Nucleic acid hydrogel formulation, and use thereof in retina reattachment

Abstract

A pH-stable nucleic acid hydrogel formulation, a preparation method therefor, and a use of the nucleic acid hydrogel in retina reattachment with indentation.

Description

A nucleic acid hydrogel preparation and its use for retinal repositioning Technical Field

[0001] The present invention relates to a nucleic acid hydrogel preparation with stable pH value, a preparation method thereof, and application of the nucleic acid hydrogel for top pressure repositioning of the retina. Background Art

[0002] The vitreous body is a transparent gel-like substance that fills the back of the eyeball. It is very important for maintaining the shape of the eyeball, supporting the retina, and conducting light. The main physiological functions of the vitreous body are to support the retina, eye refraction, cell barrier, and nutrition. However, some eye diseases, trauma, or age factors may cause problems with the vitreous body, requiring treatment or replacement. In clinical practice, pars plana vitrectomy or vitrectomy (PPV) is one of the effective means to treat serious eye diseases such as retinal detachment (RD), macular holes (MH), complications of diabetic retinopathy (DR), and posterior ocular trauma (OT).

[0003] Since PPV requires the removal of part of the vitreous body and the injection of special gases (such as sulfur hexafluoride and perfluoropropane) or silicone oil into the eyeball for filling during the operation, it is used to lift and press the detached retina, so that it can be repositioned and gradually return to normal.

[0004] Although gases offer excellent filling properties, their low refractive index can cause significant visual aberrations and a short retention time in the body. Silicone oil offers advantages such as good transparency, a refractive index close to that of natural vitreous, and high viscosity. However, silicone oil readily emulsifies in the intraocular environment, potentially causing a series of postoperative complications, limiting its clinical use. Over the past few decades, synthetic hydrogels have gained increasing attention due to their high water content, good stability, and controllable mechanical properties. However, traditional chemically cross-linked hydrogels can disrupt their network structure during injection, leading to decreased mechanical properties and uneven distribution within the vitreous cavity. Furthermore, a safe method is needed to decompose traditional synthetic hydrogels and remove them after the patient recovers. Compared to traditional chemically cross-linked hydrogels, supramolecular hydrogels possess unique shear-thinning and self-healing properties, enabling convenient direct injection and secondary removal during minimally invasive ophthalmic surgery. However, as vitreous replacement materials, supramolecular hydrogels exhibit poor mechanical strength and stability under dilute conditions.

[0005] Although various hydrogels have been shown to have potential as vitreous replacement materials, it remains a challenge to develop a more balanced vitreous replacement material that must possess reliable safety, long-term stability, good mechanical properties, and injectability.

[0006] References:

[0007] Yu Jin,Yujie Li,Sijia Song,Yuqiao Ding,Yuanchen Dong,Yao Lu,Dongsheng Liu,and Chun Zhang.,DNA Supramolecular Hydrogel as a Biocompatible Artificial Vitreous Substitute. Adv. Mater. Interfaces 2021,2101321. Summary of the Invention

[0008] Based on the excellent biocompatibility of DNA hydrogels in the eye, the inventors of the present invention have discovered that a nucleic acid hydrogel formed by a nucleic acid hydrogel preparation composed of a nucleic acid hydrogel with a certain solid content, a buffer, and an optional pH regulator has physical and chemical properties similar to those of natural vitreous. After PPV surgery for patients with retinal detachment, injection of a nucleic acid hydrogel preparation can provide sufficient pressure and support for the detached retina, thereby maintaining the shape of the eyeball. The inventors of the present invention have discovered that in the process of adjusting the nucleic acid hydrogel preparation, the purity of the nucleic acid single chain, the system pH, the gelation temperature, and different solid contents are crucial to the preparation process of the hydrogel and the mechanical properties during filling. For example, by selecting a buffer and / or adjusting the pH value, a nucleic acid hydrogel with a long-term stable pH value can be obtained. By selecting an appropriate nucleic acid solid content, a nucleic acid hydrogel preparation suitable for injection and retinal reattachment can be obtained, and so on.

[0009] The present invention thus provides a nucleic acid hydrogel preparation, which is suitable for vitreous cavity filling after PPV surgery in patients with retinal detachment, and is an auxiliary treatment for retinal reattachment in patients with various retinal detachments.

[0010] In a first aspect, the present invention relates to a nucleic acid hydrogel preparation suitable for retinal reattachment, wherein the nucleic acid hydrogel preparation comprises

[0011] A scaffold unit comprising a scaffold core and at least three single-stranded nucleic acids bound to the scaffold core, each single-stranded nucleic acid having at least one scaffold sticky end;

[0012] a cross-linking unit comprising a cross-linking core and at least two single-stranded nucleic acids bound to the cross-linking core, each single-stranded nucleic acid having at least one cross-linked sticky end; and

[0013] The scaffold unit and the cross-linking unit are cross-linked by base complementary pairing between the scaffold sticky end and the cross-linking sticky end to form a nucleic acid hydrogel with a three-dimensional spatial network structure;

[0014] buffer; and

[0015] Optional pH adjuster.

[0016] In a second aspect, the present invention relates to a method for preparing a nucleic acid hydrogel formulation, the method comprising:

[0017] a) preparing a scaffold unit assembly solution and a cross-linking unit assembly solution; and

[0018] b) assembling the prepared scaffold unit assembly solution and cross-linking unit assembly solution into a gel at a temperature higher than room temperature.

