Oxygen-releasing soluble riboflavin microneedle patch for treating keratectasia diseases and preparation method of oxygen-releasing soluble riboflavin microneedle patch

Abstract

The invention relates to the field of medical treatment, and discloses O2RF (at) MNs for treating keratectasia diseases and a preparation method of the O2RF (at) MNs. The O2RF (at) MNs comprises a patch base and a needle body array, the needle body array comprises a plurality of microneedles, and O2NE is loaded in the microneedles. The O2RF-coated MNs can form reversible micro holes in corneal epithelium, the blocking effect of hydrophobic corneal epithelium on hydrophilic RF is overcome, RF permeation is promoted, meanwhile, O2NE and PFD capable of releasing oxygen are loaded in the O2RF-coated MNs, due to the low polarity of fluorine, the oxygen can be combined and stored, after the microneedle is dissolved, the O2NE permeates into corneal stroma, oxygen is continuously released in the corneal stroma, and therefore the corneal epithelium can release oxygen continuously. In the crosslinking process, no extra device is needed to provide oxygen, so that the cornea crosslinking effect is enhanced. In addition, a tiny wound caused by the microneedle on the corneal epithelium can be completely healed within 12 hours, and corneal injury is small.

Description

Technical Field

[0001] The present application relates to the medical field, and more specifically, to an oxygen-releasing riboflavin microneedle patch (O2RF@MNs) for treating corneal ectasia and a preparation method thereof. Background Art

[0002] The incidence of corneal ectasia is about 1 / 2000-1 / 2500. The clinical manifestations are progressive refractive error, increased irregular astigmatism, and decreased corrected visual acuity in the early stage. In the late stage, the corneal posterior elastic layer will rupture, scar formation, and even perforation. Ultimately, 15-20% of patients need deep lamellar or penetrating keratoplasty. Corneal collagen cross-linking is currently the most effective method to prevent or delay the progression of corneal ectasia. It has been widely used to treat progressive keratoconus and surgery-related corneal ectasia. However, this reaction is oxygen-dependent. During cross-linking, the oxygen in the corneal stroma will drop rapidly within 10 to 15 seconds after ultraviolet light irradiation, and it takes at least 3-4 minutes for oxygen to return to the original level. Cross-linking has no effect on strengthening the cornea in an oxygen-deficient environment. At present, some studies use goggles, eyelid openers, masks, or pulse therapy to increase oxygen supply during ultraviolet light irradiation, and have found that it can significantly increase the effect of cross-linking, but it requires additional oxygen supply devices, which increases the complexity of the operation.

[0003] Currently, the commonly used corneal cross-linking methods include epithelial corneal collagen cross-linking surgery and transepithelial corneal collagen cross-linking surgery. (1) Epithelial corneal collagen cross-linking surgery (Dresden protocol) removes 8 mm of corneal epithelium before treatment, and applies 0.1% riboflavin-5'-sodium phosphate (RF) solution to the central part of the debrided cornea for 30 minutes to allow RF to fully penetrate into the corneal stroma. Then, 3 mw / cm 2 UV exposure to the cornea for 30 minutes or 9mw / cm 2 The cornea is irradiated with ultraviolet light for 10 minutes, but because the corneal epithelium needs to be removed, the damage is relatively large, the patient suffers significant pain after the operation, and the incidence of corneal opacity and infection is high. (2) Transepithelial corneal collagen cross-linking surgery is to enhance the penetration of drugs into the corneal epithelium by adding penetration enhancers such as benzalkonium chloride, tromethamine, and ethylenediaminetetraacetic acid to RF preparations while retaining the corneal epithelium. However, in clinical practice, most of these preparations have weak penetration effects, less RF enters the corneal stroma, and the cross-linking effect is poor, which cannot achieve the effect of classic epithelial removal therapy. A considerable number of patients have experienced progression of corneal ectasia after transepithelial cross-linking therapy.

[0004] Therefore, there is a need to develop a riboflavin delivery method that can achieve RF penetration comparable to that of classic epithelial removal therapy while causing less corneal damage. Summary of the invention

[0005] The purpose of the present invention is to provide an O2RF@MNs for treating corneal ectasia and a preparation method thereof. O2RF@MNs is used to improve the RF penetration into the corneal stroma and increase the oxygen supply during corneal cross-linking, thereby enhancing the cross-linking effect of corneal ectasia.

