Modified intraocular lens and preparation method and application thereof

Abstract

The invention relates to the technical field of artificial lenses, in particular to a modified artificial lens and a preparation method and application thereof. The modified intraocular lens is characterized in that a PHEMA-copolymer film-serine polymer intraocular lens is used as a carrier, and adriamycin is loaded on the surface of the carrier. The modified intraocular lens has good physical barrier and biological inhibition effects, can avoid accumulation of proteins and lipids in tears, can avoid generation of bacterial infection complications, can prevent adhesion of lens epithelial cells, can block DNA replication and transcription processes of the lens epithelial cells, can inhibit proliferation of the lens epithelial cells, and can improve the lens epithelial cell proliferation rate. The prepared nano-material can induce apoptosis of lens epithelial cells, has an excellent antibacterial effect and good biocompatibility, and can prevent the occurrence of posterior cataract.

Description

Technical Field

[0001] The present invention relates to the technical field of artificial lenses, and in particular to a modified artificial lens and a preparation method and application thereof. Background Art

[0002] Cataract is an eye disease that causes blindness and visual impairment. It is caused by factors such as aging, heredity, local nutritional disorders, immune and metabolic abnormalities, trauma or radiation, which cause lens metabolism disorders, which in turn lead to lens protein denaturation and turbidity, and eventually form cataracts. At present, the effective method for treating cataracts is surgery, that is, the original turbid lens is removed through surgery and an artificial lens is implanted.

[0003] An intraocular lens (IOL) is a precision optical component that can be implanted in the eye to replace the body's natural lens. However, after an IOL is implanted in the human body, it is prone to cause an inflammatory foreign body reaction. At the same time, the residual lens epithelium (LECs) in the capsular bag easily adheres to the surface of the IOL and undergoes proliferation, migration, and apoptosis, which in turn causes opacity on the anterior and posterior surfaces of the IOL or shrinkage of the capsular bag, leading to secondary cataracts (posterior capsularopacification, PCO). Summary of the invention

[0004] In view of the above technical problems, the present invention provides a modified artificial lens and its preparation method and application. The modified artificial lens has a physical barrier and biological inhibition effect, can prevent the adhesion of lens epithelial cells, and can also block the DNA replication and transcription process of lens epithelial cells, inhibit the proliferation of lens epithelial cells, thereby inducing apoptosis of lens epithelial cells, and can prevent the occurrence of secondary cataracts.

[0005] In order to solve the above technical problems, the present invention adopts the following technical solutions: In a first aspect, the present invention provides a modified intraocular lens, comprising: a PHEMA-copolymer film-serine polymer intraocular lens as a carrier, and doxorubicin loaded on the surface.

[0006] The modified intraocular lens obtained by grafting doxorubicin on the surface of the PHEMA-copolymer film-serine polymer intraocular lens of the present invention has physical barrier and biological inhibition effects, can avoid the accumulation of proteins and lipids in tears, avoid the occurrence of bacterial infection complications, and prevent the adhesion of lens epithelial cells; it can also block the DNA replication and transcription process of lens epithelial cells, inhibit the proliferation of lens epithelial cells, thereby inducing apoptosis of lens epithelial cells; it can also inhibit the synthesis of bacterial proteins, change the permeability of bacterial cell membranes, make it impossible for bacterial internal substances to leach out normally, hinder the growth and reproduction of bacteria, and play an antibacterial role; it has good biocompatibility and can prevent the occurrence of secondary cataracts.

[0007] In the present invention, the structural formula of doxorubicin (ADM) is shown in formula (I). The structural formula of PHEMA-copolymer membrane-serine is shown in formula (II).

[0008] Formula (I)

[0009] Formula (II) Preferably, the preparation method of the PHEMA-copolymer film intraocular lens is: placing the PHEMA intraocular lens in a modified solution, irradiating it with a 365nm ultraviolet lamp for at least 1 hour, and then immersing it in an ethanol / water solution with a volume ratio of (1-2):3 at 60-70°C for at least 3 hours to remove unreacted monomers in the copolymer film to obtain a PHEMA-copolymer film intraocular lens.

