CN116850286A - Application of initiator of alkyl free radical in preparation of scleral collagen crosslinking medicament and scleral collagen crosslinking method independent of oxygen - Google Patents
Application of initiator of alkyl free radical in preparation of scleral collagen crosslinking medicament and scleral collagen crosslinking method independent of oxygen Download PDFInfo
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- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 7
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
- A61K41/0028—Disruption, e.g. by heat or ultrasounds, sonophysical or sonochemical activation, e.g. thermosensitive or heat-sensitive liposomes, disruption of calculi with a medicinal preparation and ultrasounds
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P27/00—Drugs for disorders of the senses
- A61P27/02—Ophthalmic agents
- A61P27/10—Ophthalmic agents for accommodation disorders, e.g. myopia
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Abstract
The application of the initiator of the alkyl radical in preparing scleral collagen crosslinking medicine and the scleral collagen crosslinking method independent of oxygen utilize the initiator of the alkyl radical to crosslink the type I collagen solution and scleral tissue, compared with the ultraviolet light/riboflavin crosslinking method, the reaction is carried out independent of the participation of oxygen, and the method is more applicable to sclera in myopia and hypoxia state, and compared with the chemical crosslinking method, the crosslinking reaction is easier to control. The collagen crosslinking induced by alkyl free radicals is improved in terms of enzyme degradation resistance, thermal stability and biomechanical properties.
Description
Technical Field
The invention relates to the technical field of biological medicine, in particular to application of an initiator of alkyl free radicals in preparation of scleral collagen crosslinking medicines and an oxygen-independent scleral collagen crosslinking method.
Background
Myopia is one of the most common ophthalmic diseases worldwide, usually beginning in childhood. The prevalence of myopia is increasing, and it is expected that in 2050, the number of myopia will be as high as 47.58 hundred million, accounting for about 49.8% of the global population, while the number of high myopia will be 9.38 hundred million. And some of these highly myopic patients will develop pathological myopia. Pathological myopia is extremely harmful to vision, and is a main cause of blindness for myopes. Common complications include posterior scleral grape swelling, macular atrophy, macular cleavage, macular hole, choroidal neovascularization, retinal detachment, etc., which are often the cause of blindness in pathologically myopic patients. Pathological myopia manifests as an increasing ocular axis, with an increasing ocular axis, and with a tendency to posterior scleral uveal swelling, with consequent other complications. Regarding the etiology of myopia, the mechanism is currently unknown, and is thought to be the result of the interaction of genetic and environmental factors. It has been found that in myopia progression, the mechanical properties of the sclera decrease, the hardness decreases, the thickness becomes thinner, and the sclera elasticity increases and the collagen content decreases. Wu et al found that hypoxia promoted transdifferentiation of human scleral fibroblasts while decreasing expression of type I collagen, suggesting an important role in remodeling of scleral extracellular matrix and progression of myopia. Scleral crosslinking is a novel method for preventing and treating myopia by crosslinking scleral collagen to form a novel connection between the interior of collagen molecules and collagen molecules, thereby enhancing the mechanical properties of sclera. Current scleral crosslinking techniques are primarily physical and chemical crosslinking. Wherein the physical crosslinking is mainly ultraviolet light/riboflavin crosslinking and blue light/riboflavin crosslinking. Chemical crosslinking is mainly performed on collagen by using chemical crosslinking agents, such as glutaraldehyde, genipin, glyceraldehyde and the like.
