A lyophilized preparation of a metalloflavin complex nanomaterial, a dispersion method and a dispersed preparation

Abstract

This invention belongs to the field of ophthalmic medication technology, specifically relating to a lyophilized formulation, dispersion method, and dispersion formulation of a metalloriboflavin composite nanomaterial. This invention discovers the -HPO4 group on riboflavin sodium phosphate. ‑ Metalloriboflavin composite nanomaterials obtained by coordinating metal ions on the group exhibit excellent transepithelial riboflavin delivery capabilities. Their transepithelial corneal crosslinking effect is comparable to that of classic corneal crosslinking schemes involving epithelial ablation. Further screening of the effects of preservation methods and dispersants on their transepithelial riboflavin delivery capabilities revealed that materials prepared by lyophilization and those dispersed in deionized water both retained the material's excellent transepithelial riboflavin delivery capabilities, comparable to the crosslinking effects of clinical epithelial corneal ablation. This has significant research implications for the long-term preservation, industrial production, and even future clinical applications of metalloriboflavin composite nanomaterials.

Description

Technical Field

[0001] This invention belongs to the field of ophthalmic medication technology, specifically relating to a freeze-dried formulation of a metalloriboflavin composite nanomaterial, a dispersion method, and a dispersion formulation. Background Technology

[0002] Blinding keratoconus is a common degenerative non-inflammatory corneal disease. Riboflavin (Rf) combined with ultraviolet A (UVA) corneal cross-linking (CXL) is currently the internationally recognized primary treatment for pathologically halting and delaying the progression of keratoconus. It can improve the biomechanics and stromal instability of keratoconus, thus avoiding the need for corneal transplantation by delaying or even halting its progression and alleviating the clinical shortage of corneal donors. Furthermore, corneal cross-linking can also be used to treat various corneal diseases, such as keratoconus, infectious keratitis, corneal ulcers, and bullous keratopathy. To date, the main treatment for keratoconus uses the classic corneal cross-linking protocol (SCXL), also known as the Dresden protocol, which requires mechanical scraping of the lipophilic corneal epithelium to allow for effective penetration of hydrophilic riboflavin into the hydrophilic stroma. Although the treatment effect is relatively stable, the removal of corneal epithelium can cause common problems such as long-term postoperative pain and slow vision recovery. It can also cause a variety of serious complications such as corneal infection, edema, scarring, corneal perforation, and even blindness.

[0003] Therefore, epithelial-preserving corneal crosslinking (T-CXL) has emerged. It avoids many problems associated with epithelial-de-epithelial corneal crosslinking and boasts advantages such as lower postoperative infection risk, fewer complications, broader inclusion criteria, and higher patient comfort, making it a current research hotspot. However, the clinical efficacy of T-CXL remains controversial, with 30-50% of patients experiencing disease progression post-surgery, primarily due to insufficient riboflavin content in the stroma during the crosslinking process. How to increase the riboflavin content in the stroma while preserving the intact corneal epithelium, ensuring sufficient riboflavin enters the stroma to achieve corneal crosslinking effects comparable to classic methods, is an international research hotspot and a crucial problem urgently needing resolution. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings and deficiencies of the existing technology and to provide a freeze-dried formulation, dispersion method and dispersion formulation of metal riboflavin composite nanomaterials.

[0005] The technical solution adopted in this invention is as follows: a freeze-dried formulation of a metal riboflavin composite nanomaterial, which is obtained by freeze-drying a metal riboflavin composite nanomaterial obtained by coordinating and modifying metal ions on the -HPO4- group of sodium riboflavin phosphate.

[0006] The preparation method of the metal riboflavin composite nanomaterial includes the following steps: adding metal ions to a riboflavin sodium phosphate solution and reacting to obtain metal riboflavin nanomaterials; directly lyophilizing the prepared metal riboflavin nanomaterials to prepare lyophilized formulations.

[0007] The solvent for the riboflavin sodium phosphate solution is methanol and / or ethanol. After adding metal ions to the riboflavin sodium phosphate solution, a precipitate is formed. The precipitate is then washed to obtain the metal riboflavin nanomaterial.