[0019] In embodiments of the first or second aspect, the scaffold unit comprises a scaffold core and at least three single-stranded nucleic acids bound to the scaffold core, each single-stranded nucleic acid having at least one scaffold sticky end. Preferably, the scaffold core is a nucleic acid, specifically a D-nucleic acid or an L-nucleic acid, more specifically a D-DNA or L-DNA. In embodiments of the first or second aspect, the nucleic acid serving as the scaffold core has a complementary pairing region, the length of which can be about 4 to about 150 bp, preferably about 5 to about 50 bp, more preferably about 6 to about 30 bp, and even more preferably about 8 to about 20 bp.

[0020] In embodiments of the first or second aspects, the crosslinking unit comprises a crosslinking core and at least two single-stranded L-deoxynucleic acids bound to the crosslinking core, each single-stranded L-deoxynucleic acid having at least one crosslinked sticky end. Preferably, the crosslinking core can be a deoxynucleic acid, specifically a D-nucleic acid or an L-nucleic acid, more specifically a D-DNA or L-DNA. In embodiments of the first or second aspects, the deoxynucleic acid serving as the crosslinking core has a complementary pairing region, and the length of the complementary pairing region can be about 4 to about 150 bp, preferably about 5 to about 50 bp, preferably about 15 to about 50 bp, more preferably about 6 to about 30 bp, and more preferably about 8 to about 20 bp.

[0021] In embodiments of the first or second aspects, the length of the scaffold sticky ends or crosslinked sticky ends is 4 nt or longer, which facilitates a stable crosslinked state under physiological conditions. Preferably, the length of the scaffold sticky ends or crosslinked sticky ends is about 150 nt or less, preferably about 50 nt or less, more preferably about 30 nt or less, more preferably about 30 to about 50 nt, and more preferably about 20 nt or less; more preferably, the length of the scaffold sticky ends or crosslinked sticky ends is about 4 to about 30 nt, about 4 to about 20 nt, and more preferably about 8 to about 12 nt.

[0022] In the embodiments of the first or second aspects, when the scaffold core is identical to the crosslinking core, the single-stranded nucleic acid bound to the scaffold core and the single-stranded nucleic acid bound to the crosslinking core are identical, for example, the same L-deoxynucleic acid; and the number of single-stranded nucleic acids bound to the scaffold core and the number of single-stranded nucleic acids bound to the crosslinking core are identical (both ≥ 3), the scaffold unit is identical to the crosslinking unit. Therefore, in the embodiments of the first or second aspects, the scaffold unit is identical to the crosslinking unit. In the embodiments of the first or second aspects, the scaffold unit is different from the crosslinking unit.

[0023] In an embodiment of the first aspect or the second aspect, the molar ratio of the scaffold unit to the cross-linking unit in the nucleic acid hydrogel is about 2:1 to about 1:3, preferably about 1:1 to about 1:2, and more preferably about 1:1.5.

[0024] In an embodiment of the first or second aspect, the scaffold unit and the crosslinking unit are crosslinked by base-pairing between the scaffold sticky ends and the crosslinking sticky ends, thereby forming a three-dimensional spatial network structure. Preferably, the scaffold unit, the crosslinking unit, and the three-dimensional spatial network structure are in a stable crosslinked state under physiological conditions (e.g., 37°C, pH 7.0-7.4, 0.9 wt% NaCl, isotonic).

[0025] In embodiments of the first or second aspects, the nucleic acid solid content in the nucleic acid hydrogel formulation is about 0.3-about 5 wt% relative to the weight of the nucleic acid hydrogel formulation. Preferably, the nucleic acid solid content is about 0.3-about 3.7 wt%; more preferably, about 1-about 3.5 wt%; more preferably, about 2-about 3.5 wt%; or about 3-about 3.5 wt%. Although nucleic acid hydrogels with higher solid content have higher storage moduli, intraocular injection becomes more difficult. Furthermore, higher solid content nucleic acid hydrogels allow for longer intraocular storage. However, longer intraocular storage is not necessarily better; generally, nucleic acid hydrogels require about 3-6 months of intraocular storage before degradation. Furthermore, higher solid content can cause the nucleic acid hydrogel to become opaque after injection, generating bubbles, which can affect refraction. Therefore, considering injection difficulty, degradation time, and refraction, a nucleic acid solid content of about 3-about 3.5 wt% is suitable and more preferred for retinal reattachment applications.

[0026] In the embodiments of the first or second aspects, the single-stranded nucleic acid has a purity of about 90% or greater, preferably about 95% or greater, for example, about 95%, about 96%, about 97%, about 98%, or about 99%. The single-stranded nucleic acid used in the present invention can be synthesized using standard phosphoramidite DNA solid-phase synthesis and separated and purified by reverse-phase high-performance liquid chromatography, and its purity can be characterized by LC-MS.

[0027] In the embodiments of the first aspect or the second aspect, the scaffold unit or cross-linking unit of the nucleic acid hydrogel may include a CpG sequence. The CpG sequence is a palindromic sequence with cytosine guanine dinucleotide (CpG) as the core, with two purines at the 5' end and two pyrimidines at the 3' end, i.e., 5'-PurPur-CG-PyrPyr-3'. The CpG sequence can be recognized by mammalian cells, thereby triggering a series of body defense mechanisms, including complement activation, phagocytosis, and expression of inflammatory cytokine genes. Currently known CpG sequences with strong immunostimulatory effects include, for example, 5'-TCCATGACGTTCCTGACGTT-3'.

