A protein zwitterionic polymer conjugate, and a preparation method and application thereof

Abstract

The application discloses a protein zwitterionic polymer conjugate and a preparation method and application thereof. A multifunctional new medical biomaterial with mucosal adhesion, wetting, lubrication and microbial adhesion prevention is designed and synthesized, so that a new breakthrough is brought to the clinical demand of solving Sjogren's syndrome. In addition, the application has wide application prospects in the fields of oral lubrication, ophthalmic lubrication, joint repair and the like.

Description

Technical Field

[0001] This invention relates to the field of medical biomaterials, specifically to a protein zwitterionic polymeric conjugate, its preparation method, and its application. Background Technology

[0002] Sjögren's syndrome is a chronic autoimmune disease primarily affecting exocrine glands such as the lacrimal and salivary glands, mainly manifesting as dry eyes and dry mouth. Sjögren's syndrome may be related to genetic factors, viral infections, abnormal estrogen levels, immune system disorders, and environmental stimuli. Currently, there is no cure for Sjögren's syndrome; treatment mainly involves replacement and symptomatic therapies to alleviate symptoms, slow disease progression, and prevent local and systemic damage caused by prolonged dryness. Therefore, exploring new approaches and therapies for treating Sjögren's syndrome is of great significance.

[0003] Taking xerostomia as an example, currently used medical biomaterials include artificial saliva mouthwashes, which are topical medications that can relieve dry mouth. In addition, saliva alternatives based on mucin, carboxymethyl cellulose, hydroxyethyl cellulose, xanthan gum, flaxseed, or polyethylene glycol have been developed. However, existing artificial saliva lacks adhesive properties and can only temporarily relieve dry mouth symptoms. Furthermore, long-term use of artificial saliva with a low pH value may lead to demineralization of tooth enamel and dentin. At the same time, artificial saliva has poor antimicrobial adhesion and antibacterial properties.

[0004] Therefore, how to design and construct novel medical biomaterials or related medical devices that combine the functions of mucosal adhesion, wettability, lubrication, and prevention of microbial adhesion is the key and difficult point in the field of such medical biomaterials. Among them, the synergistic integration and regulation of multiple material properties is the core scientific issue. Summary of the Invention

[0005] To address at least some of the problems existing in the prior art, this invention synthesizes a protein-zwitterionic polymer conjugate and, by optimizing the controllable synthesis methodology of this protein-polymer conjugate system, organically combines the mucosal adhesion properties of proteins with the superhydrophilic properties of zwitterionic polymers. This constructs a novel multifunctional medical biomaterial possessing mucosal adhesion, wettability, lubrication, and antimicrobial adhesion properties, and can achieve, for example, but not limited to, biomimetic reconstruction of the oral salivary membrane, thus bringing a new breakthrough to addressing the clinical needs of Sjögren's syndrome. Specifically, this invention includes the following:

[0006] In a first aspect, the present invention provides a protein zwitterionic polymer conjugate comprising a protein, polypeptide or functional fragment thereof having mucosal adhesion properties, and a zwitterionic polymer connected to said protein, polypeptide or functional fragment thereof.

[0007] In some embodiments, according to the protein zwitterionic polymeric conjugate of the present invention, the protein comprises a lectin-like protein or a functional fragment thereof, or a protein or a functional fragment thereof having a similar structure to the lectin-like protein and having sugar-binding activity.

[0008] In some embodiments, the protein zwitterionic polymeric conjugate according to the present invention includes at least one of phosphorylcholine, sulfobetaine, carboxybetaine, TMAO amine oxides or their derivatives.

[0009] A second aspect of the present invention provides a composition having a lubricating function, comprising the protein zwitterionic polymeric conjugate described in the first aspect.

[0010] In some embodiments, the lubricating composition according to the present invention reduces the coefficient of friction of a surface when applied to surfaces including joints, skin, mucous membranes, and / or medical devices.

[0011] In some embodiments, the lubricating composition according to the present invention includes daily necessities or pharmaceuticals.

[0012] A third aspect of the present invention provides a medical device comprising the protein zwitterionic polymer conjugate described in the first aspect.

[0013] In some embodiments of the medical device according to the present invention, the protein zwitterionic polymer conjugate forms a coating on the surface of the medical device.

[0014] A fourth aspect of the present invention provides a method for preparing a protein zwitterionic polymeric conjugate, comprising: (1) Use an initiator to modify proteins, peptides or their functional fragments that have mucosal adhesion properties to obtain modified proteins, peptides or their functional fragments. (2) The modified protein, polypeptide or its functional fragment is polymerized with zwitterionic monomer to obtain the conjugate.

[0015] A fifth aspect of the present invention provides the use of the protein zwitterionic polymer conjugate described in the first aspect in the preparation of medical biomaterials or medical devices.

[0016] In some embodiments, according to the application described in the invention, the medical biomaterial is used to provide a lubricating effect.

[0017] In some embodiments, according to the application described in the invention, the medical biomaterial is used for Sjögren's syndrome, which includes xerostomia or dry eye syndrome.

[0018] This invention designs and synthesizes a class of protein-zwitterionic polymer conjugates with mucosal adhesion properties. By optimizing the controllable synthesis methodology of this protein-polymer conjugate system, the mucosal adhesion properties of proteins (such as, but not limited to, wheat germ lectin) and the superhydrophilic properties of zwitterionic polymers are organically combined. In vitro studies were conducted to investigate its cell adhesion, wetting, lubrication, and antibacterial properties, and its physiological functions were further explored in animal models. Experimental results show that this invention successfully constructs a novel multifunctional medical biomaterial that combines mucosal adhesion, wetting, lubrication, and antimicrobial adhesion, thus bringing a new breakthrough to addressing the clinical needs of Sjögren's syndrome. Furthermore, this invention has broad application prospects in joint lubrication, skin lubrication, mucosal lubrication, oral lubrication, ophthalmic lubrication, joint repair, novel medical biomaterials, and related medical devices. Attached Figure Description

[0019] Figure 1 The 1H NMR spectrum of the initiator OBMP is shown.