[0008] The reaction time after adding metal ions to the riboflavin sodium phosphate solution is 4-24 h.

[0009] The dispersion method of the lyophilized formulation of the metalloriboflavin composite nanomaterials as described above uses deionized water for dispersion.

[0010] A dispersion formulation of a metalloriboflavin composite nanomaterial is prepared by dispersing the lyophilized metalloriboflavin composite nanomaterial as described above in deionized water.

[0011] The beneficial effects of this invention are as follows: Metalloriboflavin composite nanomaterials obtained by coordinating and modifying metal ions on the -HPO4- group of riboflavin sodium phosphate exhibit excellent transepithelial riboflavin delivery capabilities. Their transepithelial corneal crosslinking effect is comparable to that of classic corneal crosslinking schemes in epithelial ablation. Further screening of the effects of preservation methods and dispersant selection on their transepithelial riboflavin delivery capabilities revealed that materials prepared by lyophilization and those dispersed with deionized water both retained the material's excellent transepithelial riboflavin delivery capabilities, comparable to the crosslinking effect of clinical epithelial corneal ablation. This has significant research implications for the long-term preservation, industrial production, and even later clinical application of metalloriboflavin composite nanomaterials. Attached Figure Description

[0012] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, obtaining other drawings based on these drawings without creative effort still falls within the scope of the present invention.

[0013] Figure 1 Slit-lamp photograph of the cornea after 30 min of infiltration of nRfpZn nanomaterials into a living cornea;

[0014] Figure 2 An experiment on the anti-enzyme dissolution of cornea after cross-linking of nRfpZn nanomaterials across the epithelial cornea;

[0015] Figure 3 Slit lamp photographs of the nRfpZn composite nanomaterials prepared in this invention at different times after being preserved in different ways.

[0016] Figure 4 These are confocal images taken at different times after the nRfpZn composite nanomaterials prepared in this invention were preserved using different methods.

[0017] Figure 5 Slit lamp photographs of the nRfpFe composite nanomaterials prepared in this invention at different times after being preserved in different ways.

[0018] Figure 6 These are confocal images taken at different times after the nRfpFe composite nanomaterials prepared in this invention were preserved using different methods.

[0019] Figure 7 The image shows an eye after the freeze-dried powder of RfpZn and nRfpFe composite nanomaterials prepared in this invention has been dispersed in different dispersants and then immersed in the cornea for 30 minutes.

[0020] Figure 8 Slit-lamp images of the cornea after immersion for 30 minutes following dispersion of the freeze-dried RfpZn and nRfpFe composite nanomaterials prepared in this invention using different dispersants. Detailed Implementation

[0021] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings.

[0022] This invention provides a freeze-dried formulation of a metalloriboflavin composite nanomaterial, which is prepared by freeze-drying a metalloriboflavin composite nanomaterial obtained by coordinating and modifying metal ions on the -HPO4- group of sodium riboflavin phosphate. The chemical formula of sodium riboflavin phosphate is as follows:

[0023] ,

[0024] The chemical formula of sodium riboflavin phosphate shows that it possesses a hydrophilic and negatively charged -HPO4- group, which can act as a ligand to complex with metal ions to form complexes. In this invention, the metal ion (Mn+) does not refer to all metal ions, but rather to all metal ions in the periodic table that can coordinate with Rfp molecules. Those skilled in the art can select suitable metal ions based on well-known theories of coordination chemistry or through a limited number of experiments. In some embodiments of this invention, the metal ions (Mn+) that can coordinate with Rfp molecules include at least Bi3+, Zn2+, Zr4+, Fe3+, Fe2+, Mn2+, Ce3+ (other rare earth ions Ln3+), Ca2+, Mg2+, Cu2+, Al3+, and Mo5+, and the metalloriboflavin composite nanomaterials prepared using the above metal ions all exhibit excellent transepithelial riboflavin delivery capabilities.

[0025] In some embodiments of the present invention, the preparation method includes the following steps: the preparation method of the metal riboflavin composite nanomaterial includes the following steps: adding metal ions to a riboflavin sodium phosphate solution and reacting to obtain metal riboflavin nanomaterials; the prepared metal riboflavin nanomaterials are directly freeze-dried to prepare freeze-dried formulations.