[0028] In an embodiment of the first aspect or the second aspect, the sequence of the scaffold unit is 5'-CGATTGACTCTCCACGCTGTCCTAACCATGACCGTCGAAG-3', 5'-CGATTGACTCTCCTTCGACGGTCATGTACTAGATCAGAGG-3' and 5'-CGATTGACTCTCCCTCTGATCTAGTAGTTAGGACAGCGTG-3'.

[0029] In an embodiment of the first aspect or the second aspect, the sequences of the cross-linking units are 5'-GAGAGTCAATCGTCTATTCGCATGAGAATTCCATTCACCGTAAG-3' and 5'-GAGAGTCAATCGCTTACGGTGAATGGAATTCTCATGCGAATAGA-3'.

[0030] In the embodiments of the first or second aspect, the nucleic acid hydrogel has suitable mechanical strength, for example, its mechanical strength can be above about 0.1 Pa, preferably above about 1 Pa, more preferably above about 10 Pa, preferably below about 10000 Pa, more preferably below about 1000 Pa.

[0031] In an embodiment of the first aspect or the second aspect, the nucleic acid hydrogel has a storage modulus G' that dominates the loss modulus G" at all strain values ​​from about 0.1 to about 100 rad / s as measured by frequency sweep measurements at about 25°C and at a strain amplitude of about 1% strain. Preferably, at strain values ​​from about 0.1 to about 100 rad / s, the ratio of the storage modulus G' to the loss modulus G" is greater than about 2; more preferably greater than about 10. In some embodiments, the storage modulus (G') of the nucleic acid hydrogel is greater than or equal to about 500 Pa and not less than about 1500 Pa.

[0032] In the embodiments of the first or second aspect, the hydrogel of the present invention can have ideal stability, for example, it can maintain its structure stably in the presence of restriction endonucleases for about 24 hours, preferably about 36 hours, preferably about 48 hours, or longer.

[0033] In an embodiment of the first or second aspect, the buffer is PBS buffer 1, which includes about 0.20 g of KCl, about 8.00 g of NaCl, about 0.20 g of KH2PO4, and about 2.08 g of Na2HPO4·12H2O. In an embodiment of the first or second aspect, the buffer is PBS buffer 2, which includes about 0.20 g of KCl, about 8.00 g of NaCl, about 0.24 g of KH2PO4, about 3.63 g of Na2HPO4·12H2O, and a pH adjuster for adjusting the pH to about 7.4.

[0034] PBS buffer 1 is prepared by dissolving approximately 0.20 g of KCl, approximately 8.00 g of NaCl, approximately 0.20 g of KH2PO4, and approximately 2.08 g of Na2HPO4·12H2O in 1 L of water, followed by sterile filtration through a 0.22 μm filter. In one embodiment, PBS buffer 1 has a pH of approximately 7.4. In one embodiment, PBS buffer 1 has an osmotic pressure of approximately 277 mOsmol / kg.

[0035] PBS buffer 2 is prepared by adding approximately 0.20 g of KCl, approximately 8.00 g of NaCl, approximately 0.24 g of KH2PO4, and approximately 3.63 g of Na2HPO4·12H2O to approximately 900 mL of water, adjusting the pH to approximately 7.4 with approximately 1 mol / L of HCl, and then making up the volume to 1 L. The solution is then sterile-filtered through a 0.22 μm filter. In one embodiment, PBS buffer 2 has a pH of approximately 7.4. In one embodiment, PBS buffer 2 has an osmotic pressure of approximately 285 to approximately 310 mOsmol / kg. In one embodiment, PBS buffer 2 has an osmotic pressure of approximately 288 mOsmol / kg. In the present invention, the pH of PBS buffer 2 remains stable after one week.

[0036] PBS buffer 1 and PBS buffer 2 have slight differences in osmotic pressure and pH stability. The pH of buffer 1 fluctuates after one week, while the pH of PBS buffer 2 is stable after one week.

[0037] In the embodiments of the first or second aspects, the pH adjuster is an HCl solution or a NaOH solution. Preferably, the pH adjuster is an HCl solution or a NaOH solution having a pH of 7-7.4, and the concentration of the HCl solution or the NaOH solution is generally 0.01 to 1 mol / L, preferably 0.01 mol / L, to maintain a suitable pH and osmotic pressure.

[0038] In an embodiment of the second aspect, the method further comprises sterile filtering the prepared scaffold unit assembly solution and the cross-linking unit assembly solution before assembling into a gel. In one embodiment, the sterile filtration comprises passing the prepared scaffold unit assembly solution and the cross-linking unit assembly solution through one or more filter membranes, respectively. In one embodiment, the sterile filtration comprises passing the prepared scaffold unit assembly solution and the cross-linking unit assembly solution through one 0.45 μm and two 0.22 μm filter membranes, respectively. In one embodiment, the filter membrane can be any suitable polymer filter membrane or ceramic filter membrane.

[0039] In an embodiment of the second aspect, the assembly gel is carried out at 40-65° C., preferably 45-55° C. (i.e., the assembly gel temperature). The inventors of the present invention have found that gelation at temperatures above room temperature can quickly produce a clear and transparent hydrogel, while at room temperature or lower, the resulting hydrogel is turbid and contains bubbles.

[0040] In an embodiment of the second aspect, the assembly into a gel comprises heating and stirring the prepared scaffold unit assembly solution at the above-mentioned gelation temperature, and then adding the prepared cross-linking unit assembly solution, and keeping warm for a period of time, for example, about 0.5 to about 5 hours or about 1 to about 2 hours. In an embodiment of the second aspect, the assembly into a gel comprises heating and stirring the prepared scaffold unit assembly solution and the prepared cross-linking unit assembly solution at the above-mentioned gelation temperature at the same time, for example, about 0.5 to about 5 hours or about 1 to about 2 hours.

