Nano ophthalmic gel preparation for treating corneal epithelium injury

Abstract

The invention discloses a nano ophthalmic gel preparation for treating corneal epithelium injury. The main component of the nano ophthalmic gel preparation is PIP nano gel; the preparation method of the PIP nanogel comprises the following steps: preparing cystamine dihydrochloride modified carboxymethyl cellulose from cystamine dihydrochloride modified carboxymethyl cellulose, taking collagen as an auxiliary material, loading a drug component PIP under the action of electrostatic self-assembly, and synthesizing the PIP nanogel. The hydrated particle size of the synthesized PIP nanogel is 50 to 500 nm. The ball-like PIP nanogel with good dispersion is proposed and successfully prepared for the first time, the blank that no suitable PIP delivery system exists in the prior art is filled, and a new technical path is provided for in-vivo application of PIP; the prepared nano ophthalmic gel preparation can prolong the PIP eye residence time to 12 hours or more, so that the cornea repair speed is increased by 50% or more compared with that of a free PIP group, and wound closure is accelerated.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a nano-ophthalmic gel formulation for treating corneal epithelial damage, the active ingredient of which is Prolactin-Induced Protein (PIP). Background Technology

[0002] Corneal epithelial injury is a common ophthalmic disease. Corneal wound healing is a complex process involving the interaction of multiple cells and molecules. Traditional treatments such as artificial tears and antibiotic eye drops have limited effectiveness and cannot address corneal scarring caused by fibrosis. While growth factors (such as EGF) and anti-inflammatory drugs (such as corticosteroids) have some effect, they suffer from drawbacks such as a single mechanism of action and significant side effects. The core function of growth factors (such as EGF) is to promote epithelial cell proliferation and migration, but it has no direct regulatory effect on inflammation or stromal remodeling. Long-term use may lead to excessive corneal epithelial proliferation, affecting vision. The core function of anti-inflammatory drugs (such as corticosteroids) is potent anti-inflammatory action, but they have no positive regulatory effect on epithelial proliferation or stromal repair; they also strongly suppress systemic and local immunity, and long-term use increases the risk of corneal infection and can lead to increased intraocular pressure.

[0003] Prolactin-induced protein (PIP) is a 17 kDa glycoprotein, initially identified as GCDFP-15 in macrocystic mastitis fluid, and is a major component of breast milk, mammary cyst fluid, and saliva. This 146-amino acid polypeptide is found in mammary glands, salivary glands, lacrimal glands, and prostate tissue; recent studies have also revealed its distribution in the tear film and cornea. PIP possesses aspartic protease activity and, as a potentially bioactive protein, plays an important role in corneal wound healing. In the early stages of trauma, PIP can promote epithelial cell proliferation and migration by activating the PI3K-AKT / mTOR pathway, accelerating wound closure. In the mid-repair stage, it can precisely regulate excessive inflammatory responses by inhibiting NF-κB pathway activity and inducing macrophage polarization towards the M2 repair phenotype. In the late stage of repair, it can guide orderly remodeling of matrix collagen by downregulating the TGF-β1 / Smad3 pathway and upregulating the expression of matrix metalloproteinases (such as MMP-2 and MMP-9). This full-cycle regulatory model enables PIP to simultaneously promote the "high efficiency" (shortening healing time) and "functionality" (maintaining corneal transparency and epithelial barrier integrity after repair) of corneal repair, effectively compensating for the technical shortcomings of traditional intervention methods.

[0004] Despite the clear advantages of PIP in corneal repair, its clinical translation remains limited by technological bottlenecks in drug delivery systems. Currently, gel-based drug delivery systems loaded with small molecule drugs are widely used in the medical field due to their advantages of slow drug release and long retention time on the ocular surface. However, gel-based drug delivery systems loaded with active proteins have seen little research and application. Traditional PIP administration methods primarily rely on simple dosage forms, often dissolving or suspending PIP directly in isotonic buffers (such as phosphate buffer) and administering it via ocular instillation. However, this approach suffers from two core problems: first, PIP proteins have poor stability in the ocular surface environment and are easily degraded and inactivated; second, the drug's retention time on the ocular surface is extremely short, preventing sustained action on the wound and resulting in very low bioavailability. In summary, the lack of a dedicated ocular gel delivery system capable of achieving stable loading, slow release, and long-lasting action of PIP proteins directly restricts the full realization of its repair efficacy. The development of slow-delivery gel products to achieve sustained action on the eye limits the clinical application of PIP. Summary of the Invention

[0005] The purpose of this invention is to provide a nano-ophthalmic gel formulation for treating corneal epithelial damage.