[0010] The modified solution preferably uses a mixed solution of glycidyl methacrylate, crosslinker triethylene glycol dimethacrylate and photoinitiator 2-hydroxy-2-methylpropiophenone in a mass ratio of 15: (0.5-1): (0.5-1). PHEMA, glycidyl methacrylate (GMA), crosslinker triethylene glycol dimethacrylate and photoinitiator 2-hydroxy-2-methylpropiophenone can undergo a reversible addition-fragmentation chain transfer free radical polymerization reaction under blue light irradiation to obtain PHEMA-copolymer film (PHEMA-co-GMA). The reaction formula is as follows:

[0011] Preferably, the preparation method of the PHEMA-copolymer film-serine polymer intraocular lens is: adding the PHEMA-copolymer film intraocular lens to an ethanol aqueous solution of serine, placing it in a constant temperature shaking incubator at 50-60°C for at least 24 hours to obtain the PHEMA-copolymer film-serine polymer intraocular lens, and then washing it with distilled water until the solution is neutral. The reaction formula is as follows:

[0012] Wherein, in the ethanol aqueous solution of serine, the ethanol concentration is 25%-30% v / v, the molar concentration of serine is 1-3 mol / L, and the pH is 10-11.

[0013] The epoxy ring of the PHEMA-copolymer membrane and the primary amino group of serine undergo a ring-opening reaction, so that serine is grafted onto the PHEMA-copolymer membrane. The resulting PHEMA-copolymer membrane-serine polymer intraocular lens has excellent hydrophilicity compared to the PHEMA intraocular lens, which can avoid protein deposition, give the modified intraocular lens good anti-cell adhesion properties, and thus prevent the adhesion of lens epithelial cells. In vitro cytotoxicity tests have shown that the PHEMA-copolymer membrane-serine polymer intraocular lens is non-cytotoxic and has good biocompatibility.

[0014] In a second aspect, the present invention also provides a method for preparing the modified intraocular lens, comprising the following steps: immersing the PHEMA-copolymer film-serine polymer intraocular lens in a 10-15ug / mL doxorubicin (ADM) aqueous solution, stirring at a low speed for at least 12 hours at 20-60°C, taking it out, washing it with distilled water for at least 6 times, and irradiating it with ultraviolet light for at least 15 minutes. The reaction formula is as follows:

[0015] Preferably, the low-speed stirring is 100-150 rpm, which is conducive to fully loading doxorubicin on the PHEMA-copolymer membrane-serine polymer.

[0016] In a third aspect, the present invention also provides the use of the modified intraocular lens or the modified intraocular lens obtained by the above preparation method in the preparation of a modified intraocular lens for preventing after-cataract. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The scanning electron microscope images are of the PHEMA-copolymer film intraocular lens (Figure a), the PHEMA-copolymer film-serine polymer intraocular lens (Figure b) and the PHEMA-copolymer film-serine-ADM intraocular lens (Figure c) obtained in Example 1 of the present invention; Figure 2 The light transmittances of the PHEMA-copolymer film intraocular lens (corresponding to the red curve), the PHEMA-copolymer film-serine polymer intraocular lens (corresponding to the blue curve) and the PHEMA-copolymer film-serine-ADM intraocular lens (corresponding to the green curve) obtained in Example 1 of the present invention at different wavelengths; Figure 3aa and b are the drug release curves of PHEMA-copolymer film-ADM intraocular lens and PHEMA-copolymer film-serine-ADM intraocular lens at different pH values, respectively; Figure 4 It is a representative agar plate for the antibacterial activity of PHEMA-copolymer membrane intraocular lens, PHEMA-copolymer membrane-serine intraocular lens and PHEMA-copolymer membrane-serine-ADM intraocular lens; Figure 5 It is the bacterial adhesion data graph of PHEMA-copolymer film-ADM intraocular lens and PHEMA-copolymer film-serine-ADM intraocular lens; Figure 6 Statistical graph of sterilization data of PHEMA-copolymer film-ADM intraocular lens and PHEMA-copolymer film-serine-ADM intraocular lens; Figure 7 Figure 2 is a graph showing the apoptosis of LECS induced by PHEMA-copolymer membrane intraocular lenses and PHEMA-copolymer membrane-serine-ADM intraocular lenses. DETAILED DESCRIPTION

[0018] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0019] The PHEMA intraocular lenses used in the following examples and comparative examples are all poly(2-hydroxyethyl methacrylate), CAS No.: 25249-16-5.