[0041] In an embodiment of the second aspect, the assembling into glue is performed under vacuum conditions.

[0042] In an embodiment of the second aspect, the method further comprises filling the formed hydrogel into a syringe at a suitable temperature after the gel is assembled. In an embodiment of the second aspect, the filling is performed at a temperature below the gelation temperature but above room temperature after the gel is stabilized. If the filling is performed at a temperature below room temperature, bubbles may form in the filled hydrogel. In an embodiment of the second aspect, the filling is performed at a temperature of about 35°C to about 40°C.

[0043] In embodiments of the first and second aspects, the nucleic acid hydrogel formulation has one or more of the following properties: 1) about 96 to about 99% water content; 2) a pH of about 7.0 to about 7.4; 3) about 1.033 to about 1.0090 g / cm 3 and 4) a refractive index of about 1.3300 to about 1.3395. In one embodiment, the nucleic acid hydrogel formulation has all of the above properties.

[0044] The third aspect relates to a method for treating an ophthalmic disease in a subject in need of treatment, the method comprising injecting a therapeutically effective amount of the nucleic acid hydrogel formulation of the present invention into the subject's eye; or relates to the use of the nucleic acid hydrogel of the present invention in the preparation of a medicament or agent for treating an ophthalmic disease.

[0045] In one embodiment, the nucleic acid hydrogel formulation is injected into the eye following vitrectomy.

[0046] In one embodiment, the ocular disease is retinal detachment, macular hole, complications of diabetic retinopathy, and posterior segment ocular trauma.

[0047] The nucleic acid hydrogel preparation of the present invention can form a hydrogel of suitable strength in the eye, which has one or more of the following advantages:

[0048] a) exhibit shear-thinning behavior, thus enabling safe injection into the eye;

[0049] b) Suitable mechanical strength and rapid recovery properties, so as to be able to be used for retinal top pressure repositioning and maintaining the shape of the eyeball;

[0050] c) physical properties similar to those of the human vitreous body, such as the aforementioned pH value, density, and refractive index;

[0051] d) Excellent biocompatibility;

[0052] e) ideal stability, for example, it can maintain its structure stably in the presence of restriction endonucleases for 48 hours, 4-8 weeks or longer, such as 3-6 months in ocular tissue filling;

[0053] f) In vivo stability and safety, such as no toxic or inflammatory response in the eye, and no complications such as vitreous opacity, hemorrhage, or retinal detachment.

[0054] Therefore, the nucleic acid hydrogel preparation of the present invention is used for injection filling of various retinal detachment patients after PPV surgery to press and support the retina, thereby assisting retinal reattachment. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] FIG1 shows the purity of the single-stranded nucleic acid of Example 1 measured by LC-MS.

[0056] FIG2 shows the measurement results of the storage modulus (G′) and loss modulus (G″) of nucleic acid hydrogel preparation 1.

[0057] FIG3 shows the transparency measurement results of nucleic acid hydrogel preparation 1.

[0058] FIG4 shows the purity of the single-stranded nucleic acid of Example 2 determined by LC-MS

[0059] FIG5 shows the measurement results of the storage modulus (G') and loss modulus (G") of nucleic acid hydrogel preparation 2.

[0060] FIG6 shows the measurement results of the storage modulus (G′) and loss modulus (G″) of nucleic acid hydrogel preparation 3.

[0061] FIG. 7 shows the transparency measurement results of nucleic acid hydrogel preparation 3.

[0062] FIG8 is a comparison of the appearance of gels formed at different temperatures.

[0063] Figure 9 Performance study of hydrogel preparations at different solid contents.

[0064] FIG10 shows an ultrasound image of a rabbit eye retinal detachment model successfully established.

[0065] FIG11 is a B-ultrasound image comparing the effects of nucleic acid hydrogel preparation and traditional silicone oil in a retinal detachment model. DETAILED DESCRIPTION

[0066] The technical terms mentioned in this specification have the same meanings as those generally understood by those skilled in the art. In case of any conflict, the definitions in this specification shall prevail.

[0067] As used in this application, "comprising" should be interpreted as indicating the presence of the stated features, integers, steps or components when mentioned, but does not exclude the presence or addition of one or more features, integers, steps or components or groups thereof. In addition, each of the terms "by", "comprising / comprises / comprised of", "including / includes / included", "involving / involves / involved" and "such as" are used in their open, non-limiting sense and are used interchangeably. In addition, the term "comprising" is intended to include instances and aspects encompassed by the terms "consisting essentially of" and "consisting of". Similarly, the term "consisting essentially of is intended to include instances encompassed by the term "consisting of.

[0068] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0069] As used herein, the term "effective amount" refers to an amount sufficient to achieve the desired modification of a physical property of a composition or material. For example, an "effective amount" of a monomer refers to an amount sufficient to achieve the desired improvement in a property regulated by a formulation component (e.g., a desired antioxidant release rate or viscoelasticity). The specific level in terms of wt% in the composition required as an effective amount will depend on a variety of factors, including the amount and type of monomer, the amount and type of polymer (e.g., acrylamide), the amount of antioxidant, and the desired release kinetics.

[0070] As used herein, the term "room temperature" refers to a temperature of about 25°C.

[0071] As used herein, the term "about" refers to the set consisting of all values ​​within the range of ±5% of the numerical value given thereafter.