[0006] The technical solution to achieve the purpose of this invention is: a nano-ophthalmic gel formulation for treating corneal epithelial damage, the main component of which is PIP (mammary gland inducible protein) nanogel; the preparation method of the PIP nanogel is as follows: cystamine dihydrochloride modified carboxymethyl cellulose (CMC-SS) is prepared by modifying carboxymethyl cellulose with cystamine dihydrochloride, and collagen with a molecular weight of 3-30 kDa is used as an excipient to load the drug component PIP under electrostatic self-assembly to synthesize the PIP nanogel; the hydrated particle size of the synthesized PIP nanogel is 50-500 nm.

[0007] Existing research largely focuses on the expression patterns, signaling pathways, and biomarker value of PIP. Related experiments mostly involve the direct application of recombinant PIP to cells or animals, with few reports on efficient PIP delivery systems. This invention breaks through the traditional method of direct PIP use, proposing and successfully preparing a well-dispersed, spherical PIP nanogel for the first time. This fills the gap in the current technology for suitable PIP delivery systems, providing a new technical pathway for the in vivo application of PIP. Furthermore, due to the poor stability and easy aggregation and inactivation of PIP protein, this patent uses cystamine dihydrochloride modified carboxymethyl cellulose as the carrier framework and collagen as the auxiliary component to load PIP protein through a mild electrostatic self-assembly method, which has multiple protective and stabilizing effects on PIP: First, collagen has good biocompatibility and protein affinity, and can form a mild interaction with PIP, reducing the self-aggregation between PIP molecules and improving its dispersion uniformity; Second, modified carboxymethyl cellulose binds to PIP through electrostatic interaction and forms a three-dimensional gel network, which can encapsulate PIP inside the gel and prevent it from being directly exposed to the external environment and causing structural instability; At the same time, the entire assembly process is under mild conditions and does not involve organic solvents, high temperature or strong shear, etc., which preserves the natural structure and bioactivity of PIP to the greatest extent; The final nanogel system can play a continuous stabilizing role for PIP. Furthermore, the hydrated particle size of the final product (PIP nanogel) is controlled within the range of 50–500 nm. Particles larger than 50 nm are not easily cleared by the aqueous humor / vitreous body, thus prolonging the intraocular retention time; particles smaller than 500 nm avoid vitreous opacity, visual interference, or local irritation caused by excessively large particle sizes. The gel matrix of the nano-ophthalmic gel formulation of this invention has good adhesion and moisturizing properties, which can extend the PIP retention time in the eye to more than 12 hours, improving bioavailability. This formulation can continuously promote corneal epithelial cell migration, increasing the corneal repair speed by more than 50% compared to the free PIP group, accelerating wound closure; at the same time, it continuously downregulates the expression of TGF-β1 and collagen III, effectively inhibiting scar formation.

[0008] Furthermore, the preparation method of the PIP nanogel includes the following steps: (1) Add collagen and PIP to deionized water, stir to dissolve them completely, and adjust the pH of the solution to 3-4 to obtain the first solution; (2) Add the modified CMC-SS to deionized water and dissolve it completely to obtain a second solution; (3) Continue stirring the second solution while slowly adding the first solution dropwise until it is evenly mixed to obtain a mixture; then, first adjust the pH of the mixture to 7.0±0.5, stir for 0.5 to 2 hours to carry out preliminary electrostatic assembly and pre-crosslinking; then adjust the pH to 4.5 to 6.0, raise the temperature to 30 to 60°C and keep it at that temperature for 0.5 to 2 hours to induce a gelation reaction to obtain a gel system; after the reaction is completed, allow it to cool naturally and adjust the pH of the gel system to 7.2±0.5 (simulating the normal pH of tears) to obtain a reaction solution; (4) Centrifuge the reaction solution obtained in step (3) and collect the supernatant; filter it through a filter membrane to obtain a uniformly dispersed transparent PIP gel nanoparticle suspension, i.e., PIP nanogel preparation.

