Composite drug delivery system based on hyaluronic acid carrier as well as preparation method and application of composite drug delivery system

Abstract

The invention 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 of the composite drug delivery system. The system is composed of outer-layer low-crosslinking-degree HA gel and inner-layer high-crosslinking-degree HA microspheres, the outer-layer gel rapidly releases anti-angiogenesis and anti-inflammatory peptide drugs to control acute lesions, and the inner-layer microspheres slowly release blood retina barrier (BRB) repair, anti-oxidation and long-acting anti-angiogenesis drugs to maintain chronic repair. And efficient treatment of diabetic retinopathy (DR) is realized through a multi-target synergistic effect. Results show that the system can effectively reduce inflammatory factors, inhibit angiogenesis and maintain BRB integrity, multifunctional peptides are effectively combined, and a novel drug delivery strategy is provided for clinical treatment of DR.

Description

TECHNICAL FIELD

[0001] 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. BACKGROUND

[0002] Diabetic retinopathy (DR) is the most common microvascular complication of diabetes, and its pathological mechanism involves abnormal proliferation of retinal neovascularization, destruction of blood-retinal barrier (BRB), vicious cycle of chronic inflammation and oxidative stress, and eventually leading to blindness. At present, the clinical treatment is mainly intravitreal injection of anti-vascular endothelial growth factor (VEGF) drugs, but it only targets the VEGF pathway, has insufficient single-target coverage, poor timeliness, high drug delivery frequency and imperfect drug delivery system. Therefore, how to load different drugs targeting pathological tissues and control the release of different drugs is the biggest difficulty and challenge at present.

[0003] Hyaluronic acid (HA) as a natural glycosaminoglycan has excellent biocompatibility, degradability and affinity for ocular tissues, and has been used as an ophthalmic drug carrier. However, the existing HA carriers are mostly single release mode, which cannot meet the sequential treatment needs of DR "acute-chronic", and lack of multi-target drug synergistic delivery design. Therefore, developing a dual-release carrier based on HA to achieve the spatiotemporal precise delivery of multi-target drugs has become a key breakthrough point for DR treatment. SUMMARY

[0004] In order to solve the problems existing in the prior art, the application provides a "microsphere-gel" composite system with hyaluronic acid as the carrier which can load multiple drugs and a preparation method and application thereof. Through the dual mode of outer layer rapid release and inner layer slow release, anti-neovascular, BRB repair, anti-inflammatory and anti-oxidation drugs are synergistically delivered to achieve multi-target and sequential treatment of DR.

[0005] The technical scheme provided by the application is a composite drug delivery system based on a hyaluronic acid carrier, which is composed of an outer layer of low cross-linking degree hyaluronic acid gel and an inner layer of high cross-linking degree hyaluronic acid microspheres. The outer layer of hyaluronic acid gel loads a first active substance; and the inner layer of hyaluronic acid microspheres loads a second active substance.

[0006] Preferably, the molecular weight of the outer layer of hyaluronic acid is 50-100 kDa, and the cross-linking degree is less than or equal to 10%; the molecular weight of the inner layer of hyaluronic acid is greater than or equal to 1000 kDa, and the cross-linking degree is greater than or equal to 30%.

[0007] Preferably, the first active substance includes an anti-neovascular peptide and an anti-inflammatory peptide; and the second active substance includes a BRB repair peptide, an anti-oxidation peptide and an NL7 peptide.

[0008] Preferably, the anti-angiogenic peptides comprise NL7 peptide and VEGF neutralizing peptide VHP; the anti-inflammatory peptides comprise IL-1Ra peptide; the BRB repair peptides comprise QHREDGS peptide and ADTC5 peptide, and the anti-oxidative peptides comprise SOD mimetic peptide.

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

[0010] Further, the application provides a preparation method of the 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) Dissolve high molecular weight hyaluronic acid in PBS buffer, add the second active substance, and prepare microspheres by emulsification-crosslinking method under the action of a crosslinking agent; (3) Disperse the microspheres obtained in step (2) in the gel precursor of step (1) to assemble a composite drug delivery system.