[0006] The O2RF@MNs of the present application can form reversible micropores in the corneal epithelium, overcome the blocking effect of the hydrophobic corneal epithelium on the hydrophilic RF, and promote the penetration of RF. At the same time, O2RF@MNs are loaded with perfluorodecalin nanoemulsion (Oxygen releasing nanoemulsion, O2NE) that can release oxygen. Perfluorodecalin (PFD) can bind and store oxygen due to the low polarity of fluorine. After the microneedle is dissolved, O2NE penetrates into the corneal stroma and continuously releases oxygen in the corneal stroma. During the cross-linking process, no additional device is required to provide oxygen, thereby enhancing the effect of corneal cross-linking. In addition, the tiny wound caused by the microneedle on the corneal epithelium can be completely healed within 12 hours, and the corneal damage is relatively small.

[0007] In order to achieve the above invention objectives, this application adopts the following technical solutions:

[0008] In a first aspect, the present application provides an O2RF@MNs for treating corneal ectasia diseases, wherein the O2RF@MNs comprises a patch base and a needle array disposed on the patch base, wherein the needle array comprises a plurality of microneedles, and the patch base and the microneedles are loaded with O2NE.

[0009] Furthermore, the O2RF@MNs is composed of a 14×14 needle array, the patch is 1 cm long and 1 cm wide, the base thickness of the microneedle patch is about 100 μm, the needle body is conical, the microneedle height is about 550 μm, the bottom width of the needle body is about 320 μm, and the spacing between needles is about 340 μm.

[0010] Furthermore, the patch base and the microneedles are loaded with O2NE, and the diameter of the O2NE is about 100-300 nm.

[0011] In a second aspect, the present application provides a method for preparing the O2RF@MNs described in the first aspect, comprising the following steps:

[0012] Step 1: Preparation of O2NE: water, PFD and alcohol ethoxylates (AEO) are mixed, ultrasonicated in an ice-water bath to form a perfluorodecalin nanoemulsion, and the perfluorodecalin nanoemulsion is oxygenated to obtain O2NE;

[0013] Step 2: Preparation of O2RF@MNs: dissolve hyaluronic acid (HA) and RF in ultrapure water, dissolve polylactic acid (PLA) in acetone, and then vortex to dissolve to obtain a PLA-HA mixture. Add the PLA-HA solution mixture containing O2NE into a polydimethylsiloxane (PDMS) mold and dry overnight to obtain O2RF@MNs.

[0014] Furthermore, in step 1, water, PFD and AEO are mixed in a ratio of 14:5:1 (w / w).

[0015] Furthermore, in step 2, HA, RF, and ultrapure water are mixed in a ratio of 20 mg:(1-5) mg:1 mL.

[0016] Furthermore, in step 2, PLA and acetone are mixed in a ratio of 6 mg:0.15 mL.

[0017] Furthermore, in step 2, O2NE and PLA-HA are mixed in a ratio of 1:10 (w / w).

[0018] In a second aspect, the present application provides the use of O2RF@MNs described in the first aspect in treating corneal ectasia diseases.

[0019] In summary, this application has the following beneficial effects:

[0020] 1. Good RF penetration effect: After O2RF@MNs delivery, the drug content in the corneal stroma exceeds that of classic epithelial removal therapy and commercial transepithelial preparation Peschek TE;

[0021] 2. Less corneal damage: Minimally invasive, corneal epithelial damage caused by O2RF@MNs can be completely recovered within 12 hours, while corneal epithelial damage caused by classic epithelial removal therapy takes 72 hours to fully recover;

[0022] 3. O2RF@MNs can release oxygen in the corneal stroma, increasing the amount of oxygen in the corneal stroma and the overall cross-linking effect during UV cross-linking. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1:Schematic diagram of the preparation process of O2NE, ordinary riboflavin microneedle (RF@MNs) and O2RF@MNs.