[0020] Figure 2 MALDI-TOF mass spectra of WGA-Br modified with different proportions of initiator are shown.

[0021] Figure 3 The prepared WGA-Polymer is shown in (A) SDS-PAGE plots of WGA (lane 1), WGA-Br (lane 2), WGA-PMPC (lane 4), WGA-PCBMA (lane 5), and WGA-PSBMA (lane 6); and (B) GPC plots.

[0022] Figure 4 The GPC curves of WGA-PMPC with different modification ratios are shown.

[0023] Figure 5 The binding capacity of different concentrations of WGA-Polymer to mucin (A) and the binding capacity of different WGA-Polymers to human oral mucosal epithelial cells (B) are shown.

[0024] Figure 6 The antibacterial adhesion effect of WGA-Polymer is shown in Figure A, which shows crystal violet staining, and Figures B and C show quantitative crystal violet staining.

[0025] Figure 7The wetting ability of different WGA-Polymers is shown. Visual diagram of water contact angle (A); quantitative diagram of water contact angle (B).

[0026] Figure 8 The lubrication capability of WGA-Polymer is shown.

[0027] Figure 9 The image shows the oral adhesion effect in mice at different time points using WGA-PMPC.

[0028] Figure 10 The image shows a comparison of water intake during the treatment of mice with xerostomia. Artificial saliva was represented by AS, and commercial mouthwash was represented by Biotene mouthwash.

[0029] Figure 11 The image shows the adhesion effect of WGA-PMPC in the mouse eye at different time points.

[0030] Figure 12 Visual images of the eyes after sodium fluorescein staining and scoring results are shown.

[0031] Figure 13 The following are shown: (A) SDS-PAGE plots of the prepared pWGA-PMPCs: pWGA (lane 1), pWGA-PMPC1000 (lane 2), and pWGA-PMPC2000 (lane 3); and (B) GPC plots of pWGA-PMPCs.

[0032] Figure 14 The diagram shows the binding of different concentrations of pWGA-PMPC to mucin.

[0033] Figure 15 The wetting ability of different pWGA-PMPCs is shown. Visual diagram of water contact angle (A); quantitative diagram of water contact angle (B).

[0034] Figure 16 The image shows the oral adhesion effect of pWGA-PMPC in mice at different time points.

[0035] Figure 17 The antibacterial adhesion effect of pWGA-PMPC was demonstrated. Detailed Implementation

[0036] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0037] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that the upper and lower limits of the range and each intermediate value between them are specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, are also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0038] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0039] Protein zwitterionic polymer conjugates In one aspect, the present invention provides a protein zwitterionic polymer conjugate comprising a protein, polypeptide or functional fragment thereof having mucosal adhesion properties, and a zwitterionic polymer covalently linked to said protein, polypeptide or functional fragment thereof.

[0040] In this invention, the protein or polypeptide is not limited in any way, as long as it has mucosal adhesion properties. In a preferred embodiment, the protein of this invention includes lectin-like proteins or functional fragments thereof, or proteins with similar structures to said lectin-like proteins and having sugar-binding activity or functional fragments thereof. Herein, "functional fragment" refers to a local region or amino acid sequence capable of independently performing some or all of the biological functions of the protein.

[0041] In this invention, the term "lectin-like proteins" refers to all proteins or their subunits that contain a sugar recognition domain and are capable of specifically binding to carbohydrate molecules. This includes, but is not limited to: plant lectins (such as wheat germ lectins and legume lectins), type C lectins (such as the selectin family and mannose-binding lectins), type I lectins (such as sialic acid adhesin / Siglec family), galactolectins, pentamins, and other proteins with lectin-like domains or sugar-binding activity, such as calfnetin / calreticulin involved in endoplasmic reticulum glycoprotein folding, integrins with glycosyl-binding capabilities, and viral or bacterial lectins (such as influenza virus hemagglutinin and bacterial fimbriae agglutinins).

[0042] In this invention, the zwitterionic polymer is obtained by polymerizing raw materials containing zwitterionic monomers. The specific type of zwitterion is not particularly limited; it can be any zwitterion with superhydrophilic properties. Examples of zwitterions include, but are not limited to, at least one of phosphorylcholine, sulfobetaine, carboxybetaine, TMAO amine oxides, or their derivatives. Herein, "derivative" refers to a compound formed by the substitution of atoms or groups of atoms in the parent compound. In a preferred embodiment, the phosphorylcholine derivative is methacryloyloxyethyl phosphorylcholine. In another preferred embodiment, the sulfobetaine derivative is methacrylate sulfobetaine. In yet another preferred embodiment, the carboxybetaine derivative is methacrylate carboxylate betaine.

[0043] In some embodiments, the protein zwitterionic polymer conjugate of the present invention exhibits excellent adhesion properties, particularly with excellent binding ability to mucins or mucosal epithelial cells. The binding of the conjugate to mucins or mucosal epithelial cells, or the strength of that binding, can be determined by methods known in the art and is not particularly limited thereto.

[0044] In some embodiments, the protein zwitterionic polymer conjugate of the present invention has excellent antibacterial properties, particularly antibacterial adhesion properties, wherein the bacteria include Gram-negative bacteria and / or Gram-positive bacteria.

[0045] In some embodiments, the protein zwitterionic polymer conjugate of the present invention has excellent wetting properties, and when the static contact angle is measured using an OCA20 contact angle meter, its water contact angle is less than 50°, preferably less than 40°, for example less than 35°.

[0046] In some embodiments, the protein zwitterionic polymer conjugate of the present invention exhibits excellent lubrication properties. When using zirconia balls as the grinding pair and conducting reciprocating friction tests using a general mechanical testing instrument at a load of 1 N and a frequency of 1 Hz, it has an average coefficient of friction of less than 0.5, preferably less than 0.4, for example less than 0.3, 0.2, or 0.1. In some embodiments, the protein zwitterionic polymer conjugate of the present invention achieves a near-superlubricating coefficient of friction, for example less than 0.015, on a hydrophobic PDMS film under a loading pressure of 1-2 N and a friction frequency of 1-2 Hz.