[0009] In the above preparation method, the pH value of the solution in each step is adjusted by conventional acid-base addition.

[0010] This invention first dissolves collagen and PIP under acidic conditions (pH 3-4) to make the protein molecules positively charged and maintain good dispersibility, avoiding aggregation and precipitation. After fully dissolving negatively charged CMC-SS, it is mixed with the positively charged protein solution (i.e., the first solution) to obtain a mixture. The pH of the mixture is adjusted to 7.0±0.5. Under neutral conditions, the positive charges of collagen and PIP and the negative charges of CMC are strongly attracted by electrostatic attraction, and the initial assembly is completed through electrostatic interaction. The pH is then adjusted to 4.5-6.0. The slightly acidic environment helps the relevant groups in the system to protonate. With the aid of gentle heating, the molecular chain rearrangement and hydrogen bonding and electrostatic interaction are synergistically enhanced to form a three-dimensional gel network, resulting in a gel system. Finally, the pH of the gel system is adjusted to 7.2±0.5 to match the human physiological environment. After removing large particulate impurities by low-speed centrifugation and membrane filtration, a PIP nanogel with uniform particle size, good physiological stability, and can be directly used in the eye is obtained. Because PIP protein is unstable and prone to aggregation and inactivation, this invention achieves stable dissolution and uniform dispersion of PIP through stepwise pH control, avoiding protein aggregation and denaturation. Furthermore, by using modified carboxymethyl cellulose and collagen as a matrix, gel assembly is completed under mild conditions, which helps to protect the structural and functional integrity of PIP.

[0011] Furthermore, the filter membrane used in step (4) is a 0.22μm filter membrane, which is a standard sterilization filter membrane commonly used in the market.

[0012] Furthermore, the mass ratio of collagen, PIP and cystamine dihydrochloride modified carboxymethyl cellulose is controlled at 2000-8000:2-10:1000-3000 to ensure that the hydrated particle size of the PIP nanogel is 50-500 nm.

[0013] The cystamine dihydrochloride-modified carboxymethyl cellulose can be purchased or prepared in-house. For in-house preparation, the process is as follows: CMC (sodium carboxymethyl cellulose) is dissolved in water and stirred thoroughly to obtain a CMC solution with a mass fraction of 0.5–2%; then EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) and NHS (N-hydroxysuccinimide) are added and stirred for 0.5–5 hours, with the EDC to NHS ratio being 1:2–1:5; then SS (cystamine dihydrochloride) is added, with the final concentration of SS controlled at 50–500 mM, and stirred for 5–30 hours; finally, dialysis is performed using a dialysis bag with a molecular weight cutoff of 3–14 kDa. After dialysis, the retained solution in the bag is freeze-dried to obtain the cystamine dihydrochloride-modified carboxymethyl cellulose (CMC-SS). Carboxymethyl cellulose (CMC) is a water-soluble natural polysaccharide derivative of cellulose. Its molecular chain contains a large number of carboxyl groups (-COOH), but pure CMC has limitations such as low mechanical strength, difficult-to-control degradation rate, and lack of active binding sites. Cystamine dihydrochloride modification introduces functional groups such as disulfide bonds (-SS-) and amino groups (-NH2), significantly improving the structural stability of CMC and promoting the cross-linking of the linear CMC molecular chain with other molecules (the collagen used in this patent) to form a three-dimensional network structure. This cross-linked three-dimensional network structure can serve as a drug carrier, exhibiting good mechanical properties and structural stability, which helps improve drug stability and sustained release. Attached Figure Description