[0024] Figure 2 : Characterization of O2NE. (A) Transmission electron microscope image of O2NE; (B) Scanning electron microscope image of O2NE; (C) Particle size distribution of O2NE; (D) Potential distribution of O2NE.

[0025] Figure 3 : Scanning electron microscope images of RF@MNs and O2RF@MNs. (A) Scanning electron microscope image of RF@MNs; (B) Scanning electron microscope image of O2RF@MNs

[0026] Figure 4 : Slit lamp microscope images of O2RF@MNs and transparent microneedle patches. (A) Slit lamp microscope image of O2RF@MNs; (B) Slit lamp microscope image of transparent microneedle patches.

[0027] Figure 5 : Confocal microscopy and inverted fluorescence microscopy images of O2RF@MNs. (A) Side view and top view of O2RF@MNs observed using 3D mode of laser confocal microscopy. (B) Images of O2RF@MNs observed using an inverted fluorescence microscope under bright field and (C) blue light excitation.

[0028] Figure 6 : Fourier transform infrared spectroscopy (FTIR) analysis of O2RF@MNs and its components (AEO, RF, PLA and HA).

[0029] Figure 7 : In vitro drug release curves of O2RF@MNs. (A) In vitro drug release curves of O2RF@MNs within 1 hour and (B) 10 hours, n=4.

[0030] Figure 8 : Mechanical strength curves of microneedles. Mechanical strength curves of (A) O2RF@MNs and (B) transparent microneedles.

[0031] Fig. 9 :In vitro oxygen release of O2RF@MNs. In vitro oxygen release curves of O2RF@MNs and RF@MNs (without oxygen release function) after adding deoxygenated PBS, with deoxygenated PBS as control. Data are expressed as mean ± SD, n = 3, and statistical analysis was performed using Two-way Anova. ****p < 0.0001;

[0032] Fig.10 : Photographs of corneal yellowing in rabbit eyes 30 minutes after application of different RF preparations.

[0033] Fig.11 : Corneal epithelial healing after application of O2RF@MNs and epi-off therapy was evaluated under cobalt blue light of a slit lamp microscope.

[0034] Fig.12 : Strain-stress curves and Young's modulus of cornea after cross-linking with different RF agents. (A) Strain-stress curves of cornea after cross-linking with different RF agents. (B) Young's modulus values ​​when the strain level was 6%, 8%, and 10%, respectively, n = 3. Data are expressed as mean ± SD, and statistical analysis was performed using Two-way ANOVA DETAILED DESCRIPTION

[0035] The technical scheme and effects of the present application are further described in detail below in conjunction with the embodiments and drawings. It should be understood that the specific embodiments described herein are only used to explain the invention, rather than to limit the invention.

[0036] Experimental materials and instruments

[0037] name source Perfluorodecalin Discovery Reagents, China Fatty alcohol polyoxyethylene ether Discovery Reagents, China Hyaluronic acid Discovery Reagents, China Polylactic acid Discovery Reagents, China acetone Discovery Reagents, China Riboflavin-5'-monophosphate sodium salt TCI, Japan Sterile PBS solution Saiweier, China Malvern Nano-ZSE Laser Particle Size Analyzer Malvern, UK Milli-Q Ultrapure Water Analyzer Millipore, USA PDMS mold Chipscreen Biopharmaceuticals, China Vacuum oven Thermo, USA Slit lamp microscope Shangbang Medical, China Inverted fluorescence microscope Zeiss, Germany Confocal laser scanning microscopy Nikon, Japan Fourier Transform Infrared Spectrometer Tensor II, Bruker, Germany Multifunctional ELISA reader Thermo Fisher Scientific, United States Universal material testing machine Jiangsu Tianyuan, China Portable dissolved oxygen meter Smart Sensor, China

[0038] Example

[0039] The O2RF@MNs of this embodiment is composed of a 14×14 needle array. The patch is 1 cm long and 1 cm wide. The thickness of the base of the microneedle patch is about 100 μm. The needle body is conical, the microneedle needle is about 550 μm high, the bottom width of the needle body is about 320 μm, the spacing between the needle bodies is about 340 μm, and the diameter of the O2NE loaded in the microneedle is about 100-300 nm.