[0020] Example 1 An embodiment of the present invention provides a method for preparing a modified intraocular lens, comprising: S1. Mix glycidyl methacrylate, crosslinker triethylene glycol dimethacrylate and photoinitiator 2-hydroxy-2-methylpropiophenone in a mass ratio of 15:0.5:0.5, stir until a transparent solution is obtained to prepare a modified solution. Then place the PHEMA intraocular lens in the above-mentioned modified solution and irradiate it with a 365nm ultraviolet lamp for 1 hour to obtain a PHEMA-copolymer film intraocular lens. Soak the PHEMA-copolymer film intraocular lens in an ethanol / water solution with a volume ratio of 1:3 at 60°C for 3 hours to remove unreacted monomers in the copolymer film to obtain a PHEMA-copolymer film intraocular lens for standby use.

[0021] S2. Add serine to a 25% v / v ethanol aqueous solution to obtain an ethanol aqueous solution of serine with a molar concentration of 1 mol / L, and add sodium hydroxide solution to a pH of 11. Then add the PHEMA-copolymer film intraocular lens to the above serine ethanol aqueous solution, place it in a 50°C constant temperature shaking incubator for reaction for 24 hours, and obtain a PHEMA-copolymer film-serine polymer intraocular lens, then wash it with distilled water until the solution pH reaches 7.0, and soak it for storage.

[0022] S3. Soak the PHEMA-copolymer membrane-serine polymer intraocular lens in a 10ug / mL doxorubicin (ADM) aqueous solution, stir at 100rpm at 60°C for 12h, take it out, wash it with distilled water for 6 times, irradiate it with ultraviolet light for 15min, and seal it in a phosphate buffered saline solution in a sterile environment, and store it at room temperature for later use, to obtain the PHEMA-copolymer membrane-serine-ADM intraocular lens.

[0023] The PHEMA-copolymer film intraocular lens, PHEMA-copolymer film-serine polymer intraocular lens and PHEMA-copolymer film-serine-ADM intraocular lens obtained in the above steps were analyzed by scanning electron microscopy, see Figure 1 The results showed that compared with the smooth surface of the PHEMA-copolymer IOL, the PHEMA-copolymer-serine polymer IOL and the PHEMA-copolymer-serine-ADM IOL showed a rougher surface with many nanoparticles.

[0024] The thickness of the copolymer film and serine on the PHEMA-copolymer film-serine polymer intraocular lens, and the thickness of the copolymer film-serine-ADM on the PHEMA-copolymer film-serine-ADM intraocular lens were measured by ellipsometry. The thickness of the polymer coating on the PHEMA-copolymer film-serine polymer intraocular lens was measured to be approximately 55.5 ± 3.2 nm; the thickness of the polymer coating on the PHEMA-copolymer film-serine-ADM intraocular lens was 60.8 ± 4.1 nm.

[0025] The rougher surface roughness and higher coating thickness may be due to the addition of ADM.

[0026] The optical transparency of PHEMA-copolymer-serine polymer intraocular lenses and PHEMA-copolymer-serine-ADM intraocular lenses was evaluated using a haze meter in the wavelength range of 400 to 700 nm. Figure 2. The spectra showed that the transparency of the PHEMA-copolymer-serine polymer IOL (92.2%, blue curve) and the PHEMA-copolymer-serine-ADM IOL (90.1%, green curve) decreased slightly compared with the PHEMA-copolymer IOL (94.5%, red curve). Nevertheless, the transmittance of the PHEMA-copolymer-serine-ADM IOL was still above 90%, showing excellent optical properties. These results indicate that the PHEMA-copolymer-serine-ADM with nanoscale thickness and rougher surface did not cause significant changes in the optical transparency of the original PHEMA-copolymer IOL.