The ultraviolet light/riboflavin crosslinking method is to irradiate the photosensitizer riboflavin with ultraviolet light with a wavelength of 370 nm, so that the riboflavin is excited to a triplet state, thereby generating active oxygen. The reactive oxygen species react with the collagen molecules to induce cross-linking between or within the amino acid molecules of adjacent collagen molecules. Wollensak et al uses a wavelength of 370 nm and an energy of 3 mW/cm 2 Is irradiated on the sclera of the equatorial portion of the rabbit eye, which is treated with 0.1% riboflavin solution, 3 days after operation, 4 months after operation, and after operationMechanical property evaluation is carried out on rabbit sclera in 8 months, and the Young's modulus of the rabbit sclera is increased by 320% in 3 days after operation, and is increased by 277% in 4 months after operation, and 502% in 8 months after operation, which shows that ultraviolet light/riboflavin crosslinking is a very effective and stable crosslinking method, and has no damage to retina or retinal pigment epithelium. However, since ultraviolet light can pass through the sclera, there is a potential for damage to the retina and other tissues within the eye. Wang et al cross-linked the sclera of rabbit eyes with 365, nm wavelength, laser of the same energy and riboflavin at the same concentration, and examined the experimental rabbit by visual electrophysiological Examination (ERG) after surgery, as a result, it was found that the amplitude of dark-adapted ERG was statistically reduced 1 week, 1 month and 3 months after surgery, and apoptotic cells and ultrastructural changes could be found in the retinal layers of the scleral cross-linked eyes. The principle of blue light/riboflavin crosslinking is similar to that of ultraviolet light/riboflavin, but blue light has longer wavelength than ultraviolet light, lower photon energy, and less damage to organisms. However, this cross-linking also has the problem of exposing the posterior sclera to light while avoiding damage to other parts of the eye. Kwok et al direct blue light to the equatorial sclera through a metal coated, pliable polymer waveguide, which minimizes leakage of light during light conduction, which may be applied to the posterior sclera. However, this photocrosslinking requires the participation of oxygen, and the myopic sclera may itself be in an anoxic state, and if oxygen between tissues is consumed during crosslinking, snow frosting may occur.
The chemical crosslinking has a higher crosslinking degree than the physical crosslinking, and a relatively uniform crosslinking effect can be obtained as compared with the instability of the physical crosslinking, but the chemical crosslinking introduces exogenous crosslinking, and thus, toxic effects may exist. Such as glutaraldehyde, is not substantially used for scleral crosslinking due to its cytotoxicity. At present, genipin and glyceraldehyde are mature in application. Genipin is a natural cross-linking agent, and several studies prove that genipin can enhance the mechanical strength of sclera and has low toxicity, but has the problems of staining sclera and even cornea and needing multiple injections. In addition, all chemical crosslinking agents can only control the degree of crosslinking by concentration dosage, and cannot control the reaction time, because the crosslinking agent cannot be taken out after being injected under the Tenon's capsules. Then, there is no scleral crosslinking method which requires neither oxygen consumption nor control of the crosslinking reaction, while obtaining relatively satisfactory mechanical properties.
Disclosure of Invention
In order to solve the defects and shortcomings in the prior art, the invention provides an application of an alkyl radical initiator in preparation of scleral collagen crosslinking drugs and an oxygen-independent scleral collagen crosslinking method.
The technical scheme adopted by the invention is as follows: the application of an initiator of alkyl free radicals in preparing scleral collagen crosslinking medicines.
The initiator of the alkyl free radical is a water-soluble azo compound which can be decomposed to generate the alkyl free radical under the heat stimulation.
The initiator of the alkyl free radical is 2,2' -aza-bis (2-imidazoline) dihydrochloride (AIBI).
The AIBI is suitable for heating at a temperature of 44-50deg.C to decompose to generate alkyl radicals under thermal stimulation.
The AIBI concentration is 0.5-5mg/mL.
A method of oxygen independent scleral collagen crosslinking comprising the steps of: the type I collagen solution or scleral tissue was added to an initiator solution capable of generating alkyl radicals, heated under light-shielding conditions for 10 minutes, and then reacted at room temperature for 24 hours.
The beneficial effects of the invention are as follows: the invention provides an application of an initiator of an alkyl radical in preparing a scleral collagen crosslinking medicament and an oxygen-independent scleral collagen crosslinking method, wherein the type I collagen and sclera are crosslinked by the initiator of the alkyl radical, compared with a photo-crosslinking method, the reaction is carried out without depending on participation of oxygen, the method is more applicable to sclera in a myopia anoxic state, compared with a chemical crosslinking method, the crosslinking reaction is easier to control, and the enzymatic degradation resistance, the thermal stability and the biomechanical property are all improved.
Drawings
FIG. 1 is a graph showing fluorescence emission spectra of phenylalanine in AIBI-modified type I collagen solutions at various concentrations; wherein the right dashed box is an enlarged portion of the left dashed box.
FIG. 2 is a graph showing fluorescence emission spectra of tyrosine in AIBI-modified type I collagen solutions at various concentrations.