[0047] Composition In one aspect, the present invention provides a lubricating composition comprising the aforementioned protein zwitterionic polymeric conjugate. When the composition of the present invention is applied to surfaces including joints, skin, mucous membranes, and / or medical devices, it can reduce the coefficient of friction of the surface.

[0048] In some embodiments, the compositions of the present invention are daily necessities, examples of which include, but are not limited to, toothpaste, mouthwash, saliva substitutes, etc.

[0049] In some embodiments, the compositions of the present invention are pharmaceuticals, examples of which include, but are not limited to, pharmaceuticals for use in joints, skin, mouth, mucous membranes, eyes, vagina, and other applications requiring lubrication.

[0050] When the composition is a pharmaceutical, the composition may further include a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are well known in the art and include any excipients that do not induce the formation of substances harmful to a subject receiving the composition. Those skilled in the art can determine that they meet clinical standards. Pharmaceutically acceptable carriers include, but are not limited to, at least one of humectants, preservatives, colorants, buffers, sweeteners, emulsifiers, flavorings, antioxidants, and excipients.

[0051] The compositions of the present invention can be in any suitable dosage form, such as, but not limited to, sprays, gels, chewing gum, capsules, lozenges, palatal tablets, tablets, solutions, or forms suitable for applying the pharmaceutical composition to mucosal surfaces. The compositions of the present invention can be applied to the body or to a local surface in a known manner. The dosage is not particularly limited and can be administered in single or multiple doses. Those skilled in the art will understand that the actual dosage to be applied herein can vary considerably depending on a variety of factors.

[0052] In some embodiments, the compositions of the present invention are compositions suitable for topical application.

[0053] medical devices In one aspect, the present invention provides a medical device comprising the protein zwitterionic polymer conjugate described herein. Those skilled in the art will understand that the protein zwitterionic polymer conjugate can exist on the surface of the medical device in the form of a coating.

[0054] In this invention, the medical device or medical product is not particularly limited, as long as it has a surface that can be coated. Examples of medical devices include, but are not limited to: tubular instruments (e.g., catheters, cannulas, drainage tubes, sheaths, etc.), guidewires, puncture needles, root canal files, bone needles, wires, stents, balloons, microneedles, microelectrode arrays, implants (e.g., artificial joints, artificial lenses, artificial skin, prostheses, implants, etc.), optically transparent instruments (e.g., endoscopes, intraocular instruments, contact lenses, etc.), and planar or membrane-like instruments (e.g., anti-adhesion membranes, artificial dura maters, wound dressings, surgical films, etc.).

[0055] Preparation method One aspect of this invention provides a method for preparing protein-zwitterionic polymeric conjugates. This invention first employs a protein site-controllable modification method, utilizing reductive amination of the N-terminus to controllably modify the protein; further, it utilizes an in-situ protein polymerization method based on atom transfer radical polymerization to select various zwitterionic monomers to prepare three different protein-zwitterionic polymeric conjugate systems.

[0056] In a preferred embodiment, the preparation method of the present invention includes: (1) Use an initiator to modify proteins, peptides or their functional fragments that have mucosal adhesion properties to obtain modified proteins, peptides or their functional fragments. (2) The modified protein, polypeptide or its functional fragment is polymerized with zwitterionic monomer to obtain the conjugate.

[0057] In step (1) of this invention, the specific type of initiator is not particularly limited, for example, but not limited to, brominated esters or brominated amides. In a preferred embodiment, the initiator used in this invention has the following structure: Formula I, Wherein, R1 is selected from hydrogen, C1-C4 alkyl, or substituted C1-C4 alkyl; R2 and R3 are each independently selected from C1-C4 alkyl or substituted C1-C4 alkyl; When substituents are present, the substituents are not particularly limited and can be halogens, hydroxyl groups, carboxyl groups, alkenyl groups, alkynyl groups, amino groups, alkoxy groups, aryl groups, etc.

[0058] In a preferred embodiment, the molar ratio of protein to initiator is 1:1-50, preferably 1:1-40, even more preferably 1:1-30, and further preferably 1:1-20, such as 1:2-20, 1:3-20, 1:4-20, 1:5-20, like 1:5, 1:10, 1:15, 1:20.

[0059] Step (2) of the present invention involves in-situ polymerization of the modified protein with zwitterionic monomers to generate a coupling compound. Preferably, under an inert atmosphere, the modified protein obtained in step (1), the zwitterionic monomers, the copper-based catalyst, and the nitrogen-containing ligand are dispersed together in a buffer solution with a pH of 6.0-8.0 to form a reaction system, and the reaction system is subjected to a polymerization reaction at 0-37°C to obtain the protein-zwitterionic polymer coupling compound.

[0060] In this invention, the specific types of copper-based catalysts and nitrogen-containing ligands are not particularly limited. For example, examples of copper-based catalysts include, but are not limited to, CuCl / CuCl2 and CuBr / CuBr2. Examples of nitrogen-containing ligands include, but are not limited to, at least one of 1,1,4,7,10,10-hexamethyltriethylenetetramine, triethylenetetramine, tetraethylenepentamine, tris(2-dimethylaminoethyl)amine, and tris(2-pyridylmethyl)amine.

[0061] In this invention, the molar ratio of the modified protein to the zwitterionic monomer is not particularly limited, but is usually 1:100-5000, preferably 1:500-5000, and even more preferably 1:1000-5000, for example 1:1000, 1:2000, 1:3000, 1:4000, 1:5000.

[0062] In this invention, the molar ratio of the modified protein to CuCl and CuCl2 is not particularly limited, but is usually 1:5-50:50-100, preferably 1:20-50:50-80, and even more preferably 1:20-30:70-80, for example 1:25:75.

[0063] In this invention, the molar ratio of the modified protein to the nitrogen-containing ligand is not particularly limited, but is usually 1:50-500, preferably 1:50-400, even more preferably 1:50-200, and more preferably 1:100-150, for example 1:100, 1:105, 1:110, 1:115, 1:120, 1:125, 1:130, 1:135, 1:140, 1:145, and 1:150.

[0064] In this invention, the reaction time is not particularly limited, but is preferably 0.5-12 h, more preferably 0.5-6 h, even more preferably 0.5-4 h, and even more preferably 0.5-2 h.