[0027] Example 2 An embodiment of the present invention provides a method for preparing a modified intraocular lens, comprising: S1. Mix glycidyl methacrylate, crosslinker triethylene glycol dimethacrylate and photoinitiator 2-hydroxy-2-methylpropiophenone in a mass ratio of 15:0.8:0.5, stir until a transparent solution is obtained to prepare a modified solution. Then place the PHEMA intraocular lens in the above modified solution and irradiate with a 365nm ultraviolet lamp for 1 hour to obtain a PHEMA-copolymer film intraocular lens. Soak the PHEMA-copolymer film intraocular lens in an ethanol / water solution with a volume ratio of 2:3 at 65°C for 3 hours to remove unreacted monomers in the copolymer film for later use.

[0028] S2. Add serine to a 28% v / v ethanol aqueous solution to obtain an ethanol aqueous solution of serine with a molar concentration of 2 mol / L, and add sodium hydroxide solution to a pH of 11. Then add the PHEMA-copolymer film intraocular lens to the above serine ethanol aqueous solution, place it in a 50°C constant temperature shaking incubator for reaction for 24 hours, and obtain a PHEMA-copolymer film-serine polymer intraocular lens, then wash it with distilled water until the solution pH reaches 7.0, and soak it for storage.

[0029] S3. Soak the PHEMA-copolymer film-serine polymer intraocular lens in a 12ug / mL doxorubicin aqueous solution, stir at 120rpm at 50°C for 12h, take it out, wash it with distilled water for 6 times, irradiate it with ultraviolet light for 15min, and seal it in a phosphate buffered saline solution in a sterile environment, and store it at room temperature for use, to obtain the modified intraocular lens.

[0030] Example 3 An embodiment of the present invention provides a method for preparing a modified intraocular lens, comprising: S1. Mix glycidyl methacrylate, crosslinker triethylene glycol dimethacrylate and photoinitiator 2-hydroxy-2-methylpropiophenone in a mass ratio of 15:1:0.5, stir until a transparent solution is obtained to prepare a modified solution. Then place the PHEMA intraocular lens in the above modified solution and irradiate with a 365nm ultraviolet lamp for 1 hour to obtain a PHEMA-copolymer film intraocular lens. Soak the PHEMA-copolymer film intraocular lens in a 1:3 volume ratio ethanol / water solution at 70°C for 3 hours to remove unreacted monomers in the copolymer film for later use.

[0031] S2. Add serine to a 30% v / v ethanol aqueous solution to obtain an ethanol aqueous solution of serine with a molar concentration of 3 mol / L, and add sodium hydroxide solution to a pH of 10. Then add the PHEMA-copolymer film intraocular lens to the above serine ethanol aqueous solution, place it in a 60°C constant temperature shaking incubator for reaction for 24 hours, and obtain a PHEMA-copolymer film-serine polymer intraocular lens, then wash it with distilled water until the solution pH reaches 7.0, and soak it for storage.

[0032] S3. Soak the PHEMA-copolymer film-serine polymer intraocular lens in a 15ug / mL doxorubicin aqueous solution, stir at 150rpm for 12h at 30°C, take it out, wash it 6 times with distilled water, irradiate it with ultraviolet light for 15min, and seal it in a phosphate buffered saline solution in a sterile environment, and store it at room temperature for use, to obtain the modified intraocular lens.

[0033] Comparative Example 1 Compared with Example 1, after step S1, the PHEMA-copolymer membrane intraocular lens is obtained, and according to the method of step S3, it is immersed in a 10ug / mL doxorubicin (ADM) aqueous solution to obtain a PHEMA-copolymer membrane intraocular lens loaded with ADM.

[0034] The following is an experimental study on PHEMA-copolymer film-serine-ADM intraocular lens.

[0035] Experimental Example 1 Physical or chemical property testing of PHEMA-copolymer film-serine-ADM intraocular lens.

[0036] (1) Average drug loading The artificial crystals prepared in Comparative Example 1 and Example 1 were placed on a magnetic stirrer and stirred at room temperature for 36 h at 12000 r·min. -1Centrifuge for 10 minutes, collect the supernatant and precipitate, the supernatant is the test solution, and the drug-loaded supernatant is transferred to a 10 mL volumetric flask by a precision instrument, filtered through a 0.45 um microporous filter membrane, injected, and the peak area is recorded. The results show that the average loading amount of ADM in each IOL of Comparative Example 1 is 8.5 ug, while the PHEMA-copolymer membrane-serine-ADM intraocular lens in Example 1 shows a higher ADM loading amount of 35.5 ug.