[0040] The oxygen-releasing O2RF@MNs are prepared by the following steps:

[0041] Step 1: Preparation of O2NE:

[0042] Water, PFD and AEO were mixed in a ratio of 14:5:1 (w / w) and then sonicated in an ice-water bath for 5 min to form perfluorodecalin nanoemulsion. The perfluorodecalin nanoemulsion was then oxygenated in a hyperbaric oxygen chamber to form O2NE, and the oxygenated O2NE was stored in a sealed glass bottle until further use.

[0043] Step 2: Preparation of O2RF@MNs:

[0044] 20 mg HA and 3 mg RF (adjustable between 1-5 mg depending on the drug loading of the microneedles) were dissolved in 1 mL of ultrapure water. 6 mg PLA was dissolved in 0.15 mL of acetone and then vortexed to dissolve. A 1 mL PLA-HA solution mixture containing 100 μL O2NE was added to the PDMS mold and then dried in a 37 °C vacuum oven overnight. After drying, the O2RF@MNs were carefully removed from the PDMS mold and visually inspected for mechanical strength and uniformity. The O2RF@MNs were stored in oxygen-enriched packaging and protected from light until use.

[0045] In addition, this embodiment also prepared RF@MNs that cannot release oxygen, and the preparation method is as follows:

[0046] 20 mg HA and 3 mg RF (adjustable between 1 and 5 mg depending on the drug loading of the microneedles) were dissolved in 1 mL of ultrapure water. 6 mg PLA was dissolved in 0.15 mL acetone and then vortexed to dissolve. 1 mL of the PLA-HA solution mixture was added to the PDMS mold and then dried in a vacuum oven at 37 °C overnight. After drying, the RF@MNs were carefully removed from the PDMS mold and visually inspected for mechanical strength and uniformity.

[0047] Performance Testing

[0048] (1) Morphological and structural characterization of O2NE

[0049] For transmission electron microscopy observation, an appropriate amount of O2NE sample was dropped onto a 300-mesh copper grid, and the excess emulsion was removed by the edge of filter paper. Its structural characteristics were observed at an accelerating voltage of 80 kV. For scanning electron microscopy analysis, an electron accelerating voltage of 3.00 kV was used to obtain surface secondary electron images in SE2 signal mode. The particle size distribution and Zeta potential of O2NE were measured using a Malvern Nano-ZSE laser particle size analyzer at 25 °C.

[0050] Please refer to the attached Figure 2 The results show that O2NE was successfully prepared in this example. O2NE presents a uniform spherical nanostructure, with a particle size of about 100-300nm under the microscope and a Zeta potential of about -0.93±0.08mV.

[0051] (2) Scanning electron microscopy observation of the morphology of RF@MNs and O2RF@MNs

[0052] The morphology and structure of RF@MNs and O2RF@MNs were characterized by scanning electron microscopy. The acceleration voltage was set to 2.50 kV, the working distance was 4.8-8.0 mm, and the SE2 signal mode was used for imaging.

[0053] Please refer to the attached Figure 3, the morphological structures of O2RF@MNs and O2RF@MNs were displayed. The results showed that both microneedles were evenly arranged in a cone shape. The surface of RF@MNs without O2NE was relatively smooth, while the surface of O2RF@MNs with O2NE was relatively rough, which may be due to the attached O2NE.

[0054] (3) Characterization of microneedle morphology using slit lamp microscopy

[0055] First, use microtweezers to clamp the microneedle sample in front of the lens, adjust the slit lamp microscope until the microneedles are clearly observed, and obtain the front and side views of the microneedle array.

[0056] Please refer to the attached Figure 4 , showing the morphology of O2RF@MNs and transparent microneedles under a slit lamp microscope. The results showed that the microneedle patch was 1 cm long and 1 cm wide, and both microneedle arrays were arranged in 14×14 with conical needle tips. In appearance, O2RF@MNs were yellow, while blank microneedles without RF loading remained transparent.

[0057] (4) Characterization of microneedle morphology using confocal laser scanning microscopy and inverted fluorescence microscopy

[0058] The morphological characterization of O2RF@MNs was performed using the 3D imaging mode of confocal laser scanning microscopy and inverted fluorescence microscopy. O2RF@MNs were first fixed to the surface of a glass slide and physically fixed using nanoglue to ensure imaging stability. Continuous tomographic scanning was performed along the Z-axis using inverted fluorescence microscopy imaging or the 3D imaging mode of confocal laser scanning microscopy.