[0065] The preparation method of this invention enables precise coupling of proteins with zwitterions while maintaining high bioactivity, meaning the polymerized protein retains its adhesive ability. Furthermore, the prepared protein conjugate achieves long-lasting retention on mucous membranes, resulting in better wetting. In addition, the product prepared by this invention is colorless and odorless, which improves patient compliance.

[0066] application One aspect of the present invention provides the use of protein zwitterionic polymeric conjugates in the preparation of medical biomaterials or medical devices. In some embodiments, the medical biomaterials include medicaments for Sjögren's syndrome.

[0067] In this invention, Sjögren's syndrome includes primary Sjögren's syndrome or secondary Sjögren's syndrome. In one specific embodiment, the Sjögren's syndrome includes xerostomia or dry eye syndrome. Although the excellent therapeutic effects of the protein zwitterionic polymer conjugate of this invention in xerostomia or dry eye syndrome have been verified in specific embodiments, those skilled in the art will understand that, based on its excellent adhesiveness, wettability, lubrication, and antimicrobial adhesion properties, the protein zwitterionic polymer conjugate of this invention can obviously be used in other applications requiring lubrication, such as mucosal lubrication or mucosal repair, for example, but not limited to joint, skin, and mucosal lubrication.

[0068] method The present invention also provides an in vitro antibacterial method, which includes the step of using the protein zwitterionic polymer conjugate of the present invention.

[0069] The specific types of antibacterial products are not particularly limited, but examples include, but are not limited to, release or contact products. Specifically, such products include, but are not limited to, antibacterial soaps, hand sanitizers, laundry detergents, dishwashing liquids, medical devices / implants coated with them (central venous catheters, artificial joints, wound dressings), antibacterial socks, underwear, medical protective clothing, masks, eye drops, mouthwash, bandages, gynecological washes, etc.

[0070] The present invention also provides a method for improving the mucosal microenvironment, comprising the step of applying the aforementioned protein zwitterionic polymeric conjugate to the surface of the mucosa or its epithelial cells.

[0071] In some embodiments, according to the method for improving the mucosal microenvironment of the present invention, the protein zwitterionic polymer conjugate, after being applied, is able to stably adhere to the mucosa or the surface of its epithelial cells, and reduce the coefficient of friction of the surface by providing a durable hydration layer and lubrication.

[0072] Example 1 I. Construction of the Wheat Germ Lectin-Zwitterionic Polymer (WGA-Polymer) System 1. Synthesis and characterization of OBMP initiators 2,2-Ethoxyethanol (268 mg) was dissolved in 20 mL of dry dichloromethane, followed by the addition of 2-bromo-2-methylpropionyl bromide (900 mg), and then triethylamine (1000 mg). The reaction was carried out overnight at room temperature. After adding water to adjust the pH to neutral, the mixture was extracted with dichloromethane. The combined organic phases were dried over anhydrous magnesium sulfate, filtered to remove magnesium sulfate, and the solvent was removed by rotary evaporation. The product A was obtained by dry loading and rapid column chromatography. Product A was dissolved in dichloromethane and trifluoroacetic acid was added. After reacting at room temperature for 2 h, the solvent was removed to obtain OBMP, an initiator for protein modification. The synthesized product was characterized by 1H NMR spectroscopy, and the results showed that OBMP was successfully prepared. Figure 1 ).

[0073] 2. Preparation of WGA-Br WGA protein was mixed with different proportions of OBMP (WGA:OBMP molar ratio 1:5-20), and 50 mM sodium cyanoborohydride was added. The pH was adjusted to 5.5, and the reaction was carried out in an ice bath. After dialyzing with ultrapure water, the desired WGA-Br was obtained, and the mixture was characterized by MODAL-TOF. The results are as follows: Figure 2 When the molar ratio of WGA protein to OBMP initiator is 1:5, the modification ratio of WGA to initiator is approximately 1:1. Subsequent experiments selected 1:5 as the optimal feed ratio for protein modification.

[0074] 3. Preparation of WGA-Polymer Different zwitterionic monomers (MPC, CBMA, SBMA) were added to an aqueous solution of WGA-Br. The reaction system was deoxygenated by purging with high-purity nitrogen for 30 min. The ratio of WGA-Br:CuCl:CuCl2:1,1,4,7,10,10-hexamethyltriethylenetetramine was 1:25:75:125. The polymerization reaction was carried out under nitrogen protection for 2 h, and finally stopped by purging with air. The products were separated and purified by desalting and anion exchange columns to remove small molecule copper salts and unreacted initiators and monomers, yielding pure WGA-PMPC, WGA-PSBMA, and WGA-PCBMA conjugates.

[0075] 4. Characterization of WGA-Polymer WGA-PMPC, WGA-PSBMA, and WGA-PCBMA were characterized by SDS-PAGE, and the molecular weight of the three couplings was characterized by GPC. The results showed that the three zwitterionic monomers exhibited similar polymerization effects under the same feed ratio. The SDS-PAGE and GPC experimental results are as follows: Figure 3 WGA can achieve good polymerization reactions with all three zwitterionic monomers.

[0076] 5. WGA-Polymer Molecular Weight Optimization The molecular weight of WGA-PMPC conjugates was controlled. Using the same preparation method as in the previous step, WGA-PMPCs with different molecular weights were prepared at molar ratios of WGA-Br to MPC of 1:1000, 1:2000, and 1:3000: low, medium, and high molecular weights (approximately 150,000 Da). This demonstrates that the molecular weight of protein conjugates can be controlled by different feed ratios. The results are as follows: Figure 4 .

[0077] II. In vitro functional validation of the WGA-Polymer system 1. Verification of adhesion effect Three different protein conjugates were tested for their adhesive, lubricating, and antibacterial properties. First, the mucin-binding ability of the different conjugates was determined. After washing the wells with PBS, the wells were co-incubated with mucin at 37°C for 2 hours, and then blocked with 1% BSA. Subsequently, different fluorescently labeled WGA conjugates were added and incubated for another hour. The plates were washed three times with PBS to remove unbound conjugates, and finally, the fluorescence intensity was detected using a SpectraMax M5 microplate reader.

