Use of alkyl radical initiators in the preparation of scleral collagen cross-linking drugs and oxygen independent methods of scleral collagen cross-linking

Abstract

The application of an alkyl radical initiator in the preparation of a scleral collagen cross-linking drug and an oxygen-independent scleral collagen cross-linking method, which utilizes an alkyl radical initiator to cross-link type I collagen solution and scleral tissue, compared with the ultraviolet light / riboflavin cross-linking method, the reaction does not depend on the participation of oxygen, and may be more suitable for the sclera under the hypoxic state of myopia, and compared with the chemical cross-linking method, the cross-linking reaction is easier to control. The alkyl radical-induced collagen cross-linking has improved in terms of anti-enzyme degradation performance, thermal stability and biomechanical performance.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of alkyl radical initiators in the preparation of scleral collagen crosslinking drugs and an oxygen-independent method for scleral collagen crosslinking. Background Technology

[0002] Myopia is one of the most common eye diseases worldwide, typically starting in childhood. The prevalence of myopia is constantly increasing; it is projected that by 2050, the number of people with myopia will reach 4.758 billion, accounting for approximately 49.8% of the global population, with 938 million having high myopia. Some of these high myopia patients will develop pathological myopia. Pathological myopia poses a significant threat to vision and is a leading cause of blindness in myopic patients. Common complications include posterior staphyloma, macular atrophy, macular schisis, macular hole, choroidal neovascularization, and retinal detachment, which are often the causes of blindness in patients with pathological myopia. Pathological myopia is characterized by continuous elongation of the axial length of the eye. Along with this elongation, the sclera is prone to posterior staphyloma, leading to other complications. The exact mechanism of myopia is currently unclear, but it is believed to be the result of the interaction between genetic and environmental factors. Studies have found that during the progression of myopia, the mechanical properties of the sclera weaken, its hardness decreases, its thickness thins, and it is accompanied by increased scleral elasticity and decreased collagen content. Wu et al. found that hypoxia can promote the transdifferentiation of human scleral fibroblasts and reduce the expression of type I collagen, indicating its important role in the remodeling of the scleral extracellular matrix and the progression of myopia. Scleral cross-linking, by cross-linking scleral collagen, creates new connections within and between collagen molecules, thereby enhancing the mechanical properties of the sclera and representing a new method for preventing and controlling the progression of myopia. Current scleral cross-linking techniques mainly include physical cross-linking and chemical cross-linking. Physical cross-linking primarily involves ultraviolet light / riboflavin cross-linking and blue light / riboflavin cross-linking. Chemical cross-linking mainly uses chemical cross-linking agents to cross-link collagen, such as glutaraldehyde, genipin, and glyceraldehyde.

[0003] The UV / riboflavin crosslinking method involves irradiating the photosensitizer riboflavin with 370 nm wavelength UV light, exciting riboflavin to the triplet state and generating reactive oxygen species (ROS). These ROS can react with collagen molecules, inducing crosslinking between or within adjacent collagen amino acid molecules. Wollensak et al. utilized a wavelength of 370 nm and an energy of 3 mW / cm². 2Ultraviolet light was used to irradiate the sclera of rabbit eyes pretreated with a 0.1% riboflavin solution at the equator. The mechanical properties of the rabbit sclera were evaluated at 3 days, 4 months, and 8 months post-surgery. The results showed that the Young's modulus of the rabbit sclera increased by 320% at 3 days, 277% at 4 months, and 502% at 8 months, indicating that ultraviolet light / riboflavin crosslinking is a very effective and stable method without damage to the retina or retinal pigment epithelium. However, since ultraviolet light can penetrate the sclera, there is a possibility of damage to the retina and other intraocular tissues. Wang et al. used a 365 nm wavelength laser with the same energy and the same concentration of riboflavin to crosslink the sclera of rabbit eyes and performed visual electrophysiological (ERG) examinations on the experimental rabbits post-surgery. The results showed that the amplitude of dark-adapted ERG decreased statistically significantly at 1 week, 1 month, and 3 months post-surgery, and apoptotic cells and changes in ultrastructure were observed in the retinal layer of the sclera-crosslinked eye. The principle of blue light / riboflavin crosslinking is similar to that of ultraviolet light / riboflavin, but blue light has a longer wavelength and lower photon energy than ultraviolet light, potentially causing less damage to organisms. However, this crosslinking method still faces the challenge of exposing the posterior sclera to light while avoiding damage to other parts of the eye. Kwok et al. used a flexible polymer waveguide with a metallic coating to guide blue light to the equatorial sclera, minimizing light leakage during transmission, which may be applicable to the posterior sclera. However, this photocrosslinking method requires oxygen, and the sclera in myopia may already be hypoxic. If the crosslinking process consumes oxygen between tissues, it could exacerbate the condition.

