Composite drug delivery system based on hyaluronic acid carrier and preparation method and application thereof

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to a composite drug delivery system based on a hyaluronic acid carrier and a preparation method and application thereof. The system is composed of an outer layer of low-cross-linking-degree HA gel and an inner layer of high-cross-linking-degree HA microspheres. The outer layer of gel rapidly releases anti-neovascular and anti-inflammatory peptide drugs to control acute lesions, and the inner layer of microspheres slowly releases blood-retinal barrier (BRB) repair, antioxidant and long-acting anti-neovascular drugs to maintain chronic repair, so that the system realizes efficient treatment of diabetic retinopathy (DR) through multi-target synergistic effect. Results show that the system can effectively reduce inflammatory factors, inhibit neovascularization and maintain BRB integrity, effectively combines multifunctional peptides, and provides a new drug delivery strategy for clinical treatment of DR.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a composite drug delivery system based on hyaluronic acid carrier, its preparation method, and its application. Background Technology

[0002] Diabetic retinopathy (DR) is the most common microvascular complication of diabetes. Its pathological mechanisms involve abnormal proliferation of retinal neovascularization, disruption of the blood-retinal barrier (BRB), and a vicious cycle of chronic inflammation and oxidative stress, ultimately leading to blindness. Current clinical treatment primarily involves intravitreal injection of anti-vascular endothelial growth factor (VEGF) drugs. However, this only targets the VEGF pathway, resulting in insufficient single-target coverage, poor timeliness, high dosing frequency, and an imperfect drug delivery system. Therefore, the greatest challenge currently faced is how to load different drugs to target pathological tissues and control the release of different drugs.

[0003] Hyaluronic acid (HA), a natural glycosaminoglycan, possesses excellent biocompatibility, biodegradability, and affinity for ocular tissues, and has been used as a drug carrier in ophthalmology. However, existing HA carriers mostly employ a single release mode, which cannot meet the needs of sequential "acute-chronic" treatment of diabetic retinopathy (DR), and lack synergistic delivery designs for multi-target drugs. Therefore, developing a dual-release HA-based carrier to achieve precise spatiotemporal delivery of multi-target drugs has become a key breakthrough in DR treatment. Summary of the Invention

[0004] To address the problems existing in the prior art, this invention provides a "microsphere-gel" composite system with hyaluronic acid as a carrier that can load multiple drugs, as well as its preparation method and application. Through a dual mode of rapid release from the outer layer and slow release from the inner layer, it synergistically delivers anti-angiogenic, BRB repair, anti-inflammatory, and antioxidant drugs, achieving multi-target, sequential treatment of DR.

[0005] The technical solution provided by this invention is: a composite drug delivery system based on hyaluronic acid carrier, which consists of an outer layer of low-crosslinked hyaluronic acid gel and an inner layer of high-crosslinked hyaluronic acid microspheres. The outer layer of hyaluronic acid gel is loaded with a first active substance, and the inner layer of hyaluronic acid microspheres is loaded with a second active substance.

[0006] Preferably, the outer layer of hyaluronic acid has a molecular weight of 50-100 kDa and a crosslinking degree of ≤10%; the inner layer of hyaluronic acid has a molecular weight of ≥1000 kDa and a crosslinking degree of ≥30%.

[0007] Preferably, the first active substance includes anti-angiogenic peptides and anti-inflammatory peptides; the second active substance includes BRB repair peptides, antioxidant peptides and NL7 peptides.

[0008] Preferably, the anti-angiogenic peptide includes NL7 peptide and VEGF neutralizing peptide VHP; the anti-inflammatory peptide includes IL-1Ra peptide; the BRB repair peptide includes QHREDGS peptide and ADTC5 peptide; and the antioxidant peptide includes SOD mimic peptide.

[0009] Preferably, the molar ratio of the active substances in the outer hyaluronic acid gel to the inner hyaluronic acid microspheres is: total NL7 peptide: VHP peptide: QHREDGS peptide: IL-1Ra peptide = 1:2:1:0.5; wherein, the content of NL7 peptide in the inner hyaluronic acid microspheres is 15%-20% of the total molar amount of NL7 peptide, and the remainder is loaded in the outer hyaluronic acid gel.