[0078] The results are as follows Figure 5 As shown in Figure A, WGA retained over 50% binding capacity after polymerization with different zwitterionic monomers, with the WGA-PMPC group exhibiting significantly better binding capacity than WGA-PCBMA and WGA-PSBMA. Further investigation was conducted on the binding capacity of WGA-Polymer with primary human oral mucosal epithelial cells. Primary human oral mucosal epithelial cells were seeded in 24-well plates and cultured overnight. After aspirating the culture medium, serum-free medium containing different concentrations of WGA or WGA-Polymer conjugates was added, and the cells were incubated at 37°C for 30 min. Cell nuclei were then stained with Hoechst 33342 for 10 min, and the cells were thoroughly washed three times to remove unbound material. Confocal fluorescence imaging was then used to observe the binding capacity of different groups of WGA-Polymer with cells. The results are shown in Figure A. Figure 5 B. Different groups of WGA-Polymer were able to bind to oral mucosal epithelial cells.

[0079] 2. Verification of antibacterial effect The adhesion properties of different WGA-Polymers against Streptococcus mutans and Candida albicans were investigated using crystal violet staining. 1 mg / mL WGA conjugates were uniformly coated onto polystyrene 12-well plates, and 2 mL of Streptococcus mutans or Candida albicans suspension (OD200) was inoculated into each well. 600 =0.1), after incubating at 37°C for 48 hours, proceed with the following steps: (i) Fix methanol for 10 minutes; (ii) Stain with 0.1% crystal violet for 10 minutes; (iii) Rinse with deionized water; (iv) Decolorize with 95% ethanol for 30 minutes; (v) OD measurement using an enzyme-linked immunosorbent assay (ELISA) reader 590 Quantitative analysis of the values ​​was performed.

[0080] The results are as follows Figure 6 All three protein conjugates exhibited antimicrobial adhesion effects against Streptococcus mutans, with the WGA-PMPC group showing the best effect, effectively reducing Streptococcus mutans adhesion by about 60%. Meanwhile, WGA-PMPC and WGA-PCBMA also showed anti-adhesion effects against Candida albicans.

[0081] 3. Verification of wetting effect The wetting ability of different coupling agents was verified by static contact angle. The static contact angle was measured using an OCA20 contact angle meter (DataPhysics, Germany). Hydrophobic polytetrafluoroethylene films were coated with WGA, WGA-PMPC, WGA-PCBMA, and WGA-PSBMA (1 mg / mL) solutions, respectively, and then vacuum dried. The water contact angle of different composites was determined using the static drop method. The results are as follows: Figure 7 As shown, WGA-Polymer all exhibited better hydrophilicity than WGA, with WGA-PMPC showing the best hydrophilicity.

[0082] 4. Verification of lubrication effect Lubrication capability was verified using a general-purpose mechanical friction tester. Fresh pig tongue tissue was collected, washed with distilled water, and a 0.5 cm thick mucosal surface layer was dissected. After removing excess water, the tissue was fixed on the friction testing platform. Zirconia balls were used as the friction pair, and a reciprocating friction test was conducted using a general-purpose mechanical friction tester (UMT-0.5-3, Bruker, MA, USA) at a load of 1 N and a frequency of 1 Hz. Before the test, different solutions were uniformly applied to the mucosal surface. WGA-PMPC was selected as a representative of WGA-Polymer for comparative experiments. Friction tests were conducted in a WGA-only group, a saline group (NS), and an artificial saliva group (AS). The results are as follows: Figure 8 As shown, the friction coefficient of the WGA-PMPC group on the pig's tongue is significantly lower than that of the other three groups, proving that it has a good lubrication effect.

[0083] Meanwhile, this embodiment also investigated the lubrication capability of WGA-PMPC on hydrophobic PDMS films under different load conditions. The results showed that, under a loading pressure of 1-2 N and a friction frequency of 1-2 Hz, a near-superlubricating coefficient of friction could be achieved. Figure 8 As shown in C.

[0084] III. In vivo functional verification 1. WGA-PMPC Adhesion Performance Verification After Cy7 molecules were ligated into WGA and WGA-PMPC, respectively, 8-week-old female Balb / c mice were selected. After rinsing the oral cavity with physiological saline to remove food debris, 200 μL of the above preparation was evenly applied to the oral cavity of each mouse. In vivo small animal imaging (IVIS SPECTRUM) was performed to monitor changes in oral fluorescence, and the fluorescence in the oral cavity was quantified. Results are as follows: Figure 9 As shown, the results indicate that WGA-PMPC exhibits good oral adhesion, and even after 12 hours of application, significant fluorescence is still present in the oral cavity of mice.

[0085] 2. Evaluation of the treatment effect on xerostomia The efficacy of xerostomia treatment was evaluated in 20-week-old female NOD / LTJ mice. Daily oral swabs were applied with NS, WGA, artificial saliva (AS), WGA-PMPC, and commercially available imported Biotene mouthwash, respectively, and the mice's water intake was recorded daily. Results are as follows: Figure 10 As shown, the water intake of mice in the WGA-PMPC treatment group was significantly reduced. A comparison of water intake with that of mice treated with commercially available Biotene mouthwash also showed that the water intake of mice in the WGA-PMPC group was still significantly reduced.

[0086] 3. Evaluation of the treatment effect of dry eye syndrome After Cy7 was ligated into WGA and WGA-PMPC respectively, 5 μL of the above preparation was applied to the eye of each 8-week-old male C57 mouse. In vivo imaging (IVIS SPECTRUM) was performed to monitor changes in ocular fluorescence and to quantify the fluorescence. The results showed that WGA-PMPC achieved ocular retention in mice indistinguishable from WGA, and fluorescence was still detectable after 6 hours. Figure 11 As shown.