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

[0012] Preferably, in step (2), the emulsification conditions are: 300 W power ultrasonic for 10 min, the oil phase is petroleum ether containing 5% Tween 80, the volume ratio of water phase to oil phase 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 application further provides the use of the composite drug delivery system in the preparation of a drug for treating diabetic retinopathy.

[0014] The application further provides a drug for treating diabetic retinopathy, and the active ingredient of the drug is the composite drug delivery system.

[0015] The application has the advantages that the "microsphere-gel" composite drug delivery system based on the hyaluronic acid carrier has good biocompatibility, the drug for treating diabetic retinopathy prepared by using the composite drug delivery system has high targeting and drug accumulation capacity on the DR model retinal tissue with high expression of VEGF and Ang2, and the loaded anti-neovascular peptide, BRB repair peptide and anti-inflammatory and anti-oxidation peptide have significant synergies; the drug can effectively reduce the levels of inflammatory factors such as TNF-alpha and IL-1beta in the DR model, inhibit the retinal neovascular area and reduce the BRB permeability; meanwhile, the double release mode can reduce the drug administration frequency, the hyaluronic acid carrier has high affinity with the ocular tissue, can reduce the risk of complications related to intravitreal injection, and provides a new efficient drug delivery strategy for the clinical treatment of DR. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 The figure is the morphology of the nanoparticles prepared in the embodiments of the application; wherein, A. inner layer microspheres; B. composite drug delivery system; Figure 2 The figure is the release curve of the "microsphere-gel" composite drug delivery system based on the hyaluronic acid carrier prepared in Example 2 of the application; wherein, A. release behavior of QHREDGS, ADTC5, SOD and NL7 peptides in the presence or absence of hyaluronidase in the inner layer microspheres; B. release behavior of NL7, VHP and IL-1Ra peptides in the outer layer gel; Figure 3 The figure is the influence of the "microsphere-gel" composite drug delivery system based on the hyaluronic acid carrier prepared in Example 2 of the application on the anti-angiogenesis of HRMECs cells; wherein, A. influence of the composite drug delivery system treatment on the survival rate of HRMECs cells; B. influence of the composite drug delivery system treatment on the number of lumens of HRMECs cells; Figure 4 The figure is the influence of the "microsphere-gel" composite drug delivery system based on the hyaluronic acid carrier prepared in Example 2 of the application on the BRB repair effect of HRMECs cells; wherein, A. change of the TEER value of HRMECs cells after the composite drug delivery system treatment; B. change of the fluorescein sodium permeability of HRMECs cells after the composite drug delivery system treatment; Figure 5 The figure is the influence of the "microsphere-gel" composite drug delivery system based on the hyaluronic acid carrier prepared in Example 2 of the application on the anti-inflammatory and anti-oxidation of BV2 cells; wherein, A. change level of TNF-alpha and IL-1beta of BV2 cells after the composite drug delivery system treatment; B. change level of ROS of BV2 cells after the composite drug delivery system treatment. DETAILED DESCRIPTION

[0017] In order for those skilled in the art to more clearly understand the technical solutions of the present application, the technical solutions of the present application will be described in detail below in conjunction with specific embodiments.

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

[0019] The present embodiment provides a composite drug delivery system based on a hyaluronic acid carrier, which is composed of an outer layer of low cross-linking degree hyaluronic acid gel and an inner layer of high cross-linking degree hyaluronic acid microspheres. The outer layer has a cross-linking degree of ≤10%, and the inner layer has a cross-linking degree of ≥30%. The outer layer of hyaluronic acid gel carries a first active substance, and the inner layer of hyaluronic acid microspheres carries a second active substance. The composite drug delivery system can be used as an active ingredient for preparing a drug for treating diabetic retinopathy.