[0010] Furthermore, the present invention provides a method for preparing the aforementioned composite drug delivery system, comprising the following steps: (1) Dissolve low molecular weight hyaluronic acid in PBS buffer, add EDC / NHS to activate carboxyl groups, add the first active substance in proportion, and stir to form a gel; (2) High molecular weight hyaluronic acid was dissolved in PBS buffer, a second active substance was added, and microspheres were prepared by emulsification-crosslinking under the action of a crosslinking agent; (3) Disperse the microspheres obtained in step (2) in the gel precursor of step (1) and assemble them to form a composite drug delivery system.

[0011] Preferably, in step (1), the molar ratio of EDC to NHS is 1.2:1, the activation time is 30 min, and the molar ratio of hyaluronic acid carboxyl group to EDC is 1:1.2.

[0012] Preferably, in step (2), the emulsification conditions are: 300 W power ultrasonication for 10 min, the oil phase is petroleum ether containing 5% Tween 80, the water-oil phase volume ratio is 1:10, the amount of crosslinking agent genipin is 20% of the mass of hyaluronic acid, the crosslinking temperature is 37℃, and the crosslinking time is 4 h.

[0013] The present invention also provides the application of the aforementioned composite drug delivery system in the preparation of a drug for treating diabetic retinopathy.

[0014] The present invention further provides a drug for treating diabetic retinopathy, the active ingredient of which is the aforementioned compound drug delivery system.

[0015] The beneficial effects of this invention are as follows: The "microsphere-gel" composite drug delivery system based on hyaluronic acid carrier provided by this invention has good biocompatibility. The drug for treating diabetic retinopathy prepared using this composite drug delivery system has high targeting and drug accumulation capacity on retinal tissue of DR model with high expression of VEGF and Ang2. The anti-angiogenic peptides, BRB repair peptides, and anti-inflammatory and antioxidant peptides loaded with this system have significant synergistic effects. This drug can effectively reduce the levels of inflammatory factors such as TNF-α and IL-1β in DR model, inhibit the area of ​​retinal neovascularization, and reduce BRB penetration. At the same time, its dual release mode can reduce the frequency of administration. The hyaluronic acid carrier has high affinity with ocular tissue and can reduce the risk of complications related to intravitreal injection, providing a novel and efficient drug delivery strategy for the clinical treatment of DR. Attached Figure Description

[0016] Figure 1 The morphology of the nanoparticles prepared in the embodiments of the present invention is shown; wherein, A. inner layer microspheres; B. composite drug delivery system; Figure 2 Release curves of the "microsphere-gel" composite drug delivery system based on hyaluronic acid carrier prepared in Example 2 of the present invention; wherein, A. the release behavior of QHREDGS, ADTC5, SOD, and NL7 peptides of the inner microspheres in the presence or absence of hyaluronidase; B. the release behavior of NL7, VHP, and IL-1Ra peptides of the outer gel; Figure 3 The effect of the "microsphere-gel" composite drug delivery system based on hyaluronic acid carrier prepared in Example 2 of the present invention on the anti-angiogenesis of HRMECs cells; wherein, A. the effect of composite drug delivery system treatment on the survival rate of HRMECs cells; B. the effect of composite drug delivery system treatment on the number of lumens in HRMECs cells; Figure 4 The effect of the "microsphere-gel" composite drug delivery system based on hyaluronic acid carrier prepared in Example 2 of the present invention on the BRB repair function of HRMECs cells; wherein, A. the change in TEER value of HRMECs cells after treatment with the composite drug delivery system; B. the change in sodium fluorescein permeability of HRMECs cells after treatment with the composite drug delivery system; Figure 5 The effects of the "microsphere-gel" composite drug delivery system based on hyaluronic acid carrier prepared in Example 2 of this invention on the anti-inflammatory and antioxidant effects of BV2 cells; wherein, A. the changes in TNF-α and IL-1β levels in BV2 cells after treatment with the composite drug delivery system; B. the changes in ROS levels in BV2 cells after treatment with the composite drug delivery system. Detailed Implementation

[0017] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0018] The experimental materials used in the embodiments of this invention are all conventional experimental materials in the art and can be purchased through commercial channels.

[0019] Example 1: This embodiment provides a composite drug delivery system based on hyaluronic acid carriers. The system consists of an outer layer of low-crosslinked hyaluronic acid gel and an inner layer of high-crosslinked hyaluronic acid microspheres. The outer layer has a crosslinking degree ≤10%, while the inner layer has a crosslinking degree ≥30%. The outer hyaluronic acid gel loads a first active substance, and the inner hyaluronic acid microspheres load a second active substance. This composite drug delivery system can be used as an active ingredient in the preparation of drugs for treating diabetic retinopathy.