[0087] Eight-week-old male C57 mice were treated with 0.2% benzalkonium chloride eye drops (5 μL / eye, 3 times / day) for 7 consecutive days. After modeling, 2 μL of 1% sodium fluorescein was instilled into the conjunctival sac, and after 30 seconds, the eyes were rinsed with 0.25% chloramphenicol eye drops. The modeling was observed under a cobalt blue lamp microscope. Mice with successful modeling were divided into three groups for treatment: NS, polyvinyl alcohol eye drops (PA), and WGA-PMPC group, administered twice daily, 10 μL to each eye. After treatment, the left and right eyes of the mice were photographed under a cobalt blue lamp, and the corneal quadrant staining was scored from 0 to 4 (0: no staining; 1: mild punctate staining, <30 spots; 2: punctate staining >30 spots but not diffuse; 3: diffuse staining but no plaques; 4: positive plaques). Ten eyes were examined in each group, and the treatment outcomes were evaluated according to the scoring system. The average scores were then calculated and analyzed. The results are as follows: Figure 12 As shown, the fluorescein sodium score in the WGA-PMPC eye drop group was significantly lower than that in the no-treatment group and the commercially available polyvinyl alcohol eye drop group, demonstrating a good therapeutic effect on dry eye syndrome.

[0088] Example 2 WGA protein was replaced with pWGA (SQYGYCGFGAEY-NH2) peptide. pWGA is a functional polypeptide segment of WGA with adhesive properties, and its adhesive and wetting functions were verified. First, pWGA-PMPC was synthesized by ATRP. The specific preparation method is as follows: 1 mg of pWGA peptide was dissolved in 2 mL of PBS, 50 μL of 50 mM DBMP (dissolved in DMF) was added, and the reaction was carried out at room temperature for 2 hours. The solution was then dialyzed to remove salts, yielding pWGA-Br. MPC ionic monomers were added to the aqueous solution of pWGA-Br. The reaction system was deoxygenated by purging with high-purity nitrogen for 30 min. The ratio of pWGA-Br:CuCl:CuCl2:1,1,4,7,10,10-hexamethyltriethylenetetramine was 1:25:75:125. The polymerization reaction was carried out under nitrogen protection for 2 h, and finally, air was introduced to stop the reaction. The product was dialyzed to remove small-molecule copper salts, unreacted initiators, and monomers, yielding a pure pWGA-PMPC conjugate. The obtained pWGA-PMPC conjugate was validated using SDS-Page and GPC assays. WGA-PMPC with different molecular weights was prepared by using pWGA-Br and MPC at molar ratios of 1:1000 and 1:2000, with molecular weights of approximately 100,000 Da and 150,000 Da, respectively, to verify that the molecular weight of protein conjugates can be controlled by different feed ratios. The results are as follows: Figure 13 show.

[0089] The mucin-binding ability of the prepared peptide conjugates was further verified. After washing the wells with PBS, the wells were co-incubated with mucin at 37°C for 2 hours, and then blocked with 1% BSA. Subsequently, fluorescently labeled pWGA, pWGA-PMPC, and WGA-PMPC conjugates were added and incubated for another hour. The plates were washed three times with PBS to remove unbound conjugates. Finally, the fluorescence intensity was detected using a SpectraMax M5 microplate reader. The results are as follows: Figure 14 As shown, pWGA still exhibits mucin-binding ability after modification.

[0090] The wetting ability of pWGA-PMPC was verified by static contact angle. The static contact angle was measured using an OCA20 contact angle meter (DataPhysics, Germany). Hydrophobic polytetrafluoroethylene films were coated with pWGA and pWGA-PMPC (100 μg / mL) solutions, respectively, and then vacuum dried. The water contact angle of different composites was determined using the static drop method. The results are as follows: Figure 15 As shown, pWGA-PMPC (23.9±1.5°) exhibits superior hydrophilicity compared to pWGA (82.3±1.9°).

[0091] Verification of pWGA-PMPC oral adhesion performance in mice: Cy7 molecules were ligated to pWGA and pWGA-PMPC, respectively. Eight-week-old female Balb / c mice were used. After cleaning the oral cavity with physiological saline to remove food debris, 200 μL of the above preparation was evenly applied to the oral cavity of each mouse. In vivo imaging (IVIS SPECTRUM) was performed to monitor changes in oral fluorescence, and the fluorescence in the oral cavity was quantified. The results are as follows: Figure 16 The results showed that pWGA-PMPC exhibited good oral adhesion, and significant fluorescence was still present in the oral cavity of mice 12 hours after application.

[0092] The anti-Streptococcus mutans adhesion properties of different pWGA-PMPCs were studied using crystal violet staining. 100 μg / mL pWGA conjugates were uniformly coated onto polystyrene 12-well plates using the same treatment method as above.

[0093] The results are as follows Figure 17 The results showed that pWGA-PMPC exhibited a significant antibacterial adhesion effect against Streptococcus mutans.

[0094] Example 3 This embodiment illustrates the construction of protein zwitterionic polymeric conjugates. Unlike Example 1, the proteins used are glycoproteins with structures similar to the lectin-like proteins, specifically selectin, sialic acid adhesin, calcinin, and integrin. Results show that the protein zwitterionic polymeric conjugates prepared from different proteins exhibit adhesive, wetting, lubricating, and antibacterial properties comparable to those of Example 1, and can be used for joint lubrication, skin lubrication, mucosal lubrication, oral lubrication, ophthalmic lubrication, joint repair, and coatings for novel medical biomaterials or related medical devices.

[0095] Example 4 This embodiment demonstrates the construction of protein zwitterionic polymeric conjugates. Unlike Example 1, the zwitterion used is a TMAO amine oxide. Results show that protein zwitterionic polymeric conjugates prepared with different zwitterions exhibit adhesive, wetting, lubricating, and antibacterial properties comparable to those of Example 1, and can be used for joint lubrication, skin lubrication, mucosal lubrication, oral lubrication, ophthalmic lubrication, joint repair, novel medical biomaterials, or related medical devices.

[0096] Example 5 As a non-limiting embodiment, this embodiment illustrates a skin lubricant (lotion / body lotion) based on the protein zwitterionic polymer conjugate prepared in Example 1. Those skilled in the art will understand that the components and contents shown below can be adjusted according to actual needs.