[0059] Please refer to the attached Figure 5 , the morphological structure and drug loading of O2RF@MNs were demonstrated. The results showed that O2RF@MNs appeared green under blue light excitation, and a large amount of RF was loaded in the needle body, which could serve as a drug reservoir for corneal cross-linking.

[0060] (5) FTIR analysis

[0061] Recording of O2RF@MNs and their components from 500 to 4000 cm at room temperature -1 FTIR spectroscopy was used to study the interactions between drugs and components.

[0062] Please refer to the attached Figure 6 , the FTIR spectra of O2RF@MNs and their constituent components were presented, and the results showed that the characteristic peaks of O2RF@MNs were similar to those of several constituent materials, indicating that RF was physically encapsulated in the microneedles.

[0063] (6) In vitro drug release curve

[0064] The microneedle patch was placed in 30 mL PBS for in vitro drug release analysis, and 100 μL of the release solution was taken every 1 hour at 1, 10, 20, 30, 40, 50 minutes and 1-10 hours for absorbance analysis. The fluorescence values ​​of the samples at different time points were measured at 37°C using a microplate reader with 435 nm and 525 nm as excitation and emission wavelengths, and a standard curve of fluorescence absorbance and RF (μg / mL) was established to calculate the drug concentration in the sample. The data are reported as the drug content of each microneedle array.

[0065] Please refer to the attached Figure 7 , demonstrated the in vitro drug release curve of O2RF@MNs. The results showed that O2RF@MNs can basically release more than 90% of the drug in PBS within half an hour, with the performance of rapid drug release, which meets the needs of clinical surgery.

[0066] (7) Microneedle mechanical properties test

[0067] The mechanical strength of the microneedle was tested using the compression mode of a universal material testing machine. The microneedle was carefully placed on the stainless steel bottom plate of the universal material testing machine with the tip facing up. The probe was then compressed at 100 μm·s -1 The microneedle is pressed down at a controlled speed and the force on the sensor is recorded as a function of the microneedle displacement.

[0068] Please refer to the attached Figure 8 , showing the mechanical strength curves of O2RF@MNs and blank microneedles. In both cases, the microneedles have sufficient mechanical strength, and the addition of RF has no significant effect on the mechanical properties of the microneedles.

[0069] (8) In vitro oxygen release performance

[0070] We measured the in vitro oxygen release of O2RF@MNs and RF@MNs. Five O2RF@MNs and five RF@MNs were added to 5 ml of PBS to fully remove oxygen from the solution using a nitrogen stream, and the oxygen concentration in the deoxygenated water of different treatment groups was measured using a portable dissolved oxygen meter at different time points.

[0071] Please refer to the attached Fig. 9 , demonstrated the in vitro oxygen release curve of O2RF@MNs, and the results showed that O2RF@MNs had a highly efficient oxygen release capacity compared with RF@MNs that could not release oxygen.

[0072] (9) Assessment of corneal yellowing

[0073] To evaluate the degree of corneal jaundice, a corneal trephine was used to obtain 8.0 mm of tissue in the center of the cornea, which was then spread out on a clean gauze to obtain a clear image of the corneal surface, which served as an intuitive basis for the subsequent assessment of the degree of jaundice.

[0074] Please refer to the attached Fig.10 , compared with corneas treated with commercial transepithelial preparation Peschke TE preparation and epi-off therapy (0.1% RF), corneas treated with O2RF@MNs with drug loading of 3mg and 5mg RF showed more obvious corneal yellowing than classic epi-off therapy (0.1% RF), indicating that the RF content in the cornea was higher after treatment with 3mg and 5mg O2RF@MNs.

[0075] (10) Evaluation of corneal epithelial repair rate after O2RF@MNs and epi-off therapy

[0076] New Zealand white rabbits were selected to receive O2RF@MNs treatment and epi-off therapy intervention (without corneal cross-linking). The corneal epithelial healing process was dynamically observed by sodium fluorescein staining at different time points after surgery to evaluate the effects of the two intervention methods on the corneal epithelial repair rate.