FIG. 3 is SDS-PAGE of type I collagen before and after AIBI modification; wherein 1: a control group; 2-6, AIBI concentration: 0. 0.5, 1, 3, 5 mg/mL;7: and (5) Marker.
FIG. 4 shows the biomechanical parameters of AIBI processing of porcine sclera with normal mechanical properties; a: stress-strain curves for different groups (strain ranges 0-8%); b. c, d: the sclera thickness, the elastic modulus at 8% strain, and the ultimate stress were compared for each of the different groups. P <0.01
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
EXAMPLE 1 preparation of modified type I collagen solution
AIBI solutions of different concentrations were prepared with PBS and stored at 4℃for further use. Experimental group: 150 mu L of type I collagen solution (3 mg/mL) is taken, 1050 mu L of AIBI solution is added, the working concentration of the AIBI solution is 1mg/mL, and the AIBI solution is heated for 10 minutes at the temperature of 44 ℃ in the absence of light. Control group: the same amount of type I collagen solution (3 mg/mL) was taken and added to an equal volume of PBS solution, eventually keeping the total volume of the solution consistent with the experimental set.
To study the modification effect of AIBI solutions of different concentrations on type I collagen solutions, different concentration experimental groups were set up: and taking 150 mu L of type I collagen solution (3 mg/mL), adding 1050 mu L of AIBI solution, and heating the AIBI solution for 10 minutes under the light-shielding condition, wherein the working concentration of the AIBI solution is 0.5mg/mL, 1mg/mL, 3mg/mL and 5mg/mL. The control group was as described above, and both the experimental group and the control group were allowed to react at room temperature for 24 hours and then used for detection by fluorescence spectrophotometer and sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
Example 2 preparation and treatment of ex vivo porcine scleral strips
Fresh pig eyes with death times within 6 hours were obtained from a local slaughterhouse. The 50 eyes were randomly divided into 5 groups of 10 eyes each, which were control group, PBS group and AIBI group, respectively. Separating conjunctiva and subconjunctival tissue above pig eye, exposing sclera, removing muscle tissue, preparing 5mm×15 mm sclera strip along sagittal plane toward optic nerve with scalpel after 12-point angle sclera edge 6-7 mm (muscle attachment point), and storing in PBS solution at 4deg.C.
1mg/mL AIBI solution was prepared with PBS and stored at 4deg.C for use. At the beginning of the experiment, AIBI solution was placed in an oil bath at 44-50deg.C for 5min, then scleral strips were added for 10 min, and 3 scleral strips were added per 10 mL AIBI solution. Scleral strips of the PBS group except for PBS as the reaction solution, the remaining treatment was identical to that of the AIBI group, and scleral strips of the control group were kept in a refrigerator at 4 ℃.
Example 3 ex vivo porcine scleral strips were used for scanning electron microscope observation and biomechanical performance testing.
The results were as follows:
in the experiment, the influence of AIBI on the tertiary structure of the type I collagen solution is reflected by detecting endogenous fluorescence, namely, mainly measuring fluorescence emission spectra of phenylalanine and tyrosine. As shown in FIG. 1, fluorescence emission spectra of phenylalanine in AIBI-modified type I collagen solutions at various concentrations are shown: the type I collagen solution has an absorption peak at 278nm and the fluorescence intensity decreases or disappears significantly with increasing concentration of AIBI, but no blue shift or red shift, indicating that AIBI changes phenylalanine, which may be consumed or the collagen structure is changed and then phenylalanine is embedded, resulting in a decrease in fluorescence intensity without changing the hydrophobic environment of the type I collagen hydrophobic cavity.
FIG. 2 is a fluorescence emission spectrum of tyrosine corresponding to AIBI after modification of type I collagen solutions at various concentrations. As can be seen, the type I collagen solution has an absorption peak at 308nm, and the fluorescence intensity of tyrosine gradually decreases as the AIBI concentration increases. However, the amount of decrease in fluorescence intensity of tyrosine was not significant as compared with phenylalanine, and similarly, the absorption peak of tyrosine was not blue-shifted or red-shifted. In summary, AIBI modification has been shown to have an effect on both phenylalanine and tyrosine, which have been altered, e.g., consumed or entrapped, but not their hydrophobic environment of the hydrophobic cavity, and thus the tertiary structure of type I collagen solutions may not be altered.