[0020] Example 2 This example provides a method for preparing a composite drug delivery system based on hyaluronic acid carrier, including the following steps: (1) NL7 peptide, VHP peptide, IL-1Ra peptide, QHREDGS peptide, ADTC5 peptide and SOD mimic peptide were synthesized and purified by solid-phase synthesis. The amino acid sequence of NL7 peptide is Asn-Leu-Leu-Met-Ala-Ala-Ser; the amino acid sequence of VHP peptide is His-Asp-Val-Phe-Met-Arg-Gly-Phe; the amino acid sequence of QHREDGS peptide is Gln-His-Arg-Glu-Asp-Gly-Ser; the amino acid sequence of ADTC5 peptide is Ala-Asp-Thr-Cys-Cys; the amino acid sequence of IL-1Ra peptide is Ala-Pro-Gln-Gly-Leu-Glu-Val; and the amino acid sequence of SOD mimic peptide is His-His-Val-Glu-Arg.

[0021] (2) Preparation of outer layer low cross-linking drug-loaded HA gel The outer low-crosslinking HA gel uses hyaluronic acid with a molecular weight of 50-100 kDa as raw material and achieves covalent bonding with peptide drugs through amide bonds.

[0022] The preparation method is as follows: Weigh HA, EDC and NHS in a molar ratio of 1:1.2:1 and dissolve them in PBS buffer (pH=7.4), stir and activate for 30 min.

[0023] After activation, NL7 peptide (outer layer), VHP peptide, and IL-1Ra peptide were added in a molar ratio of 0.85:2:0.5, respectively. The mixture was stirred for 6 h, and the reaction solution was collected and purified by dialysis to obtain the outer drug-loaded HA gel.

[0024] (3) Preparation of highly cross-linked HA microspheres in the inner layer The inner layer of highly cross-linked HA microspheres is prepared from hyaluronic acid with a molecular weight ≥1000 kDa through an emulsification-cross-linking method.

[0025] The preparation method is as follows: Weigh 0.5 g of high molecular weight HA, dissolve it in 50 mL of PBS buffer (pH=7.4), stir overnight at 4℃ until completely dissolved to form a viscous solution, weigh QHREDGS peptide:ADTC5 peptide:SOD mimic peptide:NL7 peptide (inner layer part) in a molar ratio of 1:1:1:0.15, add it to the high molecular weight HA solution, and stir to mix evenly.

[0026] Using the above solution as the aqueous phase, a petroleum ether containing 5% Tween 80 was prepared as the oil phase. The aqueous phase and oil phase were mixed at a volume ratio of 1:10, and ultrasonically emulsified at 300 W for 10 min (3 s on, 2 s off) to form a W / O emulsion. 0.1 g of the crosslinking agent genipin was weighed, dissolved in a small amount of DMSO, and added to the W / O emulsion. The mixture was stirred and crosslinked at 37℃ for 4 h. The microspheres were collected by centrifugation, washed three times alternately with petroleum ether and distilled water, and then lyophilized to obtain drug-loaded HA microspheres in the inner layer.

[0027] (4) Assembly of the “microsphere-gel” composite drug delivery system Weigh the inner HA microspheres and outer HA gel at a mass ratio of 1:5, stir and mix evenly, let stand at room temperature for 2 hours, and the outer gel will completely solidify to form an "inner microsphere-outer gel" composite system.

[0028] Example 3: Morphological observation of the "microsphere-gel" composite drug delivery system The experimental method is as follows: The above-mentioned inner HA microspheres and "microsphere-gel" composite drug delivery system were dissolved in an aqueous solution. An appropriate amount of solution was taken out, dropped onto a copper grid, stained, and observed using a transmission electron microscope.

[0029] The inner HA microspheres and the "microsphere-gel" composite drug delivery system exist in aqueous solution as nanospheres, exhibiting good dispersibility, such as... Figure 1 As shown.

[0030] Example 4: Validation of the slow drug release behavior of the inner HA microspheres The experimental method is as follows: the inner HA microspheres were dissolved in a dialysis bag containing PBS. The dialysis bag was placed in release media with and without hyaluronidase. The release solution was collected at specific time points and the concentrations of QHREDGS peptide, ADTC5 peptide, SOD mimic peptide and NL7 peptide in the solution were detected by HPLC. The cumulative drug release was calculated.