[0072] As used herein, the terms "optional" or "optionally" mean that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

[0073] Example

[0074] Material:

[0075] 1. Single-stranded DNA: The DNA sequence was synthesized using a standard phosphoramidite DNA solid-phase synthesis method and purified by reverse-phase high-performance liquid chromatography. Its purity was characterized by LC-MS. The specific DNA sequence is shown in Table 1 below.

[0076] Table 1. DNA sequence information used to construct DNA hydrogels

[0077] ε represents the absorption coefficient, L / mol cm. Sequences Y1, Y2, and Y3 form the Y-scaffold unit; sequences L1C and L2C form the cross-linking unit; underlines indicate the sticky ends of the DNA sequence, and bold indicates the EcoR I restriction endonuclease recognition sequence.

[0078] 2. Buffer

[0079] The present invention uses the following PBS buffer 1 and PBS buffer 2.

[0080] PBS buffer 1 is prepared by adding 0.20 g of KCl, 8.00 g of NaCl, 0.20 g of KH2PO4, and 2.08 g of Na2HPO4·12H2O into 1 L of water, mixing and dissolving, and sterilizing by filtration through a 0.22 μm filter membrane to obtain a PBS solution with a pH value of 7.4. The osmotic pressure of the solution is measured to be approximately 277 mOsmol / kg.

[0081] PBS buffer 2 is prepared by adding approximately 0.20 g of KCl, approximately 8.00 g of NaCl, approximately 0.24 g of KH2PO4, and approximately 3.63 g of Na2HPO4·12H2O to approximately 900 mL of water, adjusting the pH to approximately 7.4 with approximately 1 mol / L HCl, and then making up the volume to 1 L. The solution is sterile-filtered through a 0.22 μm filter membrane and has an osmotic pressure of approximately 288 mOsmol / kg.

[0082] Example 1: Nucleic Acid Hydrogel Preparation 1

[0083] The method for preparing the nucleic acid hydrogel preparation of the present invention comprises the following standard steps:

[0084] 1) Using the scaffold unit Y and cross-linking unit L shown in Table 1, wherein the purity of the single-stranded nucleic acid is 90% as determined by LC-MS, as shown in FIG1 .

[0085] 2) Preparation of assembly solution.

[0086] Table 2: Nucleic Acid Hydrogel Standard Step Prescription Information

[0087] Weigh the prescribed amounts of Y1, Y2, and Y3 into tank A. Then add the prescribed amount of PBS solution 1 and stir at room temperature to completely dissolve. Monitor the system pH using an online pH monitor and adjust the pH to 7.2 with NaOH solution. Prepare the Y assembly solution at room temperature.

[0088] Weigh the prescribed amounts of L1C and L2C into tank B. Then add the prescribed amount of PBS solution 1 and stir at room temperature to completely dissolve. Monitor the system pH using an online pH monitor and adjust the pH to 7.2 with NaOH solution. Prepare the L assembly solution at room temperature.

[0089] 3) Sterile filtration

[0090] The Y assembly solution and the L assembly solution were sterilized by filtration through one 0.45 μm and two 0.22 μm PES (polyethersulfone) filters, respectively.

[0091] 4) Assemble into glue

[0092] The filtered Y assembly solution was transferred to a mixing tank, heated to 55° C., and the L assembly solution was added under stirring. The mixture was kept warm for 2 hours, during which vacuum degassing was performed.

[0093] 5) Canned

[0094] After the gel is stabilized, it is cooled to 40°C, filled into pre-filled syringes, and then packaged.

[0095] The properties of nucleic acid hydrogel preparation 1 are as follows

[0096] 1. The nucleic acid solid content was found to be 3.0 wt% relative to the nucleic acid hydrogel preparation.

[0097] 2. Upon visual inspection, the nucleic acid hydrogel preparation 1 was a colorless and transparent gel;

[0098] 3. The storage modulus (G') and loss modulus (G") of nucleic acid hydrogel formulation 1 were measured using a rotational rheometer. The results are shown in Figure 2: Within the frequency range of 0.01 Hz to 10 Hz, the storage modulus (G') and loss modulus (G") were measured. G' was greater than G", indicating successful hydrogel formation. Meanwhile, G' exceeded 500 Pa, indicating that the gel was a strong gel.

[0099] 4. The transparency of nucleic acid hydrogel preparation 1 was measured by UV spectrophotometry: the transmittance T% at a scanning range of 400-800 nm was greater than 90%, indicating that the hydrogel was transparent in the visible light region; as shown in FIG3 .

[0100] 5. pH value of nucleic acid hydrogel preparation 1:

[0101] a) pH value after preparation: 7.36;

[0102] b) Gradually decreases to 6.84 after two weeks.

[0103] Example 2: Nucleic Acid Hydrogel Preparation 2

[0104] Nucleic acid hydrogel 2 was prepared in a manner similar to Example 1, except that the purity of the single-stranded nucleic acid used was 95% as determined by LC-MS, as shown in FIG4 ; and more NaOH solution was used to adjust the pH to 7.2 when preparing the Y assembly solution at room temperature.

[0105] The properties of nucleic acid hydrogel preparation 2 are as follows

[0106] 1. The nucleic acid solid content was found to be 3.0 wt% relative to the nucleic acid hydrogel preparation.

[0107] 2. Upon visual inspection, the nucleic acid hydrogel preparation 2 was a colorless and transparent gel;

[0108] 3. The storage modulus (G') and loss modulus (G") of nucleic acid hydrogel formulation 2 were measured using a rotational rheometer. The results are shown in Figure 5 : Within the frequency range of 0.01 Hz to 10 Hz, the storage modulus (G') and loss modulus (G") were measured. G' was greater than G", indicating successful hydrogel formation. Meanwhile, G' exceeded 500 Pa, indicating that the gel was a strong gel.

