency was >85% and the loading was about 19 wt% as determined by ultrafiltration.

[0023] Table 1. Raw material usage and preparation parameters for each group

[0024] Example 2: Effect Verification

[0025] 1. Characterization and detection

[0026] 1.1 Particle size characterization

[0027] The particle size determination steps are as follows: Dynamic light scattering (DLS) was used to determine the particle size of trehalose nanogels in empty groups 1-3 (groups 1-3 prepared after step 7 was removed in Example 1) and sample groups 1-3 (groups 1-3 prepared in Example 1). The equipment used was a Nicomp Z3000 nanoparticle size and potential meter. 1000 μL of sample was diluted to 2000 μL with PBS buffer of the same concentration, and the particle size of the sample was determined at room temperature with an instrument frequency of 300 kHz. The results are shown in Table 2.

[0028] Table 2 Particle size detection results

[0029] As shown in Tables 1 and 3, the optimal ratio of phenyl-2,4,6-trimethylbenzoyl lithium phosphinate (LAP): disulfide crosslinking agent (bisacrylamide, BAC) is 2.31 mg: 0.050 mmol (empty 1). The average particle size of the trehalose nanogel samples prepared under this ratio is between 80 and 120 nm. Among them, the average particle size of the trehalose nanogel sample carrying rhElastin in sample 1 is 156.11 ± 0.29 nm, which is the optimal particle size.

[0030] 1.2 Ultrafiltration and HPLC methods were used to determine the encapsulation efficiency and drug loading: 1.2.1 Solution Preparation Drug-loaded nanogel suspension (S-loaded): Samples 1-3 above were prepared into nanogel suspensions with a known total rhElastin dosage (m_total).

[0031] Free rhElastin standard solutions: Prepare a series of rhElastin standard solutions of different concentrations and plot an HPLC standard curve.

[0032] 1.2.2 Separation of free drug (ultrafiltration)

[0033] Take a certain volume (V_loaded, such as 1 mL) of S-loaded sample and place it in an ultrafiltration centrifuge tube (MilliporeAmicon Ultra, molecular cutoff is usually selected as 30 kDa or 100 kDa).

[0034] Centrifuge at the specified speed (e.g., 10000×g) for 15 minutes.

[0035] Collect the filtrate, which contains unencapsulated free rhElastin.

[0036] HPLC determination of drug concentration

[0037] Chromatographic conditions: Chromatographic column: Reversed-phase C18 column (e.g., Agilent ZORBAX SB-C18, 4.6 × 250 mm, 5 μm); Mobile phases: Phase A (0.1% TFA aqueous solution), Phase B (0.1% TFA acetonitrile solution); Gradient elution: 0-15 min, phase B increases linearly from 20% to 60%; Flow rate: 1.0 mL / min; Column temperature: 30°C; Detection wavelength: 220 nm; Injection volume: 20 μL; Plotting the standard curve: RhElastin standard solutions of different concentrations were injected and analyzed. Linear regression was performed on the peak area (A) against the concentration (C) to obtain the standard curve equation Y=2960.19X+81.69, R2=0.9981, and the detection limit was 0.01mg / mL.

[0038] Determine the concentration of the filtrate: After appropriately diluting the filtrate collected in step two, inject it for analysis, record the peak area (A_free), substitute it into the standard curve equation, and calculate the concentration of free rhElastin in the filtrate (C_free).

[0039] Calculate the mass of free rhElastin in the sample (m_free): m_free = C_free × V_loaded Calculate the encapsulated rhElastin mass (m_encapsulated): m_encapsulated = m_total - m_free Calculation formula: Encapsulation rate = (Total drug amount - Free drug amount) / Total drug amount × 100%.

[0040] The encapsulation efficiency test results are shown in Table 3.

[0041] Table 3 Encapsulation efficiency test results

[0042] Example 3: Efficacy Testing

[0043] The trehalose nanogel carrier systems described below were all prepared according to the method in Example 1, and the raw material amounts were as in Group 1.