[0037] (2) Drug release profile Under the conditions of pH 5.5 and 7.4, the drug release curves of Example 1 and Comparative Example 1 within 30 days were tested, as shown in Figure 3. As can be seen from the figure: under the condition of pH 5.5, in the IOL directly loaded with ADM in Comparative Example 1, ADM showed a burst release phenomenon and reached the limit of drug amount (about 90%, 7.6 μg) within 5 days. The ADM in the PHEMA-copolymer film-serine-ADM intraocular lens showed a two-stage drug release pattern: a burst release phase was also found in the first 5 days, and about 53% (18.8μg) of ADM was released, and then the ADM in the HEMA-copolymer film-serine-ADM intraocular lens entered the sustained release phase in the next 25 days, and showed a long-term effective release time, which has a positive effect on the continuous suppression of post-cataract PCO throughout the postoperative recovery period.

[0038] It can be seen that the intraocular lens grafted with serine and then loaded with ADM has a high drug loading and a drug release curve that is more conducive to inhibiting secondary cataracts.

[0039] Experimental Example 2 Detection of antibacterial, bactericidal and antibiofilm activities of PHEMA-copolymer membrane-serine-ADM intraocular lenses.

[0040] (1) Antibacterial test PHEMA-copolymer film IOL, PHEMA-copolymer film-serine polymer IOL, and PHEMA-copolymer film-serine-ADM IOL were co-cultured with Escherichia coli and Staphylococcus aureus for 48 h, respectively. Representative agar plates are shown in Figure 4 The results showed that the PHEMA-copolymer membrane-serine-ADM intraocular lens had a significant inhibitory effect on Escherichia coli and Staphylococcus aureus.

[0041] (2) Bacterial adhesion rate experiment Experimental process: ① Coating: Add 100ul of coating solution to each well of a 96-well plate, place the plate at 2-8°C overnight, remove the coating solution, and wash 2-3 times with washing solution. ② Bacterial inoculation: After the bacteria to be tested are processed, 5×104 Cells / well are inoculated in a 96-well plate. It is recommended to set up 3-5 replicate wells and a control group. Incubate in a 37° incubator for 1 hour, remove the culture plate, discard the culture medium, and then wash 2-3 times with the corresponding culture medium. Add 100ul of fresh culture medium to each well. ③ Adhesion rate determination: Add 10ul staining solution to each well of the 96-well plate, incubate at 37° for 30 minutes, and measure the OD value of the sample well.

[0042] Test results see Figure 5 The results showed that the PHEMA-copolymer membrane intraocular lens directly loaded with ADM prepared in Comparative Example 1 could resist bacterial adhesion to a certain extent, but the effect was significantly lower than the bacterial adhesion of the PHEMA-copolymer membrane-serine-ADM intraocular lens.

[0043] (3) Sterilization rate The PHEMA-copolymer film intraocular lens of ADM and the PHEMA-copolymer film-serine-ADM intraocular lens prepared in Comparative Example 1 were co-cultured with typical pathogenic bacteria S. aureus and E. coli (150 μL, 105 CFU / mL) for 24 h, respectively.

[0044] Draw lines on the culture dish and count the areas under a microscope. The results show that (see Figure 6 ): Direct loading of ADM on PHEMA-copolymer film intraocular lens can only kill 55.5% of Staphylococcus aureus and 71.2% of Escherichia coli, while the killing rate of both bacteria in PHEMA-copolymer film-serine-ADM intraocular lens is above 99.0%. It proves that PHEMA-copolymer film-serine-ADM intraocular lens has good killing effect on Staphylococcus aureus and Escherichia coli.

[0045] Experimental Example 3 PHEMA-copolymer membrane-serine-ADM was implanted intraocularly to evaluate its biocompatibility and anti-PCO.