[0109] The experimental results show that there is no obvious difference in the nucleic acid hydrogel preparations made from single-stranded nucleic acids of different purities to the naked eye. However, when single-stranded nucleic acids with a purity of more than 95% are prepared into an assembly solution, the pH is acidic and needs to be adjusted with more NaOH aqueous solution. The storage modulus of the hydrogel prepared is also larger, increasing to 1200Pa, which can be predicted to have better filling and supporting performance.

[0110] Example 3: Nucleic Acid Hydrogel Preparation 3

[0111] Nucleic acid hydrogel 3 was prepared in a similar manner to Example 1, except that PBS buffer 2 was used instead of PBS buffer 1.

[0112] The properties of nucleic acid hydrogel preparation 3 are as follows

[0113] 1. pH value of nucleic acid hydrogel preparation 3:

[0114] a) pH value after preparation is 7.25;

[0115] b) Gradually decreases to 7.36 after two weeks.

[0116] 2. The storage modulus (G') and loss modulus (G") of nucleic acid hydrogel formulation 3 were measured using a rotational rheometer. The results are shown in Figure 6: Within the frequency range of 0.01 Hz to 10 Hz, the storage modulus (G') and loss modulus (G") were measured. G' was greater than G", indicating successful hydrogel formation. Meanwhile, G' exceeded 500 Pa, indicating that the gel was a strong gel.

[0117] 3. The transparency of nucleic acid hydrogel preparation 3 was measured by UV spectrophotometry: the transmittance T% at 400-800 nm was greater than 90%, indicating that the hydrogel was transparent in the visible light region; as shown in FIG7 .

[0118] By measuring the pH of the hydrogel at different times, it can be seen that the nucleic acid hydrogel preparation 3 prepared using buffer 2 can maintain a pH value between 7.0 and 7.4 for a long time, which meets the standards for ophthalmic preparations; while the pH value of the nucleic acid hydrogel preparation 1 prepared using buffer 1 gradually decreased from 7.36 to 6.84 two weeks after preparation.

[0119] Example 4: Effects of gelation at different temperatures on the properties of nucleic acid hydrogels

[0120] The following nucleic acid hydrogel preparations were prepared in a similar manner to Example 3, except for the gelation temperature:

[0121] Example 4a: Gluing at 5°C

[0122] Example 4b: Gluing at 25°C

[0123] Example 4c: Gel formation at 37°C

[0124] Example 4d: Example 3 was gelled at 55°C

[0125] Example 4e: Gel formation at 65°C

[0126] The final physical and rheological properties of nucleic acid hydrogel preparations prepared at different temperatures did not change significantly. However, the experimental results showed that the lower the temperature, the slower the gelation time. In the 5°C and 25°C experimental groups, after the assembly was mixed, the gel was turbid and contained a large number of bubbles. After standing for 24 hours, it gradually became clear and transparent, but the bubbles did not disappear on their own. In the 37°C experimental group, after the assembly was mixed, the gel was turbid and contained a large number of bubbles. After standing for 1-2 hours, it gradually became clear and transparent, and the bubbles disappeared. At 55°C and 65°C, after the assembly was mixed, the system was almost liquid with no bubble formation. As the temperature decreased to below 55°C, a clear and transparent gel was formed. Figure 8 shows the two states of hydrogels prepared at different gelation temperatures: turbid (Example 4a, left) and clear (Example 4d, right). The hydrogel formed at room temperature was turbid, while the hydrogel formed at temperatures above room temperature, such as 55°C or 65°C, was clear and transparent.

[0127] From the above data, it can be seen that appropriately increasing the temperature can accelerate the stabilization of the gelation system and eliminate bubbles. However, considering that nucleic acids may be degraded under long-term high temperature, the final choice is to carry out the gelation at 40-65°C, preferably 45-55°C.

[0128] Example 5: Effect of Nucleic Acid Solid Content on the Performance of Nucleic Acid Hydrogel Formulations

[0129] The following nucleic acid hydrogel preparations were prepared in a similar manner to Example 3, except that the nucleic acid solid content was different:

[0130] Example 5a: Nucleic acid solid content is 0.385 wt%;

[0131] Example 5b: Nucleic acid solid content is 0.77 wt%;

[0132] Example 5c: The nucleic acid solid content is 1.5% wt%;

[0133] Example 5d: The nucleic acid solid content is 2.0 wt%;

[0134] Example 5e: Example 3, the nucleic acid solid content is 3.0 wt%;

[0135] Example 5f: Nucleic acid solid content is 3.8 wt%;

[0136] Example 5g: The nucleic acid solid content is 4.6 wt%.

[0137] The effects of varying solid content on the performance of the prepared nucleic acid hydrogel formulations were then tested. As shown in Figure 9, as the solid content of nucleic acid increases from 0.385wt% to 4.6wt%, the storage modulus of the prepared nucleic acid hydrogel increases, from 20Pa to 1371Pa. During the initial stages of intravitreal injection, support performance is optimal. However, as the aqueous humor secretes and dilutes, the hydrogel in contact with the aqueous humor may be diluted and dissolved by the aqueous humor and carried away through the aqueous humor circulation. The DNA solid content gradually decreases, and so does the storage modulus of the hydrogel, until it becomes completely liquid and no longer gels.