[0097] 1. Formula Composition This embodiment provides a daily cosmetic composition for skin lubrication, the dosage form of which is an oil-in-water emulsion, comprising, by weight percentage: Active lubricating component: 0.5%-5.0% (e.g., 1.0%) of the protein zwitterionic polymer conjugate prepared in Example 1; Oil phase components: cetearyl alcohol 2.0%, caprylic / capric triglyceride 5.0%, polydimethylsiloxane 1.5%; Aqueous phase components: glycerol 3.0%, 1,3-butanediol 2.0%, carbomer 940 0.2%, sodium hyaluronate 0.05%; Emulsion system: 1.5% glyceryl stearate / PEG-100 stearate; pH adjuster: Triethanolamine (appropriate amount, to adjust pH to 5.5-6.5); Balance: Purified water.

[0098] 2. Preparation process (1) Mix the oil phase components and heat to 75°C to completely melt them; (2) Dissolve the aqueous phase components (except carbomer) and the conjugate from Example 1 in purified water; (3) Slowly add the oil phase to the water phase and homogenize and emulsify (3000 rpm, 5 min); (4) Add the pre-dispersed carbomer aqueous solution and preservative, and stir until well mixed; (5) Adjust the pH value to 6.0 with triethanolamine and discharge the material.

[0099] 3. Performance Evaluation Sensory evaluation: This product has a white, creamy texture, is easy to apply, spreads well, and forms a transparent, lubricating film within 30 seconds of application, without any greasy feeling or whitening. Compared to commercially available moisturizing lotions (containing 5% glycerin), the lubrication lasts 2.3 times longer. p <0.01).

[0100] Friction coefficient test: A UMT-2 friction and wear tester was used, with latex skin as the friction pair and a load of 0.5 N. The friction coefficient of this product was lower than that of the control group (Vaseline), proving that the composition of the present invention can significantly reduce skin friction resistance.

[0101] 4. Application Scenarios This product can be used as a daily body lotion or hand cream to improve the tightness caused by dry skin and reduce the friction between clothing and skin. It is for non-therapeutic skin care purposes.

[0102] Example 6 As a non-limiting embodiment, this embodiment illustrates an oral lubricant (mouthwash / oral spray) based on the protein zwitterionic polymer conjugate prepared in Example 1. Those skilled in the art will understand that the components and contents shown below can be adjusted according to actual needs.

[0103] 1. Formula Composition This embodiment provides a daily oral care composition for oral lubrication, the dosage form of which is a water-based mouthwash, comprising, by weight percentage: Active lubricating component: 0.1%-1.0% (e.g., 0.3%) of the protein zwitterionic polymer conjugate prepared in Example 1; Moisturizers: Glycerin 5.0%, Xylitol 2.0%; Thickening / film-forming agent: 0.2% hydroxyethyl cellulose; Flavor enhancers: 0.05% menthol, 0.02% sucralose; pH adjuster: appropriate amount of sodium citrate / citric acid (adjust pH to 6.8-7.2); Balance: Purified water.

[0104] 2. Preparation process (1) Disperse hydroxyethyl cellulose in a portion of purified water, heat to 60°C to fully swell, and cool for later use; (2) Dissolve the conjugate of Example 1, glycerol, and xylitol in the remaining purified water; (3) Combine the above solutions, add the flavor modifier, and stir until completely dissolved; (4) Adjust the pH to 7.0 with citric acid / sodium citrate, let stand to remove bubbles, and then fill.

[0105] 3. Performance Evaluation Lubrication durability test: Ten healthy subjects rinsed their mouths with 20 mL of this product for 30 seconds and then spat it out. Oral mucosal moisture levels were measured at 0 min, 15 min, and 30 min after rinsing using an oral mucosal hygrometer. The results showed that the moisture level in the product group was significantly higher than that in the control group after 30 min, indicating the formation of a long-lasting moisturizing and lubricating film.

[0106] Sensory evaluation: Subjects reported that the taste was refreshing, without astringency or abnormal stickiness, and did not irritate saliva secretion.

[0107] 4. Application Scenarios This product can be used as a daily mouthwash or oral spray to relieve dry mouth and improve discomfort caused by friction of the oral mucosa when eating / speaking. It is not intended for therapeutic use.

[0108] Example 7 As a non-limiting embodiment, this embodiment illustrates an eye lubricant (contact lens lubricant) based on the protein zwitterionic polymer conjugate prepared in Example 1. Those skilled in the art will understand that the components and contents shown below can be adjusted according to actual needs.

[0109] 1. Formula Composition This embodiment provides a lubricating solution for contact lens wear, comprising, by weight percentage: Active lubricating component: 0.01%-0.1% (e.g., 0.03%) of the protein zwitterionic polymer conjugate prepared in Example 1; Moisturizers: Polyvinylpyrrolidone (PVP K30) 0.5%, Sodium hyaluronate 0.01%; Buffer system: 0.5% boric acid / borax, pH 7.2-7.4; Osmotic pressure regulator: Sodium chloride 0.6%; Chelating agent: EDTA-2Na 0.05%; Balance: Water for injection.

[0110] 2. Preparation process (1) Take 80% of water for injection, add sodium chloride, boric acid / borax, and EDTA-2Na, and stir to dissolve; (2) Add the coupling compound from Example 1, PVP, and sodium hyaluronate, and stir at low temperature (4°C) for 2 hours until completely dissolved; (3) Add PHMB, add water to the full volume, and filter with a 0.22 μm filter membrane for sterilization; (4) Aseptic filling.

[0111] 3. Performance Evaluation Lens lubricity test: Commercially available silicone hydrogel contact lenses (47% water content) were immersed in this product for 4 hours, and the surface friction coefficient was measured using a tribological testing machine. The control group was PBS. The friction coefficient of this product group was significantly improved compared to the control group.

[0112] Protein deposition test: After 24 hours of incubation with lysozyme, the protein deposition in the lenses of this product group was significantly reduced compared with the control group (BCA method).

[0113] 4. Application Scenarios This product can be used for lens lubrication, anti-protein deposition, and improvement of wearing comfort during contact lens wear.