SDS-PAGE was used to verify that alkyl radicals have a crosslinking effect on type I collagen. SDS-PAGE can obtain the molecular weight of collagen based on the rate of movement of collagen molecules in the gel. Type I collagen has a triple helix feature comprising 2 α1 chains and 1 α2 chain, wherein each chain is a left-handed helix and 3 chains are intertwined to form a right-handed helix. FIG. 3 is a SDS-PAGE of type I collagen before and after AIBI modification, wherein the control group of lanes shows that the type I collagen has an alpha chain of about 130kDa and an alpha 1 chain of greater molecular weight than the alpha 2 chain in a ratio of about 2: the 1, beta chain is its dimer and the gamma chain is its trimer. Lane 2 is 0mg/mL AIBI, which corresponds to heating type I collagen for 10 minutes, with no significant difference compared to the control, indicating that the heating process does not affect the molecular weight of type I collagen. From the graph, the AIBI modified type I collagen shows that alpha and beta chains are reduced, and gamma chains are increased, which indicates that the type I collagen is crosslinked after the AIBI acts, collagen molecules with higher molecular weight are formed, and the process is related to the acting concentration of the AIBI, and the higher the concentration is, the more obvious the reaction is. Lanes 5 and 6 show no significant increase in gamma chain compared to lane 4, probably due to cross-linking between collagen molecules to form a polymer of greater molecular weight, which is too large to enter the lane.
Tables 1-5 in FIG. 4 are tables of biomechanical parameters measured after AIBI treatment on porcine sclera with normal mechanical properties, and corresponding statistical graphs of parameters in FIG. 4. As can be seen from the table, the sclera thicknesses of AIBI group, PBS group and control group were (0.78+ -0.07), 0.79+ -0.06 and 0.80+ -0.03) mm, respectively, and there was no statistical difference between the three groups, indicating that the modification effect of AIBI had no significant effect on sclera thickness. The limiting stresses of 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, and there was no statistical difference between the groups. There was a statistical difference in stress at 8% strain between the three groups (p < 0.01), and the stress-strain curves in fig. 4 were compared between the groups two by two, and the difference between the AIBI group and the control group was more pronounced 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. The difference between the three groups is also statistically significant (p < 0.01) in terms of modulus of elasticity at 8% strain, and a pairwise comparison between groups can be seen in graph c of fig. 4: AIBI group was statistically different from control group, PBS group, p <0.01. In conclusion, after AIBI modifies pig sclera with normal mechanical property, the elastic modulus can be increased, the stress of 8% strain is improved, but the limit stress is not changed obviously.
Experimental results show that the invention can generate crosslinking effect on the I-type collagen solution and the isolated pig sclera by using the initiator of the alkyl free radical, and the invention has improved enzymatic degradation resistance, thermal stability and biomechanical property.
The skilled person will know: while the invention has been described in terms of the foregoing embodiments, the inventive concepts are not limited to the invention, and any modifications that use the inventive concepts are intended to be within the scope of the appended claims.
The above description is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above examples, and all technical solutions belonging to the concept of the present invention belong to the protection scope of the present invention. It should be noted that modifications and adaptations to the present invention may occur to one skilled in the art without departing from the principles of the present invention and are intended to be within the scope of the present invention.
Claims (6)
1. The application of an initiator of alkyl free radicals in preparing scleral collagen crosslinking medicines.
2. The use according to claim 1, wherein the alkyl radical initiator is a water-soluble azo compound which decomposes under thermal stimulation to generate alkyl radicals.
3. The use according to claim 1, wherein the alkyl radical initiator is 2,2' -aza-bis (2-imidazoline) dihydrochloride (AIBI).
4. The use according to claim 3, wherein the AIBI is heated to a suitable temperature of 44-50 ℃ for decomposition to alkyl radicals under thermal stimulation.
5. The use according to claim 3, wherein the concentration of 2,2' -aza-bis (2-imidazoline) dihydrochloride (AIBI) is 0.5-5mg/mL.
6. A method of oxygen independent scleral collagen cross-linking comprising the steps of: the type I collagen solution or scleral tissue was added to an initiator solution capable of generating alkyl radicals, heated under light-shielding conditions for 10 minutes, and then reacted at room temperature for 24 hours.
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