[0077] Please refer to the attached Fig.11 The results of sodium fluorescein staining showed that the rabbit corneal epithelium could heal completely within 12 hours after the application of O2RF@MNs; while after using epi-off therapy, it took 72 hours for the rabbit corneal epithelium to heal completely, indicating that O2RF@MNs caused less damage to the cornea.

[0078] (11) Measurement of corneal biomechanical properties after cross-linking

[0079] Normal New Zealand white rabbits were anesthetized with different RF preparations for 30 minutes. 2 The cornea was cross-linked with UVA for 10 minutes. After the cross-linking was completed, the experimental rabbits were euthanized, and samples from the central part of the cornea and sclera were taken. A custom double-edged knife was used to cut a corneal strip with a width of 4 mm and a length of 25 mm along the 12 o'clock and 6 o'clock directions. The thickness of the corneal strip was measured, and the corneal strip was vertically clamped between the two fixtures of the universal material testing machine. The distance between the fixtures was measured and input. Before the formal test, pre-loading was performed to eliminate pre-stress. The pre-load amount was 0.05N and the speed was 2mm / min. When the stress of 0.05N was reached, the program automatically returned to zero. Then the corneal strip was stretched at a speed of 3mm / min, and the stress-strain curve was recorded using a tensile sensor, and the Young's modulus was calculated.

[0080] Please refer to the attached Fig.12At the same strain level, the stress and Young's modulus values ​​of the 5mg O2RF@MNs group were significantly higher than those of the commercial transepithelial preparation Peschke TE group, the 5mg RF@MNs group, and the 0.5% epi-off group, indicating that O2RF@MNs had a significant improvement in corneal biomechanics after oxygen supplementation compared with RF@MNs that could not release oxygen and epi-off therapy (p<0.05).

[0081] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make modifications to the present embodiment without any creative contribution as needed, but such modifications are protected by the patent law as long as they are within the scope of the claims of the present application.

Claims

1. An O2RF@MNs, characterized in that: The O2RF@MNs includes a patch base and a needle array disposed on the patch base, wherein the needle array includes a plurality of microneedles, and the microneedles are loaded with O2NE.

2. The O2RF@MNs according to claim 1, characterized in that: The needle array is a 14×14 needle array, the patch is 1 cm long and 1 cm wide, the thickness of the patch base is 100 μm, the microneedles are conical, the microneedle height is 550 μm, the bottom width is 320 μm, and the spacing between microneedles is 340 μm.

3. The O2RF@MNs according to claim 1, characterized in that: The diameter of the O2NE is about 100-300 nm.

4. The method for preparing O2RF@MNs according to any one of claims 1 to 3, characterized in that: The following steps are involved: Step 1: Preparation of O2NE, water, PFD and AEO are mixed, ultrasonicated in an ice water bath to form a perfluorodecalin nanoemulsion, and the perfluorodecalin nanoemulsion is oxygenated to obtain O2NE; Step 2: Preparation of O2RF@MNs: HA and RF were dissolved in ultrapure water, PLA was dissolved in acetone, and then vortexed to dissolve to obtain a PLA-HA mixture. The PLA-HA solution mixture containing O2NE was added to the PDMS mold and dried overnight to obtain O2RF@MNs.

5. The method for preparing O2RF@MNs according to claim 4, characterized in that: In step 1, water, PFD and AEO are mixed in a ratio of 14:5:1 (w / w).

6. The method for preparing O2RF@MNs according to claim 4, characterized in that: In step 2, HA, RF, and ultrapure water are mixed in a ratio of 20 mg:(1-5) mg:1 mL.

7. The method for preparing O2RF@MNs according to claim 4, characterized in that: In the step 2, PLA and acetone were mixed in a ratio of 6 mg:0.15 mL.

8. The method for preparing O2RF@MNs according to claim 4, characterized in that: In step 2, O2NE and PLA-HA are mixed in a ratio of 1:10 (w / w).

9. Use of the O2RF@MNs according to any one of claims 1 to 3 in treating corneal ectasia diseases.

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