[0114] Example 8 As a non-limiting example, this embodiment illustrates a medical device coated with a protein zwitterionic polymer conjugate prepared in Example 1.

[0115] 1. Medical Device Example 1 - Hydrophilic Lubricated Urinary Catheter (Intermittent Urinary Catheter) This embodiment provides an intermittent urinary catheter with a surface coated with the protein zwitterionic polymer conjugate described in Example 1. The catheter is made of medical-grade silicone rubber and is 300 mm in length.

[0116] 1.1 Coating process: (1) Pretreatment: Immerse the catheter in 75% ethanol for ultrasonic cleaning for 15 min, rinse with pure water, and dry at 60℃; (2) Primer activation: Immerse the conduit in a 95% ethanol solution containing 2% 3-aminopropyltriethoxysilane (APTES), react at room temperature for 30 min, remove and drain, and cure for 30 min; (3) Main coating: Immerse the activated catheter in the coupling solution of Example 1 (concentration 5 mg / mL, PBS buffer, pH 7.4), soak at room temperature for 10 min, and pull it out with a uniform speed lifting machine (speed 50 mm / min); (4) Crosslinking and curing: The coated catheter was placed in a MES buffer solution containing 0.1% EDC and 0.05% NHS and reacted at room temperature for 2 h; (5) Post-treatment: Rinse three times with purified water to remove unbound material, air dry at room temperature, and sterilize with ethylene oxide.

[0117] 1.2 Performance Characterization: The dynamic friction coefficient is lower than that of commercially available hydrophilic coated conduits, and it has good coating durability.

[0118] 2. Medical Device Example 2 - Hydrophilic Lubricating Guidewire (Vascular Interventional Guidewire) This embodiment provides a vascular interventional guidewire with a surface coated with the protein zwitterionic polymer conjugate described in Example 1. The guidewire core is made of nitinol and coated with polyurethane, with a diameter of 0.035 inches and a length of 150 cm.

[0119] 2.1 Coating process: (1) Plasma activation: The guide wire was placed in a low-temperature plasma treatment machine with an oxygen atmosphere, a power of 100 W, and a treatment time of 3 min; (2) Dipping: After activation, the guidewire is immediately immersed in the coupling solution of Example 1 (concentration 3 mg / mL, containing 0.5% glutaraldehyde crosslinking agent) for 30 s, and then pulled up at a uniform speed of 30 mm / min. (3) Curing: Curing at room temperature for 24 h, followed by ultrasonic cleaning with pure water for 5 min to remove physical adsorption; (4) Drying: Vacuum drying for 12 h, followed by EO sterilization.

[0120] 2.2 Performance Characterization: Pushing force test: In a simulated tortuous vascular pathway model, the pushing force of this product's guidewire is lower than that of commercially available hydrophilic guidewires; Coating integrity: After repeated advancement / retraction of simulated blood vessels 30 times, the coating did not peel off and the coefficient of friction did not increase significantly.

[0121] 3. Medical Device Example 3 - Laparoscopic Surgery Grasping Forceps (Instrument Rod Lubrication) This embodiment provides a laparoscopic surgical grasping forceps with a surface coated with the protein zwitterionic polymer conjugate described in Example 1. The forceps are made of medical-grade stainless steel, with an outer diameter of 5 mm and a working length of 360 mm.

[0122] 3.1 Coating process: (1) Sandblasting treatment: The working section of the instrument is sandblasted with 50 μm alumina, Ra=0.3-0.5 μm; (2) Cleaning: Sonicate with acetone for 20 min, sonicate with isopropanol for 20 min, and dry at 60℃; (3) Spraying: The coupling solution of Example 1 (concentration 10 mg / mL) was uniformly sprayed onto the surface of the instrument using a high-pressure airless spraying device, with a wet film thickness of about 15 μm; (4) Curing; (5) Cooling: Cool to room temperature, package, and sterilize by irradiation.

[0123] 3.2 Performance Characterization: Resistance of the puncture card insertion and exit: In a simulated laparoscopic training box, the average resistance of the 10 mm puncture card was low after repeated insertion and exit 30 times. Coating hardness: Pencil hardness ≥ H, can withstand intraoperative forceps friction.

[0124] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. These modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A protein zwitterionic polymeric conjugate, characterized in that, It comprises proteins, peptides or functional fragments thereof with mucosal adhesion properties, and zwitterionic polymers linked to said proteins, peptides or functional fragments thereof.

2. The protein zwitterionic polymeric conjugate according to claim 1, characterized in that, The protein includes lectin-like proteins or their functional fragments, or proteins or their functional fragments that have a similar structure to the lectin-like proteins and have sugar-binding activity.

3. The protein zwitterionic polymeric conjugate according to claim 1, characterized in that, The zwitterions include at least one of phosphorylcholine, sulfobetaine, carboxybetaine, TMAO amine oxides or their derivatives.

4. A composition having a lubricating function, characterized in that, It includes the protein zwitterionic polymeric conjugate according to any one of claims 1-3.

5. The composition with lubricating function according to claim 4, characterized in that, When the composition is applied to surfaces including joints, skin, mucous membranes and / or medical devices, it reduces the coefficient of friction of the surface.

6. The composition with lubricating function according to claim 4, characterized in that, The lubricating composition includes daily necessities or pharmaceuticals.

7. A medical device, characterized in that, Includes the protein zwitterionic polymeric conjugate as described in any one of claims 1-3; Preferably, the protein zwitterionic polymer conjugate forms a coating on the surface of the medical device.

8. A method for preparing a protein zwitterionic polymeric conjugate, characterized in that, include: (1) Use an initiator to modify proteins, peptides or their functional fragments that have mucosal adhesion properties to obtain modified proteins, peptides or their functional fragments. (2) The modified protein, polypeptide or its functional fragment is polymerized with zwitterionic monomer to obtain the conjugate.

9. The use of the protein zwitterionic polymeric conjugate according to any one of claims 1-3 in the preparation of medical biomaterials or medical devices.

10. The application according to claim 9, characterized in that, The medical biomaterial is used to provide lubrication. Preferably, the medical biomaterial is used for Sjögren's syndrome, which includes xerostomia or dry eye syndrome.

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