[0044] 1. In vitro sustained-release properties: An appropriate amount of the rhElastin-carrying trehalose nanogel system was resuspended in a small amount of PBS (pH 7.4). The sample solution was placed into a pre-treated dialysis bag (MWCO 100 kDa), sealed, and immersed in the corresponding pH release medium (20 mL PBS) at 37°C and 100 rpm shaking. At predetermined time points (6, 8, 12, 24, 36, 48, 72 h), 1 mL of external release solution was aspirated, and an equal volume of fresh medium was immediately added at the same temperature. The concentration of rhElastin in the samples taken at each time point was determined by HPLC (C18 column, 220 nm detection), and the cumulative release rate was calculated. The highest release rate reached 63% after 72 hours. The results are as follows: Figure 1 As shown.

[0045] 2. In vitro transdermal absorption performance: Transdermal experiments were conducted using a Franz diffusion cell and fresh Bama miniature pig skin on nanogels with and without hyaluronic acid (HA) surface modification (HA modification) and nanogels without HA surface modification (step 6 was omitted in the preparation process). The specific procedures are as follows: 1. Preparation before the experiment Reagent and Material Preparation Receiving solution: Prepare according to the solubility of the components (use PBS pH 7.4 for water-soluble solutions, and add 5% ethanol-PBS for lipid-soluble solutions). Preheat to 32±0.5℃ to avoid skin irritation due to temperature differences.

[0046] Fresh Bama miniature pig skin: Take back skin (with little hair and a stratum corneum thickness similar to that of humans) within 1 hour after slaughter, and transport it in an ice box containing 4°C saline solution (must be processed within 24 hours).

[0047] Samples to be tested: Before the experiment, the samples to be tested (such as moisturizing facial mask liquid) need to be pretreated. If necessary, centrifugation should be performed to remove air bubbles, and the sample should be preheated to 32±0.5℃.

[0048] Instrument parameter settings

[0049] Thermostatic water bath circulator: Start the machine 1 hour in advance, set the temperature to 32±0.5℃ (error ≤0.1℃), and connect the circulating water to the diffusion tank jacket to ensure stable temperature in the receiving chamber.

[0050] Magnetic stirrer: Set the speed to 400 rpm (avoid excessive speed that could damage the skin structure, and excessive speed that could lead to uneven concentration of the receiving fluid).

[0051] Analytical instruments: Based on the properties of the analyte (HPLC), turn on the instrument in advance, calibrate it, and establish a standard curve to ensure that concentration analysis can be performed immediately after sampling.

[0052] 2. Skin pretreatment of Bama miniature pigs

[0053] Subcutaneous tissue dissection

[0054] Use surgical scissors to gently separate the subcutaneous fat along the skin edge (retaining only the epidermis and dermis, with a thickness of about 0.3~0.5mm). During the procedure, gently lift the skin with your fingers to avoid pulling and causing the stratum corneum to peel off. Immediately after peeling, soak the skin in 4℃ saline solution (5 minutes, keeping the skin moist).

[0055] Integrity Detection

[0056] Take 100 μL of 0.5% sodium fluorescein solution, apply it evenly to the skin surface, and leave it at room temperature for 5 minutes; Rinse three times with 4℃ saline solution and observe under a UV lamp (365nm): If there is no fluorescent leakage on the epidermis (only a small amount of residue at the edge), it indicates that the stratum corneum is intact; if punctate fluorescence appears, it indicates that the skin is damaged and needs to be recut.

[0057] Record the skin samples that pass the integrity test and discard those that fail.

[0058] Cutting and Fitting

[0059] Cut the skin using a punch (2.5cm in diameter, effective area of ​​approximately 4.9cm²) that matches the diameter of the diffusion pool. Each piece of skin should only be used once to avoid repeated use that could damage the barrier.

[0060] 3. Assembly of Franz diffusion cell

[0061] Disinfection and assembly of diffusion tank

[0062] Soak the supply chamber and receiving chamber in 75% alcohol for 20 minutes, rinse three times with distilled water, and assemble after drying. First, inject 3 mL of receiving liquid preheated to 32°C into the receiving chamber, place a stir bar, and (supplement) carefully check and ensure that there are no air bubbles on the surface of the liquid in contact with the dermal layer of the skin.