[0031] The inner HA microspheres can responsively release peptide drugs, such as hyaluronidase, in an environment containing hyaluronidase. Figure 2 As shown in Figure A. The inner HA microspheres slowly release the loaded drug over 1-3 months, with 20%-30% released in the first 2 weeks.

[0032] Example 5: Validation of rapid drug release behavior of the outer drug-loaded HA gel The experimental method is as follows: the outer drug-loaded HA gel was dissolved into a dialysis bag containing PBS, the dialysis bag was placed in PBS, the release solution was collected at specific time points, and the concentrations of NL7 peptide, VHP peptide and IL-1Ra peptide in it were detected by HPLC, and the cumulative drug release was calculated.

[0033] The outer drug-loaded HA gel can rapidly absorb water and swell in solution, spontaneously releasing peptide drugs, such as... Figure 2 As shown in Figure B, the outer HA gel releases more than 80% of the loaded drug within 1-7 days.

[0034] Example 6: Inhibitory effect of the "microsphere-gel" composite drug delivery system on human retinal microvascular endothelial cells (HRMECs) The experimental method is as follows: HRMECs cells in the logarithmic growth phase were collected and then adjusted to an appropriate concentration, at 1×10⁶ cells per well. 4 Cells were seeded in 96-well plates and cultured overnight in a cell culture incubator. Different concentrations of the "microsphere-gel" composite drug delivery system were added to the cell culture plates for 48 h, and CCK-8 was added to detect cell viability.

[0035] like Figure 3 As shown in Figure A, the "microsphere-gel" composite drug delivery system can effectively inhibit the abnormal proliferation of HRMECs cells leading to angiogenesis. As the drug concentration increases, the cell survival rate decreases significantly.

[0036] Example 7: Verification of the "microsphere-gel" composite drug delivery system's inhibition of lumen formation The experimental method is as follows: HRMECs cells in the logarithmic growth phase were collected and then adjusted to an appropriate concentration, at 2 × 10⁶ cells per well. 4 Cells were seeded in 24-well plates and cultured overnight in a cell culture incubator. Different concentrations of the "microsphere-gel" composite drug delivery system were added to the cell culture plates for 6 hours, and the number of lumens was counted and photographed under a microscope.

[0037] like Figure 3 As shown in Figure B, the "microsphere-gel" composite drug delivery system can effectively inhibit the formation of lumens.

[0038] Example 8: Verification of the repair effect of the "microsphere-gel" composite drug delivery system on BRB. The experimental method is as follows: HRMECs cells in the logarithmic growth phase were collected and then adjusted to an appropriate concentration, at 1×10⁶ cells per well. 5 Cells were seeded in the wells of Transwell cells and cultured in a cell culture incubator for 7 days to form a complete monolayer barrier. During this period, transepithelial resistance (TEER) was measured using an epithelial resistance meter. Successful barrier model construction was confirmed when the TEER value remained stable at a high level for 3 consecutive days. To establish a damage model, the upper and lower chambers were replaced with high-glucose medium containing 30 mM glucose and cultured for another 24 hours. Then, a microsphere-gel composite drug delivery system was added to treat the cells for 48 hours. Subsequently, TEER values ​​were measured using an epithelial resistance meter to assess the integrity of the barrier system. The culture medium from both chambers was then aspirated. A solution containing 100 μM sodium fluorescein was added to the upper chamber, and an equal volume of culture medium was added to the lower chamber. The cells were incubated at 37°C for 2 hours, and the fluorescence intensity in the lower chamber was measured using a microplate reader to calculate the barrier permeability.

[0039] like Figure 4 As shown, after treatment with the "microsphere-gel" composite drug delivery system, the TEER value of HRMECs was significantly higher than that of the damage model group, while the sodium fluorescein permeability was significantly lower than that of the damage model group. Therefore, it can be demonstrated that the composite drug delivery system can repair retinal barrier damage caused by high glucose.