[0014] Figure 1 TEM images (scale bar: 500 nm) of the PIP nanogels prepared in Examples 1-3, where Figures A, B, and C correspond to Examples 1, 2, and 3, respectively; Figure 2 The in vitro release curve of the PIP nanogel formulation described in Example 1 is shown, where PIP and CCP correspond to the PIP group and CCP group, respectively. Figure 3 The PIP nanogel formulation described in Example 1 is shown to have a proliferative effect on human corneal fibroblasts (HCFS), where Control, PIP, and CCP correspond to the PBS group, PIP group, and CCP group, respectively. Figure 4 The scratch test results of the PIP nanogel formulation described in Example 1 on human corneal fibroblasts HCFS are shown, where Control, PIP, and CCP correspond to the PBS group, PIP group, and CCP group, respectively. Figure 5The effect of the PIP nanogel formulation described in Example 1 on wound healing in animal models was investigated. Control, PIP, and CCP corresponded to the PBS group, PIP group, and CCP group, respectively. Figure A shows representative fluorescence imaging images of animals in the three treatment groups (Control, PIP, and CCP) during the wound healing process, displaying the fluorescence distribution in the wound area at baseline, 24h, and 48h. Figure B shows the quantitative statistical analysis results of the wound healing rate of each group over time (Baseline, 24h, and 48h). Data are expressed as mean ± standard deviation (mean ± SD). ns indicates no significant difference, and ** indicates P < 0.01. Detailed Implementation

[0015] The embodiments of the nano-ophthalmic gel formulation for treating corneal epithelial damage of the present invention are described in detail below with reference to the accompanying drawings. Example 1

[0016] A nano-ophthalmic gel formulation for treating corneal epithelial damage is a PIP nanogel. The PIP nanogel is prepared by modifying carboxymethyl cellulose with cystamine dihydrochloride and using collagen with a molecular weight of 3-30 kDa as an excipient, loading the drug component PIP under electrostatic self-assembly to synthesize the PIP nanogel. The specific preparation steps are as follows: (1) Add collagen powder and PIP powder to deionized water, stir to dissolve them completely, and adjust the pH of the solution to 3-4 to obtain the first solution; (2) Add cystamine dihydrochloride modified carboxymethyl cellulose powder to deionized water and dissolve it completely to obtain a second solution; (3) Continue stirring the second solution while slowly adding the first solution dropwise, mix thoroughly to obtain a mixture, wherein the mass ratio of collagen, PIP, and cystamine dihydrochloride modified carboxymethyl cellulose is 2000:2:1000; then, first adjust the pH of the mixture to 7.0±0.5, stir for 1 h, and carry out preliminary electrostatic assembly and pre-crosslinking; then adjust the pH to 4.5~6.0, raise the temperature to 30~60℃ and keep it at that temperature for 1 h to induce gelation reaction and obtain a gel system; after the reaction is completed, cool naturally and adjust the pH of the gel system to 7.2±0.5 (simulating the normal pH of tears) to obtain the reaction solution; (4) Centrifuge the reaction solution obtained in step (3) and collect the supernatant; filter it through a 0.22 μm filter membrane to obtain a uniformly dispersed transparent PIP gel nanoparticle suspension, i.e., PIP nanogel preparation, and store it at 4℃; In the above preparation method, the pH value of the solution in each step is adjusted by conventional acid-base addition.

[0017] TEM characterization showed ( Figure 1 (Figure A) The PIP nanogel prepared in Example 1 has good dispersibility, is approximately spherical, and has an average particle size of about 72±21nm (scale bar: 500nm).

[0018] The preparation process of Examples 2 and 3 is the same as that of Example 1, except that the mass ratio of collagen, PIP, and cystamine dihydrochloride modified carboxymethyl cellulose, the stirring time of the mixture at pH 7.0±0.5, and the heat preservation reaction time at 30-60℃ are different, as shown in Table 1 below.

[0019] Table 1

[0020] From Table 1 and Figure 1 It is evident that when the mass ratio of collagen, PIP, and cystamine dihydrochloride modified carboxymethyl cellulose was controlled at 2000–8000:2–10:1000–3000 (Examples 1–3), PIP nanogel formulations were successfully prepared. The PIP nanogel particle sizes in Examples 1–3 were 72±21 nm, 136±43 nm, and 357±124 nm, respectively. The gel particle sizes of the three formulations were all within the ideal range of 50–500 nm. This range ensures that the formulations are not easily cleared by the aqueous humor / vitreous body, thereby prolonging the intraocular retention time and avoiding problems such as vitreous opacity, visual interference, or local irritation caused by excessively large particle sizes. The stirring time at pH 7.0±0.5 and the incubation reaction time at 30–60°C were both controlled within 0.5–2 hours, which met the process requirements while avoiding unnecessary time consumption.