[0063] Lay the skin flat at the interface with the epidermis facing the supply chamber and the dermis facing the receiving chamber, and secure it with a silicone sealing ring (to avoid direct pressure from metal clamps on the skin, which could cause local barrier damage).

[0064] Edge sealing and temperature balance

[0065] Apply a thin layer of medical petroleum jelly to the skin edges (only seal the gaps, do not cover the effective diffusion area) to prevent leakage of the receiving fluid; Place the assembled diffusion tank into a constant temperature water bath, allowing the jacketed circulating water (32℃) to surround the receiving chamber. Turn on the stirring (400rpm) and equilibrate for 30 minutes (to allow the skin temperature to match the receiving liquid, at which point the moisture content of the stratum corneum of the skin will return to its physiological state, approximately 15%-20%).

[0066] 4. Transdermal test procedure

[0067] Sample loading

[0068] After equilibration, use a pipette to draw 0.5 mL of the sample to be tested (preheated to 32°C in advance) and slowly inject it into the supply chamber (inject along the wall, without directly impacting the skin surface), and record the start time (t=0); seal the opening of the supply chamber with a parafilm membrane (to prevent water evaporation from causing an increase in sample concentration).

[0069] Gently shake the diffusion cell to ensure the sample is evenly distributed on the skin surface of the supply chamber.

[0070] Regular sampling and fluid replacement

[0071] Define specific sampling time points and conduct sampling at t=6, 8, 12, 24, 36, 48, and 72h.

[0072] At each preset time point, accurately aspirate a certain volume (e.g., 0.5 mL) of receiving liquid from the sampling port of the receiving chamber using a pipette, and immediately store it in the labeled sample tube.

[0073] After sampling, an equal volume of fresh receiving liquid at the same temperature (32°C) is immediately added to the receiving chamber to maintain a constant volume of the receiving chamber and the conditions for leakage.

[0074] Record the volume of each sample taken and the volume replenished, for volume correction in subsequent concentration calculations.

[0075] The results are as follows Figure 2 As shown, the results indicate that the nanogel group modified with hyaluronic acid (HA) exhibits a significant transdermal enhancement effect, with a cumulative rhElastin penetration rate of up to 68% after 24 hours, which is significantly better than the unmodified group of 29%, demonstrating that HA modification can effectively promote the penetration of active ingredients into the stratum corneum of the skin.

[0076] 3. Cell safety evaluation

[0077] The survival rate of human immortalized keratinocytes (HaCAT) was assessed using the CCK-8 assay for both the trehalose nanogel carrier without rhElastin (step 7 omitted in the preparation process) and the trehalose nanogel carrier with rhElastin (Trehalose nanogel carrier+rhElastin) to evaluate systemic toxicity. Results are as follows: Figure 3 As shown, after 24 hours of treatment with the rhElastin-carrying trehalose nanogel carrier within the effective range of 0.1–1 mg / mL, the HaCAT cell survival rate of the trehalose nanogel carrier with a concentration of 0.3 mg / mL carrying rhElastin was higher than 90%, and the HaCAT cell survival rate of the trehalose nanogel carrier with a concentration of 0.8 mg / mL carrying rhElastin was close to 70%, indicating that the carrier system has good biocompatibility and safety.

[0078] 4. Storage and physiological stability

[0079] Experimental methods and procedures

[0080] Sample preparation

[0081] Experimental group: Group 1: Trehalose nanogel dispersion carrying rhElastin Group 2: Trehalose nanogel dispersion without rhElactinin (blank carrier) Dispersion medium preparation: Medium I: Phosphate-buffered saline (PBS, pH 7.4) Medium II: PBS solution containing 10% (v / v) fetal bovine serum (FBS) (simulating body fluid environment) Incubation system construction The nanogel dispersions of Group 1 and Group 2 were diluted with medium I (PBS) and medium II (10% FBS-PBS), respectively.