[0004] Chemical crosslinking achieves a higher degree of crosslinking than physical crosslinking. Compared to the instability of physical crosslinking, it can produce a more uniform crosslinking effect. However, chemical crosslinking introduces exogenous crosslinking, which may lead to toxicity. For example, glutaraldehyde is rarely used in scleral crosslinking due to its cytotoxicity. Currently, genipin and glyceraldehyde are more mature crosslinking agents. Genipin is a natural crosslinking agent, and multiple studies have confirmed that it can enhance the mechanical strength of the sclera and has low toxicity. However, it has the problems of scleral and even corneal staining and the need for multiple injections. In addition, all chemical crosslinking agents can only control the degree of crosslinking through concentration and dosage, but cannot control the reaction time, because the crosslinking agent cannot be removed after being injected under Tenon's capsule. So, is there a scleral crosslinking method that does not require oxygen consumption, can control the crosslinking reaction, and can also obtain satisfactory mechanical properties? Summary of the Invention

[0005] To address the shortcomings and deficiencies of existing technologies, this invention provides the application of alkyl radical initiators in the preparation of scleral collagen crosslinking drugs and an oxygen-independent method for scleral collagen crosslinking.

[0006] The technical solution adopted in this invention is the application of alkyl radical initiators in the preparation of scleral collagen crosslinking drugs.

[0007] The initiator of the alkyl radical is a water-soluble azo compound that can decompose to generate alkyl radicals under thermal stimulation.

[0008] The initiator for the alkyl radical is 2,2'-azabis(2-imidazoline) dihydrochloride (AIBI).

[0009] The suitable heating temperature for AIBI to decompose and generate alkyl free radicals under thermal stimulation is 44-50℃.

[0010] The concentration of AIBI is 0.5-5 mg / mL.

[0011] A method for crosslinking scleral collagen without relying on oxygen includes the following steps: adding a type I collagen solution or scleral tissue to an initiator solution that can generate alkyl free radicals, heating for 10 minutes under light-protected conditions, and then reacting at room temperature for 24 hours.

[0012] The beneficial effects of this invention are as follows: This invention provides an application of alkyl free radical initiators in the preparation of scleral collagen crosslinking drugs and an oxygen-independent scleral collagen crosslinking method. By using alkyl free radical initiators to crosslink type I collagen and sclera, compared with photocrosslinking, the reaction does not depend on the participation of oxygen, which may be more suitable for sclera in myopic hypoxic conditions. Compared with chemical crosslinking, it is also easier to control the crosslinking reaction, and it has improved in terms of anti-enzymatic degradation performance, thermal stability and biomechanical properties. Attached Figure Description

[0013] Figure 1 The fluorescence emission spectra of phenylalanine in type I collagen solutions modified with AIBI at different concentrations are shown; the right dashed box is the magnified portion of the left dashed box.

[0014] Figure 2 The fluorescence emission spectra of tyrosine in type I collagen solutions modified with AIBI at different concentrations are shown.

[0015] Figure 3 SDS-PAGE of type I collagen before and after AIBI modification; where 1: control group; 2-6, AIBI concentrations: 0, 0.5, 1, 3, 5 mg / mL; 7: marker.

[0016] Figure 4Biomechanical parameters of porcine sclera with normal mechanical properties were processed using AIBI; a: Stress-strain curves for different groups (strain range 0-8%); b, c, and d: Comparison of scleral thickness, elastic modulus at 8% strain, and ultimate stress for different groups, respectively. (**p<0.01) Detailed Implementation

[0017] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0018] Example 1: Preparation of Modified Type I Collagen Solution

[0019] AIBI solutions of different concentrations were prepared using PBS and stored at 4°C for later use. Experimental group: 150 µL of type I collagen solution (3 mg / mL) was added to 1050 µL of AIBI solution to achieve a working concentration of 1 mg / mL. The solution was heated at 44°C for 10 minutes in the dark. Control group: The same amount of type I collagen solution (3 mg / mL) was added to an equal volume of PBS solution, ensuring the total volume of the solution was consistent with the experimental group.