[0138] Although nucleic acid hydrogels with higher solid content have higher storage modulus, intraocular injection will be more difficult; and the higher the solid content of nucleic acid hydrogels, the longer the nucleic acid hydrogels can be stored in the eye. However, the longer the storage time of nucleic acid hydrogels in the eye is not necessarily better. Generally, the nucleic acid hydrogels need to be stored in the eye for about 3-6 months before they degrade; and a higher solid content will make the nucleic acid hydrogels opaque after injection and produce bubbles, which will affect refraction. Therefore, considering the difficulty of injection, degradation time and refraction, a nucleic acid solid content of 3-3.5wt% is suitable and more preferred for retinal reattachment applications.

[0139] Example 6: Nucleic acid hydrogel preparation filling aids retinal reattachment

[0140] This example studies the nucleic acid hydrogel preparation 3 prepared in Example 3, injects it into the retina after PPV surgery in a retinal detachment model, and evaluates its effect on retinal reattachment.

[0141] A cyanotic rabbit model of retinal detachment was constructed. Figure 10 shows the rabbit model. Figure 10A shows a detached retina under water flow. After the retina was temporarily reattached and the tear was sealed with laser therapy, no hydrogel or traditional silicone oil filling was used. A follow-up ultrasound examination one month after modeling revealed that the retina was still detached (Figure 10B).

[0142] Figure 10A. Retinal detachment (the white area is the detached retina, and the central red dot is the hole); Figure 10B. B-ultrasound at 1 month after surgery showed that the retina was still detached (the white arrow is the detached retina).

[0143] Twelve cyanotic rabbits were randomly divided into a hydrogel group and a silicone oil group, with six rabbits in each group. The right eye was used for surgery. A retinal detachment model was first established, followed by retinal reattachment. Approximately 1 ml of hydrogel or silicone oil was then injected into the vitreous cavity. As shown in Figure 11, ultrasound examinations were performed 1, 2, 3, and 6 months after surgery. The retinas of all rabbits in both the hydrogel and silicone oil groups remained in situ at all observation time points.

[0144] Retinal reattachment was observed by injecting a hydrogel formulation into a rabbit model of retinal detachment. Results showed that the retinas remained in place in all rabbit eyes in both the hydrogel and traditional silicone oil groups within six months. Compared to silicone oil, the use of nucleic acid hydrogel formulations eliminates the need for patients to maintain a prone position for 2-3 weeks, significantly reducing pain. Furthermore, because nucleic acids are degraded by nucleases in the body, secondary surgical removal is unnecessary.

[0145] From these two experimental results, it can be seen that the nucleic acid hydrogel preparation achieves the same top pressure support effect as silicone oil, providing an alternative for the clinical treatment of retinal detachment.

[0146] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A nucleic acid hydrogel preparation suitable for retinal reattachment, the nucleic acid hydrogel preparation comprising A scaffold unit, comprising a scaffold core and at least three single-stranded nucleic acids bound to the scaffold core, each single-stranded nucleic acid having at least one scaffold sticky end; A cross-linking unit, the cross-linking unit comprising a cross-linking core and at least two single-stranded nucleic acids bound to the cross-linking core, each single-stranded nucleic acid having at least one cross-linked sticky end; as well as The scaffold unit and the cross-linking unit are cross-linked by base complementary pairing between the scaffold sticky end and the cross-linking sticky end to form a nucleic acid hydrogel with a three-dimensional spatial network structure; Buffer; and Optional pH adjuster.

2. A method for preparing a nucleic acid hydrogel preparation, the method comprising: a) preparing a scaffold unit assembly solution and a cross-linking unit assembly solution; as well as b) assembling the prepared scaffold unit assembly solution and cross-linking unit assembly solution into a gel at a temperature higher than room temperature.

3. The nucleic acid hydrogel preparation of claim 1 or the method of claim 2, wherein the scaffold unit comprises a scaffold core and at least three single-stranded nucleic acids bound to the scaffold core, each single-stranded nucleic acid having at least one scaffold sticky end; preferably, the scaffold core is a nucleic acid, specifically a D-nucleic acid or an L-nucleic acid, more specifically a D-DNA or an L-DNA; preferably, the nucleic acid serving as the scaffold core has a complementary pairing region, the length of which may be 4 to 150 bp, preferably 5 to 50 bp, more preferably 6 to 30 bp, more preferably 8 to 20 bp.

4. The nucleic acid hydrogel preparation of claim 1 or the method of claim 2, wherein the cross-linking unit comprises a cross-linking core and at least two single-stranded L-deoxynucleic acids bound to the cross-linking core, each single-stranded L-deoxynucleic acid having at least one cross-linked sticky end; preferably, the cross-linking core can be a deoxynucleic acid, specifically a D-nucleic acid or an L-nucleic acid, more specifically a D-DNA or an L-DNA.

5. The nucleic acid hydrogel preparation of claim 4 or the method of claim 4, wherein the deoxynucleic acid as the cross-linked core has a complementary pairing region, and the length of the complementary pairing region can be 4 to 150 bp, preferably 5 to 50 bp, preferably 15 to 50 bp, more preferably 6 to 30 bp, and more preferably 8 to 20 bp.

6. The nucleic acid hydrogel preparation of claim 1 or the method of claim 2, wherein the length of the scaffold sticky ends or the cross-linked sticky ends is 4-30 nt, 4-20 nt, more preferably 8-12 nt.

7. The nucleic acid hydrogel formulation of claim 1 or the method of claim 2, wherein the scaffold unit is the same as the cross-linking unit.