[0082] Ensure that the final concentration of the nanogel is consistent across all samples (e.g., 1 mg / mL).

[0083] The well-mixed sample was incubated in a 37°C constant temperature shaker to simulate the core temperature of the human body and to avoid precipitation.

[0084] Typical total incubation volume: 1 mL / sample (for easy subsequent sampling and measurement).

[0085] To ensure the statistical significance of the data, parallel samples (n≥3) are set up.

[0086] Timed sampling and testing

[0087] Samples were taken from each incubation system at preset time points 24 hours later.

[0088] Detection methods and indicators: a. Particle size and polydispersity index (PDI) measurement: The hydrodynamic particle size and PDI of the samples at each time point were measured using a dynamic light scattering (DLS) instrument. Increased particle size or broadened PDI usually indicates nanoparticle aggregation, protein adsorption, or degradation.

[0089] b. Zeta potential measurement: The zeta potential of the sample is measured using a laser Doppler electrophoresis system. An absolute value of the potential approaching zero (e.g., from -30 mV to -10 mV) indicates that serum proteins are adsorbed on the surface of the nanoparticles.

[0090] The results are as follows Figure 4 As shown in Figure A (the results of the blank carrier detection and Figure B (the results of the rhElastin carrier detection)), the results show that the particle size change rate of the nanogel is less than 10%, and no obvious aggregation or precipitation was observed, which proves that the system has excellent stability in complex physiological environments, is easy to store, and ensures in vivo delivery efficiency.

[0091] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a trehalose nanogel carrier system for delivering recombinant human elastin, characterized in that, Includes the following steps: 1) Dissolve trehalose acrylate, acrylamide, 3-acrylamidopropyltrimethylammonium chloride, bisacryloylcysteine ​​and photoinitiator in buffer solution to form an aqueous phase; 2) The surfactant is dissolved in cyclohexane to form an oil phase; 3) The aqueous phase is added to the oil phase, and ultrasonic emulsification is performed to form a microemulsion; 4) The microemulsion is subjected to a polymerization reaction under ultraviolet light irradiation to obtain the initial nanogel product; 5) The nanogel primary product was subjected to acetone precipitation, dialysis purification and freeze-drying in sequence to obtain trehalose nanogel carrier; 6) After activating hyaluronic acid by EDC / NHS method, it reacts with the amino groups on the surface of the trehalose nanogel carrier to perform HA surface grafting modification, and obtain HA modified nanogel. 7) Recombinant human elastin and glutathione are linked by disulfide bonds to form a complex, which is then loaded into HA-modified nanogels to obtain the carrier system.

2. The preparation method according to claim 1, characterized in that, The molar ratio of trehalose acrylate, acrylamide, 3-acrylamidopropyltrimethylammonium chloride, and bisacryloylcysteine ​​in step 1) is 0.31~0.51:0.45~0.56:0.081~0.098:0.050~0.

061.

3. The preparation method according to claim 1, characterized in that, The amount of photoinitiator used in step 1) is 2.2~2.4 mg, and the amount of buffer solution used is 1~2 mL.

4. The preparation method according to claim 1, characterized in that, The surfactant mentioned in step 2) is Span80, and the mass-to-volume ratio of the surfactant to cyclohexane is 0.5~0.6 g: 8~12 mL.

5. The preparation method according to claim 1, characterized in that, In step 3), the volume ratio of the oil phase to the water phase is 1~2:7~15; the conditions for ultrasonic emulsification are 50~60% amplitude and ultrasonication for 5~10 min.

6. The preparation method according to claim 1, characterized in that, In step 4), the wavelength of ultraviolet light irradiation is 395 nm, and the polymerization time is 25~30 min.

7. A trehalose nanogel carrier system prepared by the preparation method according to any one of claims 1 to 6.

8. The application of the trehalose nanogel carrier system according to claim 7 in the preparation of anti-aging skin care products.

9. The application of the trehalose nanogel carrier system according to claim 7 in the preparation of medical dressings.

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