[0020] To investigate the modifying effect of different concentrations of AIBI solution on type I collagen solution, experimental groups with different concentrations were set up: 150 µL of type I collagen solution (3 mg / mL) was added to 1050 µL of AIBI solution to make the working concentrations of AIBI solution 0.5 mg / mL, 1 mg / mL, 3 mg / mL, and 5 mg / mL, respectively, and heated for 10 minutes under light-protected conditions. The control group was treated similarly. Both experimental and control groups were allowed to react at room temperature for 24 hours before detection by fluorescence spectrophotometry and sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

[0021] Example 2: Preparation and treatment of isolated porcine scleral strips

[0022] Fresh pig eyes from pigs that had died within 6 hours were obtained from a local slaughterhouse. Fifty pig eyes were randomly divided into five groups of ten each: a control group, a PBS group, and an AIBI group. The conjunctiva and subconjunctival tissue above the pig eye were separated to expose the sclera. Muscle tissue was removed, and a 5mm × 15mm scleral strip was prepared at the 12 o'clock position, 6-7 mm from the limbus (muscle attachment point), along the sagittal plane towards the optic nerve. The strip was stored in PBS solution at 4°C for later use.

[0023] Prepare a 1 mg / mL AIBI solution using PBS and store it at 4°C. At the start of the experiment, preheat the AIBI solution in an oil bath at 44-50°C for 5 minutes, then add scleral strips and react for 10 minutes. Add 3 scleral strips per 10 mL of AIBI solution. The scleral strips in the PBS group were treated the same as the AIBI group, except that the reaction solution was PBS. The scleral strips in the control group were stored at 4°C throughout the experiment.

[0024] Example 3: Isolated porcine sclera strips were used for scanning electron microscopy observation and biomechanical performance testing.

[0025] The results are as follows:

[0026] In this experiment, the effect of AIBI on the tertiary structure of type I collagen solution was reflected by detecting endogenous fluorescence, primarily by measuring the fluorescence emission spectra of phenylalanine and tyrosine. For example... Figure 1 The fluorescence emission spectra of phenylalanine in type I collagen solutions modified with different concentrations of AIBI are shown. Type I collagen solutions have an absorption peak at 278 nm, and the fluorescence intensity decreases or disappears significantly with increasing AIBI concentration, but there is no blue shift or red shift. This indicates that AIBI alters phenylalanine. This process may consume phenylalanine or embed phenylalanine after altering the collagen structure, thus leading to a decrease in fluorescence intensity, but without changing the hydrophobic environment of the hydrophobic cavity of type I collagen.

[0027] Figure 2 The figure shows the fluorescence emission spectra of tyrosine in type I collagen solutions modified with different concentrations of AIBI. As can be seen, the type I collagen solution exhibits an absorption peak at 308 nm. The fluorescence intensity of tyrosine gradually decreases with increasing AIBI concentration. However, compared to phenylalanine, the decrease in fluorescence intensity of tyrosine is not significant, and similarly, the absorption peak of tyrosine does not show a blue shift or red shift. In summary, AIBI modification affects both phenylalanine and tyrosine, causing certain alterations such as consumption or encapsulation, but it does not change the hydrophobic environment of their hydrophobic cavities. Therefore, the tertiary structure of the type I collagen solution may remain unchanged.