[0026] In some embodiments of the present invention, the preparation method includes the following steps: the solvent of the riboflavin sodium phosphate solution is methanol and / or ethanol, Rfp molecules can be dissolved in methanol and ethanol, and the reaction product nRfpM obtained by modifying the -HPO4- group with metal ions (Mn+) will precipitate out. The lipophilicity of the precipitate is greatly improved by the coordination of Mn+ with the -HPO4- group that improves the hydrophilicity of Rfp molecules, thereby improving the transepithelial delivery ability of the product nRfpM nanomaterial, and greatly improving the transepithelial corneal crosslinking effect of the obtained product.

[0027] To select a suitable preservation method, this invention employs the following approach: The obtained aqueous solution of the metalloriboflavin composite nanomaterial is divided into three portions. One portion is stored at room temperature, one portion is stored at 4 degrees Celsius, and the remaining portion is freeze-dried into powder and then dispersed in deionized water to prepare a dispersion of equal concentration before use. The transepithelial delivery capacity of the material is evaluated after different storage times. The evaluation confirms that, compared to room temperature storage and 4-degree Celsius storage, the material freeze-dried into powder and then dispersed in deionized water exhibits the strongest transepithelial delivery capacity of riboflavin.

[0028] Suitable reagents were selected for dispersing the material using the following method: The lyophilized metalloriboflavin composite nanomaterial powder, divided into three equal portions, was dispersed in three solutions: one portion in an aqueous solution, one portion in a PBS solution, and one portion in physiological saline. The concentration of the metalloriboflavin composite nanomaterial was the same in all solutions. The transepithelial delivery capability of the material for riboflavin was then evaluated.

[0029] Example 1:

[0030] A methanol solution of Zn(NO3)2 and Rfp was rapidly added together and stirred thoroughly at room temperature for 2–8 h, then allowed to stand overnight. After centrifugation, washing, and sieving, an aqueous dispersion of the nRfpZn composite nanomaterial was obtained. A corneal infiltration experiment was performed on day 0, and the transepithelial delivery capability of the material was evaluated using a slit lamp and confocal microscopy. The remaining solution was divided into three equal portions: one portion was stored at room temperature, one portion was stored at 4°C, and one portion was lyophilized into powder and dispersed in deionized water to prepare an aqueous dispersion of the same concentration before use. Corneal infiltration experiments were performed on days 3, 5, and 7, and the transepithelial delivery capability of the material for riboflavin was evaluated using a slit lamp and confocal microscopy.

[0031] Example 2:

[0032] The zinc salt (Zn(NO3)2) was replaced by FeCl3 as the raw material, and nRfpFe composite nanomaterials were prepared using the same method. The same treatments and tests as in Example 1 were then performed.

[0033] Example 3:

[0034] A methanol solution of Zn(NO3)2 and Rfp was rapidly added together and stirred thoroughly at room temperature for 2-8 hours, then allowed to stand overnight. After centrifugation, washing, and sieving, an aqueous dispersion of the nRfpZn composite nanomaterial was obtained. This dispersion was lyophilized into powder, divided into three equal portions, and centrifuged to obtain precipitates. One portion was dispersed in deionized water, one in PBS solution, and one in physiological saline. Subsequently, corneal infiltration experiments were performed, and the infiltrated rabbit eyes were photographed using both physical and slit-lamp methods.

[0035] Example 4:

[0036] By replacing FeCl3 with zinc salt (Zn(NO3)2), the same method was used to prepare lyophilized powder of nRfpFe composite nanomaterials. The powder was then grouped and dispersed in different solutions for corneal infiltration and evaluation.

[0037] Figure 1 Slit-lamp images of the cornea after 30 min of in vivo immersion in nRfpFe3+ nanomaterials. For the same immersion time, the cornea of ​​the nRfpM nanomaterial group was more yellow than that of the positive experimental group, indicating a higher riboflavin content.