[0040] Example 9: Validation of the synergistic anti-inflammatory and antioxidant effects of the "microsphere-gel" composite drug delivery system The experimental method is as follows: BV2 cells in the logarithmic growth phase were collected and then adjusted to an appropriate concentration, at a ratio of 5 × 10⁶ cells per well. 5 Cells were seeded in 6-well plates and cultured overnight in a cell culture incubator. The next day, each well was treated with 1 μg / ml LPS for 24 hours to establish an inflammation model. Subsequently, the liquid in each well was discarded, and different drugs were added to treat the cells for 24 hours. Finally, the cell supernatant was collected, and the levels of TNF-α and IL-1β were detected according to the ELISA kit instructions. Simultaneously, the level of intracellular ROS was detected using the DCFH-DA fluorescent probe method.

[0041] like Figure 5 As shown, the "microsphere-gel" composite drug delivery system can effectively reduce the levels of TNF-α and IL-1β in the inflammation model group, while also reducing the intracellular ROS fluorescence intensity.

[0042] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A composite drug delivery system based on hyaluronic acid carrier, characterized in that: This system consists of an outer layer of low-crosslinked hyaluronic acid gel and an inner layer of high-crosslinked hyaluronic acid microspheres. The outer layer of hyaluronic acid gel is loaded with a first active substance; the inner layer of hyaluronic acid microspheres is loaded with a second active substance. The molecular weight of the outer layer of hyaluronic acid is 50-100 kDa, and the degree of crosslinking is ≤10%; the molecular weight of the inner layer of hyaluronic acid is ≥1000 kDa, and the degree of crosslinking is ≥30%. The first active substance is an anti-angiogenic peptide and an anti-inflammatory peptide; the second active substance is a BRB repair peptide, an antioxidant peptide, and an NL7 peptide. The anti-angiogenic peptide is an NL7 peptide and a VEGF neutralizing peptide (VHP); the anti-inflammatory peptide is an IL-1Ra peptide; the BRB repair peptide is a QHREDGS peptide and an ADTC5 peptide; the antioxidant peptide is an SOD mimic peptide; the molar ratio of the active substances in the outer layer of hyaluronic acid gel to the inner layer of hyaluronic acid microspheres is: total NL7 peptide : VHP peptide : QHREDGS peptide : IL-1Ra peptide = The ratio is 1:2:1:0.5; wherein the content of NL7 peptide in the inner layer of hyaluronic acid microspheres is 15%-20% of the total molar amount of NL7 peptide, and the remainder is loaded in the outer layer of hyaluronic acid gel; the amino acid sequence of NL7 peptide is Asn-Leu-Leu-Met-Ala-Ala-Ser; the amino acid sequence of VHP peptide is His-Asp-Val-Phe-Met-Arg-Gly-Phe; the amino acid sequence of QHREDGS peptide is Gln-His-Arg-Glu-Asp-Gly-Ser; the amino acid sequence of ADTC5 peptide is Ala-Asp-Thr-Cys-Cys; the amino acid sequence of IL-1Ra peptide is Ala-Pro-Gln-Gly-Leu-Glu-Val; and the amino acid sequence of SOD mimic peptide is His-His-Val-Glu-Arg. The composite drug delivery system is prepared by the following method: (1) Dissolve low molecular weight hyaluronic acid in PBS buffer, add EDC / NHS to activate the carboxyl group, and add the outer layer NL7 peptide, VHP peptide and IL-1Ra peptide in a molar ratio of 0.85:2:0.5 respectively; the molar ratio of EDC to NHS is 1.2:1, the activation time is 30 min; the molar ratio of hyaluronic acid carboxyl group to EDC is 1:1.2; (2) High molecular weight hyaluronic acid was dissolved in PBS buffer, and QHREDGS peptide, ADTC5 peptide, SOD mimic peptide and NL7 peptide of the inner layer were added in a molar ratio of 1:1:1:0.

15. Microspheres were prepared by emulsification-crosslinking under the action of crosslinking agent. The emulsification conditions were: 300 W power sonication for 10 min, oil phase was petroleum ether containing 5% Tween 80, water phase-oil phase volume ratio was 1:10, the amount of crosslinking agent genipin was 20% of the mass of hyaluronic acid, the crosslinking temperature was 37℃, and the crosslinking time was 4 h. (3) Disperse the microspheres obtained in step (2) in the gel precursor of step (1) and assemble them to form a composite drug delivery system; The combined drug delivery system is used to treat diabetic retinopathy.

2. The use of the composite drug delivery system according to claim 1 in the preparation of a medicament for treating diabetic retinopathy.

3. A drug for treating diabetic retinopathy, characterized in that: The active ingredient is the composite drug delivery system as described in claim 1.

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