8. The nucleic acid hydrogel formulation of claim 1 or the method of claim 2, wherein the scaffold unit is different from the cross-linking unit.

9. The nucleic acid hydrogel preparation of claim 1 or the method of claim 2, wherein the molar ratio of the scaffold unit to the cross-linking unit in the nucleic acid hydrogel is 2:1-1:3, preferably 1:1-1:2, and more preferably 1:1.

5.

10. The nucleic acid hydrogel preparation of claim 1 or the method of claim 2, wherein the scaffold unit and the cross-linking unit are cross-linked by base complementary pairing between the scaffold sticky end and the cross-linking sticky end, thereby forming a three-dimensional spatial network structure.

11. The nucleic acid hydrogel preparation of claim 1 or the method of claim 2, wherein the nucleic acid solid content in the nucleic acid hydrogel preparation is 0.3-5wt%, relative to the weight of the nucleic acid hydrogel preparation; preferably, the nucleic acid solid content is 0.3-3.7wt%; more preferably 1-3.5wt%; more preferably 2-3.5wt%; or 3-3.5wt%.

12. The nucleic acid hydrogel preparation of claim 1 or the method of claim 2, wherein the single-stranded nucleic acid has a purity of more than 90%, preferably more than 95%, for example, 95%, 96%, 97%, 98% or 99%.

13. The nucleic acid hydrogel preparation of claim 1 or the method of claim 2, wherein the scaffold unit or cross-linking unit of the nucleic acid hydrogel may comprise a CpG sequence.

14. The nucleic acid hydrogel formulation of claim 1 or the method of claim 2, wherein the sequences of the scaffold units are 5'-CGATTGACTCTCCACGCTGTCCTAACCATGACCGTCGAAG-3', 5'-CGATTGACTCTCCTTCGACGGTCATGTACTAGATCAGAGG-3', and 5'-CGATTGACTCTCCCTCTGATCTAGTAGTTAGGACAGCGTG-3'; and The sequences of the cross-linking units are 5'-GAGAGTCAATCGTCTATTCGCATGAGAATTCCATTCACCGTAAG-3' and 5'-GAGAGTCAATCGCTTACGGTGAATGGAATTCTCATGCGAATAGA-3'.

15. The nucleic acid hydrogel preparation according to claim 1 or the method according to claim 2, wherein the mechanical strength of the produced nucleic acid hydrogel is 0.1 Pa or more, preferably 1 Pa or more, more preferably 10 Pa or more, preferably 10000 Pa or less, more preferably 1000 Pa or less.

16. The nucleic acid hydrogel formulation of claim 1 or the method of claim 2, wherein the produced nucleic acid hydrogel has a dominant storage modulus G' relative to the loss modulus G" at all strain values ​​from 0.1 to 100 rad / s as measured by frequency sweep measurements at 25°C and at a strain amplitude of 1% strain; preferably, at strain values ​​from 0.1 to 100 rad / s, the ratio of the storage modulus G' to the loss modulus G" is greater than 2; more preferably greater than 10.

17. The nucleic acid hydrogel preparation of claim 1 or the method of claim 2, wherein the buffer is PBS buffer 1, which includes about 0.20 g of KCl, about 8.00 g of NaCl, about 0.20 g of KH2PO4, and about 2.08 g of Na2HPO4·12H2O; or the buffer is PBS buffer 2, which includes about 0.20 g of KCl, about 8.00 g of NaCl, about 0.24 g of KH2PO4, about 3.63 g of Na2HPO4·12H2O, and a pH adjuster for adjusting the pH to about 7.

4.

18. The nucleic acid hydrogel preparation of claim 1 or the method of claim 2, wherein the pH adjuster is an HCl solution or a NaOH solution; preferably, the pH adjuster is an HCl solution or a NaOH solution having a pH value of 7-7.4, and the concentration of the HCl solution or the NaOH solution is generally 0.01 to 1 mol / L, preferably 0.01 mol / L.

19. The method of claim 2, wherein the method further comprises sterilizing and filtering the prepared scaffold unit assembly solution and cross-linking unit assembly solution before assembling into gel.

20. The method of claim 2, wherein the assembling gel is carried out at 40-65°C, preferably at 45-55°C.

21. The method of claim 2, wherein the assembling into gel comprises heating and stirring the prepared scaffold unit assembly solution at the above-mentioned gelation temperature, and then adding the prepared cross-linking unit assembly solution and keeping warm for a period of time, such as 0.5-5 hours or 1-2 hours.

22. The method of claim 2, wherein the assembling into gel comprises heating and stirring the prepared scaffold unit assembly solution and the prepared cross-linking unit assembly solution at the above-mentioned gelation temperature simultaneously, for example, for 0.5-5 hours or 1-2 hours.

23. The method of claim 2, wherein the assembling into glue is performed under vacuum conditions.

24. The method of claim 2, wherein the method further comprises filling the formed hydrogel into a syringe at a suitable temperature after assembly into a gel.

25. A method for treating an ophthalmic disease in a subject in need of treatment, the method comprising injecting a therapeutically effective amount of the nucleic acid hydrogel formulation of the present invention into the subject's eye; or involving use of the nucleic acid hydrogel of the present invention in the preparation of a medicament or agent for treating an ophthalmic disease.

26. The method of claim 25, wherein the nucleic acid hydrogel formulation is injected into the eye following vitrectomy.

27. The method of claim 25, wherein the ocular disease is retinal detachment, macular hole, complications of diabetic retinopathy, and posterior segment ocular trauma.

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