[0021] The PIP nanogels prepared in Example 1 (CCP group) were used in an in vitro release experiment under artificial tear conditions (containing lysozyme 1.4 ± 0.5 mg / mL). Quantitative analysis was performed using a PIP (ELISA) kit, and the results were compared with free PIP (PIP group). The initial PIP concentration for both groups was uniformly set at 1.0 μg / mL. Figure 2 As shown, the cumulative release rate of PIP nanogel prepared in Example 1 was greater than 60% after 12 hours. Free PIP is easily decomposed by lysozyme and has poor stability. Therefore, PIP nanogel can significantly improve the stability of PIP. After 12 hours of in vitro treatment, the concentration of active protein in PIP nanogel was more than 50% higher than that in free PIP nanogel.

[0022] Human corneal fibroblasts (HCFS) were treated with PIP nanogel (CCP group) prepared in Example 1 and free PIP (PIP group) for 24 h. The initial PIP concentration in both groups was uniformly 0.5 μg / mL. The cell viability of PIP nanogel was 146.8 ± 9.2%, which was significantly higher than that of free PIP group (115.3 ± 11.4%), indicating that PIP nanogel has higher proliferative activity than free PIP (e.g., PIP nanogel). Figure 3 (As shown).

[0023] At the same time, such as Figure 4 As shown, the cell scratch assay showed that the PIP nanogel prepared in Example 1 (CCP group) had a greater effect on promoting the migration of human corneal fibroblasts (HCFS) than free PIP (PIP group).

[0024] In addition, the applicant has also conducted animal experiments to verify the nano-ophthalmic gel formulation of Example 1: (1) A mouse corneal epithelial injury model was established and randomly divided into PBS group (PBS group, also known as blank control group), PIP solution group (PIP group), and PIP nanogel group (CCP group). (2) The administration regimen was once daily. At baseline, corneal fluorescein staining was used for assessment. The corneal epithelial healing was evaluated as the percentage of fluorescein-stained area to the total corneal area. The sample size for each group was n=4. Results are as follows: Figure 5 As shown. The results show: Figure 5As shown, at baseline, the percentages of corneal epithelial staining area in the PBS group, PIP group, and CCP group were 93.75±2.98%, 96.0±2.94%, and 94.5±1.91%, respectively. Statistical analysis showed no significant difference among the three groups (p=0.504). Corneal fluorescein sodium staining was performed at 48 and 72 hours post-administration to assess ocular surface repair. The results showed that at 24 hours post-administration, the differences among the three groups were statistically significant (p=0.001). Intergroup comparisons showed that the fluorescein staining area in both the PIP group (staining area percentage 21.36% ± 1.74%) and the CCP group (staining area percentage 16.54% ± 1.68%) was significantly less than that in the PBS group (staining area percentage 25.78% ± 2.82%) (p = 0.001 for CCP group vs. PBS group; p = 0.011 for PIP group vs. PBS group), and the corneal fluorescein staining area in the CCP group was significantly less than that in the PIP group (p = 0.017). After 48 hours of drug administration, the differences among the three groups were statistically significant (p = 0.001). Intergroup comparisons showed that the fluorescein staining area in both the PIP group (4.64%±1.98%) and the CCP group (3.41±1.90%) was significantly reduced compared to the control group (9.94%±2.27%) (CCP group vs. PBS group: p=0.002; PIP group vs. PBS group: p=0.005), while there was no statistically significant difference in corneal fluorescein staining area between the PIP and CCP groups (p=0.424). These results indicate that both the PIP and CCP groups significantly enhance corneal epithelial healing capacity in mice, and the CCP group demonstrates a stronger ability to promote corneal epithelial regeneration than the PIP group in the early stages of epithelial injury.