[0020] The present embodiment provides a preparation method of a composite drug delivery system based on a hyaluronic acid carrier, which comprises the following steps: (1) NL7 peptide, VHP peptide, IL-1Ra peptide, QHREDGS peptide, ADTC5 peptide and SOD mimetic peptide are 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 mimetic peptide is His-His-Val-Glu-Arg.

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

[0022] The preparation method is as follows: HA, EDC and NHS are weighed according to a molar ratio of 1:1.2:1 and dissolved using PBS buffer (pH=7.4), stirred and activated for 30 min.

[0023] After the activation is completed, NL7 peptide (outer layer part), VHP peptide, IL-1Ra peptide are added according to a molar ratio of 0.85:2:0.5, and stirring is continued for 6 h. The reaction solution is collected and purified by dialysis to obtain the outer layer 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.

[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 layer HA microspheres can release polypeptide drugs in response to hyaluronidase environment, such as Figure 2 The inner layer HA microspheres can release polypeptide drugs in response to hyaluronidase environment, such as

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

[0033] The outer layer drug-loaded HA gel can quickly absorb water and swell in solution, and spontaneously release polypeptide drugs, such as Figure 2 The outer layer HA gel can release more than 80% of the loaded drugs within 1-7 days.

[0034] Example 6 Inhibition of "microsphere-gel" composite drug delivery system on human retinal microvascular endothelial cells (HRMECs) The experimental method is as follows: the HRMECs cells in the logarithmic growth phase are collected, then adjusted to a suitable concentration, and seeded in a 96-well plate at a number of 1×10 4 The experimental method is as follows: the HRMECs cells in the logarithmic growth phase are collected, then adjusted to a suitable concentration, and seeded in a 96-well plate at a number of 1×10

[0035] As shown in Figure 3 The "microsphere-gel" composite drug delivery can effectively inhibit the abnormal proliferation of HRMECs cells leading to the growth of new blood vessels, and the cell survival rate decreases significantly with the increase of drug concentration.

[0036] Example 7 Verification of "microsphere-gel" composite drug delivery system inhibiting lumen formation The experimental method is as follows: the HRMECs cells in the logarithmic growth phase are collected, then adjusted to a suitable concentration, and seeded in a 96-well plate at a number of 1×10 4 The experimental method is as follows: the HRMECs cells in the logarithmic growth phase are collected, then adjusted to a suitable concentration, and seeded in a 96-well plate at a number of 1×10

[0037] As shown in Figure 3 The "microsphere-gel" composite drug delivery system can effectively inhibit the formation of lumen.

[0038] Example 8 Verification of the repair effect of "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: 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 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.

2. The composite drug delivery system according to claim 1, characterized in that: The outer layer of hyaluronic acid has a molecular weight of 50-100 kDa and a cross-linking degree of ≤10%; the inner layer of hyaluronic acid has a molecular weight of ≥1000 kDa and a cross-linking degree of ≥30%.

3. The composite drug delivery system according to claim 1, characterized in that: The first active substances include anti-angiogenic peptides and anti-inflammatory peptides; the second active substances include BRB repair peptides, antioxidant peptides, and NL7 peptides.

4. The drug according to claim 3, characterized in that: The anti-angiogenic peptides include NL7 peptide and VEGF neutralizing peptide VHP; the anti-inflammatory peptides include IL-1Ra peptide; the BRB repair peptides include QHREDGS peptide and ADTC5 peptide; and the antioxidant peptides include SOD mimic peptide.

5. The drug according to claim 4, characterized in that: The molar ratio of 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.

6. A method for preparing the composite drug delivery system according to any one of claims 1-5, characterized in that, Includes 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.

7. The preparation method according to claim 6, characterized in that, In step (1), the molar ratio of EDC to NHS is 1.2:1, and the activation time is 30 min; the molar ratio of hyaluronic acid carboxyl group to EDC is 1:1.

2.

8. The preparation method according to claim 6, characterized in that, 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.

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

10. A drug for treating diabetic retinopathy, characterized in that: The active ingredient is the composite drug delivery system as described in any one of claims 1-5.

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