[0046] Experimental process: (1) Twenty 2-month-old New Zealand white rabbits (3-3.5 kg) were divided into two groups: ① a simple soft IOL group; ② a PHEMA-copolymer membrane-serine-ADM coated IOL group. Before implantation, all rabbit eyes underwent ophthalmological examinations to ensure health. The random eyes of the rabbits underwent phacoemulsification combined with IOL sample implantation. The cortical polishing step was omitted during the operation to facilitate the establishment of a posterior capsule opacification (PCO) model, ensuring that PCO was achieved within one month. high incidence and sufficient samples for analysis.

[0047] (2) Tropicamide phenylephrine eye drops (norepinephrine hydrochloride 5 mg / mL, tropicamide 5 mg / mL) were given to the operated eye to reduce anterior chamber inflammation, and the pupil was dilated once a day for 0-3 days. Levofloxacin lactate and tobramycin dexamethasone ointment were applied externally to prevent infection for 0-7 days, 4 times a day. After implantation, the rabbit's operated eye was examined and observed with a slit lamp at regular intervals.

[0048] Slit-lamp images (see Figure 7 ) was used to evaluate PCO in the rabbit model at 15 days, 1 month, and 2 months after surgery. A small amount of fibrotic tissue appeared on the posterior capsule of the PHEMA-copolymer intraocular lens at 2 months, and local LEC proliferation was seen in the peripheral capsular bag, indicating fibrotic proliferation of residual LECs (a, c).

[0049] With the PHEMA-copolymer membrane-serine-ADM IOL, the posterior capsule remained transparent at 2 months, with minimal fibrotic proliferation on the posterior capsule.

[0050] In summary, the embodiments of the present invention provide a PHEMA-copolymer film-serine-ADM intraocular lens, which can simultaneously provide a physical barrier and a biological inhibitory effect, prevent the adhesion of LECs, block the DNA replication and transcription process to induce cell apoptosis, block the migration and proliferation of lens epithelial cells LECs, and inhibit the development of PCO within 2 months without affecting normal eye tissues, showing excellent ocular biocompatibility.

[0051] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modification, equivalent substitution or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A modified intraocular lens, characterized in that: PHEMA-copolymer membrane-serine polymer intraocular lens was used as a carrier and doxorubicin was loaded on the surface.

2. The modified intraocular lens according to claim 1, characterized in that: The preparation method of the PHEMA-copolymer film-serine polymer intraocular lens is as follows: adding the PHEMA-copolymer film intraocular lens to an ethanol aqueous solution of serine, placing it in a constant temperature shaking incubator at 50-60°C for reaction for at least 24 hours to obtain the PHEMA-copolymer film-serine polymer intraocular lens, and then washing it with distilled water until the solution is neutral.

3. The modified intraocular lens according to claim 2, characterized in that: In the ethanol aqueous solution of serine, the ethanol concentration is 25%-30% v / v, the molar concentration of serine is 1-3 mol / L, and the pH is 10-11.

4. The modified intraocular lens according to claim 2, characterized in that: The preparation method of the PHEMA-copolymer film intraocular lens is as follows: placing the PHEMA intraocular lens in a modified solution, irradiating it with a UV lamp for at least 1 hour, and then soaking it in an ethanol / water solution with a volume ratio of (1-2):3 at 60-70°C for at least 3 hours to obtain the PHEMA-copolymer film intraocular lens.

5. The modified intraocular lens according to claim 4, characterized in that: The modified solution is a mixed solution of glycidyl methacrylate, crosslinking agent triethylene glycol dimethacrylate and photoinitiator 2-hydroxy-2-methylpropiophenone in a mass ratio of 15:(0.5-1):(0.5-1).

6. The method for preparing the modified intraocular lens according to any one of claims 1 to 5, characterized in that: The method comprises the following steps: immersing the PHEMA-copolymer film-serine polymer intraocular lens in a 10-15ug / mL doxorubicin aqueous solution, stirring at a low speed for at least 12 hours at 20-60°C, taking it out, washing it with distilled water for at least 6 times, and irradiating it with ultraviolet light for at least 15 minutes.

7. The method for preparing the modified intraocular lens material according to claim 6, characterized in that: The low speed stirring is 100-150 rpm.

8. Use of the modified intraocular lens according to any one of claims 1 to 5 or the modified intraocular lens obtained by the preparation method according to any one of claims 6 to 7 in the preparation of an intraocular lens for preventing after-cataract.

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