[0025] The cystamine dihydrochloride-modified carboxymethyl cellulose can be purchased or made in-house. The cystamine dihydrochloride-modified carboxymethyl cellulose used in the above embodiments of the present invention is a self-made product. Its manufacturing process is as follows: CMC (sodium carboxymethyl cellulose) was dissolved in water and stirred thoroughly to obtain a CMC solution with a mass fraction of 0.5–2%. Then, EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) and NHS (N-hydroxysuccinimide) were added and stirred for 0.5–5 hours, with the EDC to NHS ratio being 1:2–1:5. Next, SS (cystamine dihydrochloride) was added, with the final concentration of SS controlled at 50–500 mM, and stirred for 5–30 hours. Finally, dialysis was performed using a dialysis bag with a molecular weight cutoff of 3–14 kDa. After dialysis, the retained solution was collected and freeze-dried to obtain the cystamine dihydrochloride-modified carboxymethyl cellulose (CMC-SS). The method for preparing cystamine dihydrochloride-modified carboxymethyl cellulose is existing technology and will not be described in detail here.

[0026] The above description is merely an embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent transformations made based on the content of the present invention specification, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. A nano-ophthalmic gel formulation for treating corneal epithelial damage, characterized in that: Its main component is PIP nanogel; the preparation method of the PIP nanogel is as follows: cystamine dihydrochloride modified carboxymethyl cellulose is obtained by modifying carboxymethyl cellulose with cystamine dihydrochloride, and collagen with a molecular weight of 3 to 30 kDa is used as an excipient to load the drug component PIP under electrostatic self-assembly to synthesize the PIP nanogel; the hydrated particle size of the synthesized PIP nanogel is 50 to 500 nm.

2. The nano-ophthalmic gel formulation for treating corneal epithelial damage according to claim 1, characterized in that, The preparation method of the PIP nanogel includes the following steps: (1) Add collagen and PIP to deionized water, stir to dissolve them completely, and adjust the pH of the solution to 3-4 to obtain the first solution; (2) The modified cystamine dihydrochloride modified carboxymethyl cellulose was added to deionized water and dissolved completely to obtain a second solution; (3) Continue stirring the second solution while slowly adding the first solution dropwise until it is homogeneous and a mixture is obtained. Then, adjust the pH of the mixture to 7.0±0.5 and stir for 0.5 to 2 hours to perform preliminary electrostatic assembly and pre-crosslinking. Then, adjust the pH to 4.5 to 6.0, raise the temperature to 30 to 60°C and keep it at that temperature for 0.5 to 2 hours to induce a gelation reaction and obtain a gel system. After the reaction is completed, allow it to cool naturally and adjust the pH of the gel system to 7.2±0.5 to obtain a reaction solution. (4) Centrifuge the reaction solution obtained in step (3) and collect the supernatant; filter it through a filter membrane to obtain a uniformly dispersed transparent PIP gel nanoparticle suspension, i.e., PIP nanogel preparation.

3. The nano-ophthalmic gel formulation for treating corneal epithelial damage according to claim 2, characterized in that: The filter membrane used in step (4) of the preparation method of the PIP nanogel is a 0.22 μm filter membrane.

4. The nano-ophthalmic gel formulation for treating corneal epithelial damage according to claim 2, characterized in that: In the preparation method of the PIP nanogel, the mass ratio of collagen, PIP and cystamine dihydrochloride modified carboxymethyl cellulose is controlled at 2000-8000:2-10:1000-3000.

5. The nano-ophthalmic gel formulation for treating corneal epithelial damage according to claim 1, characterized in that, The preparation process of the cystamine dihydrochloride modified carboxymethyl cellulose is as follows: sodium carboxymethyl cellulose is dissolved in water and stirred thoroughly to obtain a sodium carboxymethyl cellulose solution with a mass fraction of 0.5-2%; then EDC and NHS are added and stirred for 0.5-5 hours, with the addition ratio of EDC to NHS being 1:2-1:5; then cystamine dihydrochloride is added, with the final concentration of cystamine dihydrochloride controlled at 50-500 mM, and stirred for 5-30 hours; finally, dialysis is performed using a dialysis bag with a molecular weight cutoff range of 3-14 kDa, and after dialysis, the retained solution in the bag is taken and freeze-dried to obtain the cystamine dihydrochloride modified carboxymethyl cellulose.

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