[0028] This invention uses SDS-PAGE to verify the cross-linking effect of alkyl radicals on type I collagen. SDS-PAGE can determine the molecular weight of collagen based on the migration speed of collagen molecules in the gel. Type I collagen has a triple helix structure, which contains two α1 chains and one α2 chain. Each collagen chain is a left-handed helical structure, and the three collagen chains intertwine to form a right-handed helical structure. Figure 3These are SDS-PAGE images of type I collagen before and after AIBI modification. The control group lanes show that the molecular weight of the α-chain of type I collagen is approximately 130 kDa, with the α1 chain having a larger molecular weight than the α2 chain, in a ratio of approximately 2:1. The β-chain is its dimer, and the γ-chain is its trimer. Lane 2 contains 0 mg / mL AIBI, equivalent to heating type I collagen for 10 minutes. Compared to the control group, there is no significant difference, indicating that the heating process does not affect the molecular weight of type I collagen. The graph shows that AIBI-modified type I collagen exhibits a decrease in α and β chains and an increase in γ chains, indicating that cross-linking occurs after AIBI treatment, forming collagen molecules with higher molecular weights. This process is related to the concentration of AIBI; the higher the concentration, the more significant the reaction. Lanes 5 and 6, compared to lane 4, show no significant increase in γ chains. This may be due to cross-linking between collagen molecules forming larger molecular weight polymers, which are too large to enter the lanes.

[0029] Figure 4 Table 1-5 shows the biomechanical parameters measured by AIBI after treating the sclera of pigs with normal mechanical properties. Figure 4 The corresponding parameter statistics are shown in the table. As can be seen from the table, the scleral thicknesses of the AIBI group, PBS group, and control group were (0.78±0.07) mm, (0.79±0.06) mm, and (0.80±0.03) mm, respectively. There was no statistically significant difference among the three groups, indicating that the modification effect of AIBI had no significant impact on scleral thickness. The ultimate stresses of the AIBI group, PBS group, and control group were (2.11±0.40) MPa, (2.19±0.28) MPa, and (1.95±0.46) MPa, respectively, with no statistically significant difference among the groups. Regarding the stress at 8% strain, there was a statistically significant difference among the three groups (p<0.01). Pairwise comparisons between groups are shown in the table. Figure 4 The stress-strain curves showed more significant differences between the AIBI group and the control group compared to the PBS group, with p-values ​​of 0.001 (AIBI group vs. control group) and 0.006 (AIBI group vs. PBS group), respectively. There were also statistically significant differences among the three groups in the elastic modulus at 8% strain (p<0.01), and pairwise comparisons between groups showed... Figure 4 Figure c shows statistically significant differences between the AIBI group and the control group and the PBS group (p < 0.01). In conclusion, AIBI modification of the sclera of pigs with normal mechanical properties increases its elastic modulus and improves the stress at 8% strain, but has no significant effect on the ultimate stress.

[0030] Experimental results show that the present invention utilizes alkyl free radical initiators to crosslink type I collagen solutions and isolated porcine sclera, thereby improving their anti-enzymatic degradation performance, thermal stability, and biomechanical properties.

[0031] Please note to all technical personnel: Although the present invention has been described according to the specific embodiments above, the inventive concept of the present invention is not limited to this invention. Any modifications that utilize the inventive concept will be included within the scope of protection of this patent.

[0032] The above description is merely a preferred embodiment of the present invention. The scope of protection of the present invention is not limited to the above embodiments. All technical solutions falling within the scope of the present invention's concept are within the scope of protection of the present invention. It should be noted that for those skilled in the art, any improvements and modifications made without departing from the principles of the present invention should also be considered within the scope of protection of the present invention.

Claims

1. Use of an initiator of alkyl radicals for the preparation of a scleral collagen cross-linking medicament, characterized in that, The alkyl radical initiator is a water-soluble azo compound that can decompose to generate alkyl radicals under thermal stimulation, the alkyl radical initiator is 2,2'-azobis(2-imidazoline) dihydrochloride (AIBI), a suitable heating temperature for the AIBI to decompose to generate alkyl radicals under thermal stimulation is 44-50°C, and the concentration of the 2,2'-azobis(2-imidazoline) dihydrochloride (AIBI) is 0.5-5 mg / mL.

2. An ex vivo scleral collagen cross-linking method independent of oxygen, characterized in that, The method comprises the following steps: The collagen type I solution or scleral tissue is added to an alkyl radical initiator solution, heated for 10 minutes under light shielding, and then reacted at room temperature for 24 hours, the alkyl radical initiator is 2,2'-azobis(2-imidazoline) dihydrochloride (AIBI), and the concentration of the 2,2'-azobis(2-imidazoline) dihydrochloride (AIBI) is 0.5-5 mg / mL.

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