[0038] Figure 2 The study investigated the corneal anti-enzyme dissolution experiment after cross-linking of nRfpFe3+ nanomaterials across the epithelium and cornea. Compared with the negative control group and the positive control group, after 48 hours, the remaining corneal area and dry weight in the nRfpFe3+ nanomaterial group were higher than those in the positive control group. The negative control group completely dissolved after 8 hours. This indicates that the nRfpFe3+ nanomaterial group has a corneal cross-linking effect comparable to that of the positive experimental group under the premise of cross-epithelial drug delivery.

[0039] Figure 3 , Figure 4 This study examines the effects of different preservation methods and storage times on the transepithelial delivery capacity of nRfpZn composite nanomaterials. Regardless of slit-lamp images (…), Figure 3 ) or confocal fluorescence image ( Figure 4 All studies found that, compared to room temperature and 4-degree refrigerator storage, lyophilized powder samples exhibited good transepithelial riboflavin delivery ability when redispersed in aqueous solution before use. The riboflavin content in the corneal stroma was higher than that in the room temperature and 4-degree refrigerator samples at the same time, and was comparable to that in the fresh solution group on day 0. This indicates that lyophilized samples have a better ability to deliver riboflavin quickly through the epithelium, and the preservation effect is minimally affected by time.

[0040] Figure 5 , Figure 6 This study examines the effects of different preservation methods and storage times on the transepithelial delivery of riboflavin in nRfpFe composite nanomaterials. Regardless of the slit-lamp photograph (… Figure 5 ) or confocal fluorescence image ( Figure 6 All studies found that, compared with room temperature storage and 4-degree refrigerator storage, the freeze-dried nRfpFe composite nanomaterials exhibited good transepithelial riboflavin delivery ability when redispersed in aqueous solution before use. The riboflavin content in the corneal stroma was higher than that in the room temperature and 4-degree sample groups at the same time, and was comparable to that in the fresh solution group on day 0, indicating that the freeze-dried sample has a better ability to deliver riboflavin quickly to the epithelium.

[0041] Figure 7 Photos of corneal samples after lyophilized nRfpZn and nRfpFe composite nanomaterials were dispersed in different solutions. Compared with the PBS control group, the eye with the composite nanomaterials dispersed in deionized water was the yellowest, indicating that the corneal riboflavin content was the highest. The sample dispersed in physiological saline had the worst wetting effect, close to the PBS control group.

[0042] Figure 8Slit-lamp images of the cornea after lyophilized powders of nRfpZn and nRfpFe composite nanomaterials were dispersed in different solutions. Compared with the composite nanomaterials dispersed in PBS and saline, the composite nanomaterials dispersed in deionized water showed bright yellow corneal bands, indicating that the composite nanomaterials dispersed in water have a better ability to deliver riboflavin across the epithelium.

[0043] The above description discloses only preferred embodiments of the present invention and should not be construed as limiting the scope of the present invention. Therefore, equivalent variations made in accordance with the claims of the present invention are still within the scope of the present invention.

Claims

1. The application of a lyophilized formulation of a metalloriboflavin composite nanomaterial in the preparation of a formulation that promotes the cross-linking effect of riboflavin across the epithelial cornea, characterized in that: The lyophilized formulation of the metalloriboflavin composite nanomaterial is a -HPO4-on-riboflavin sodium phosphate. - Metal riboflavin composite nanomaterials obtained by coordination modification of metal ions on a functional group were prepared by freeze-drying. The metal ion is Bi. 3+ Zn 2+ Zr 4+ Fe 3+ Fe 2+ Mn 2+ Ce 3+ Ca 2+ Mg 2+ Cu 2+ Al 3+ Mo 5+ One of them.

2. The application according to claim 1, characterized in that: The preparation method of the metal riboflavin composite nanomaterial includes the following steps: adding metal ions to a riboflavin sodium phosphate solution and reacting to obtain metal riboflavin nanomaterials; directly lyophilizing the prepared metal riboflavin nanomaterials to prepare lyophilized formulations.

3. The application according to claim 2, characterized in that: The solvent for the riboflavin sodium phosphate solution is methanol and / or ethanol. After adding metal ions to the riboflavin sodium phosphate solution, a precipitate is formed. The precipitate is then washed to obtain the metal riboflavin nanomaterial.

4. The application according to claim 2, characterized in that: The reaction time after adding metal ions to the riboflavin sodium phosphate solution is 4-24 h.

Images