Nanogel for rapid permeation of articular cartilage and responsive release of growth factors

Abstract

The invention provides a nanogel for rapid permeation of articular cartilage and responsive release of a growth factor, which is prepared by polymerizing a peptide fragment which can be cut by a substance (mainly enzyme) which can be specifically expressed on the surface of osteoarthritis cells or secreted into a matrix with the growth factor, an acrylamide monomer and a cationic micromolecule monomer. Unreacted monomer molecules are filtered out through an ultrafiltration tube or other filtering systems, so that the nanogel which can rapidly permeate into the deep part of articular cartilage and can be recognized and cut by enzyme highly expressed on the surface of osteoarthritis cells or secreted enzyme to release growth factors is prepared, and the effect of the growth factors can be more effectively exerted; the nanogel has strong cartilage infiltration capacity, can completely infiltrate cartilage with the thickness of 1mm within less than 3 days, can be cut by MT1-MMP protein specifically expressed on the surfaces of osteoarthritis cells, releases growth factors, is specifically combined with growth factor receptors on the surfaces of cartilage cells, and activates downstream signal channels, so that the proliferation of the cartilage cells is promoted, and the cartilage cell proliferation rate is increased. The cartilage matrix secretion is promoted, so that the cartilage matrix thickening effect is realized, and the osteoarthritis is prevented and treated.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical materials, and in particular relates to a nanogel that rapidly penetrates articular cartilage and responsively releases growth factors. Background Art

[0002] Current research on local injection therapy for articular cartilage focuses on increasing the lubrication between articular cartilage and promoting rapid drug penetration deep into the joint to promote matrix regeneration. Many naturally occurring and synthetic macromolecular / supramolecular systems possess excellent biolubrication properties and have been extensively studied for joint lubrication. While this approach can alleviate pain, improve movement, and slow or prevent further cartilage degeneration, it has no therapeutic effect on established cartilage lesions. Therefore, current research is not only focused on increasing the lubrication function of the joint surface itself, but also on promoting rapid drug penetration deep into the cartilage.

[0003] To promote rapid drug penetration deep into cartilage, delivery systems including nanoparticles, microparticles, and hydrogels have been developed. However, these delivery systems are larger than the articular cartilage space and therefore cannot effectively penetrate the cartilage. Nanoparticles, on the other hand, have the advantage of being smaller; those with a diameter less than 20 nm can freely penetrate the cartilage matrix.

[0004] Osteoarthritis (OA) is the most common joint disease characterized by damage to articular cartilage and involvement of the entire joint tissue, ultimately leading to degeneration, fibrosis, fracture, defect, and damage to the entire joint surface. Growth factors participate in the proliferation, differentiation, and migration of chondrocytes and play a crucial role in the maintenance and degradation of the cartilage matrix. Various growth factors, such as fibroblast growth factor 2 (FGF2), transforming growth factor β (TGF-β), insulin-like growth factor 1 (IGF-1), and bone morphogenetic protein 7 (BMP-7), have been shown to induce or stimulate chondrocyte differentiation. These factors can be used to enhance cartilage healing and prevent the development and progression of degenerative osteoarthritis. To enable rapid penetration of growth factors deep into the cartilage, nanoparticle delivery is a viable option.

[0005] However, current research has focused on the design and preparation of growth factor nanogels, and there is no method for preparing nanogels that can responsively release growth factors on the surface of osteoarthritis chondrocytes to achieve targeted osteoarthritis chondrocyte treatment.

[0006] Therefore, it is urgent to find a new method for preparing growth factor nanoparticles that can enable the growth factor to quickly penetrate the entire articular cartilage and be responsively released on the surface of osteoarthritis chondrocytes, thereby achieving the thickening of the cartilage matrix and achieving the effect of preventing and treating osteoarthritis. Summary of the Invention

[0007] To remedy the deficiencies of the prior art, the present invention provides a nanogel that rapidly penetrates articular cartilage and responsively releases growth factors, as well as a preparation method and application thereof. The method increases the overall positive charge level of the growth factor preparation by in situ polymerizing the growth factor with a positively charged cationic monomer molecule, controls the particle size to be less than 40 nm, and polymerizes the growth factor and acrylamide with a peptide segment that can be cleaved by a substance specifically expressed on the surface of osteoarthritis cells, and then ultrafilters the unreacted monomer molecules using an ultrafiltration tube, thereby producing a nanogel carrier that can quickly penetrate deep into the articular cartilage and can be recognized and cleaved by enzymes on the surface of osteoarthritis cells or enzymes secreted into the matrix by the cells, thereby releasing the growth factor loaded therein and more effectively exerting the growth factor effect. The nanogel prepared by this method has a strong cartilage penetration ability and can completely penetrate 1 mm thick cartilage in osteoarthritis cartilage explants in less than 3 days. It can also be cleaved by the MT1-MMP protein specifically expressed on the surface of osteoarthritis cells, releasing the growth factor, which specifically binds to the growth factor receptor on the surface of the chondrocytes, activates downstream signaling pathways, and thus promotes chondrocyte proliferation and cartilage matrix secretion, thereby achieving the effect of cartilage matrix thickening, and realizing the prevention and treatment of osteoarthritis.

[0008] On the one hand, the present invention provides a nanogel that rapidly penetrates articular cartilage and responsively releases growth factors. The nanogel includes growth factors, responsive polypeptides, neutral small molecules with double bonds, and cationic small molecules with double bonds. The nanogel is first prepared by a mixed reaction of growth factors, responsive polypeptides, neutral small molecules with double bonds, and cationic small molecules, and then in situ cross-linking.

[0009] Furthermore, the growth factors include any one or more of fibroblast growth factor 2 (FGF2), recombinant human transforming growth factor β (TGF-β), insulin-like growth factor 1 (IGF-1), and bone morphogenetic protein 7 (BMP-7).

[0010] It is understandable that any growth factor can be used to prepare nanogels that rapidly permeate articular cartilage and responsively release growth factors.

[0011] Furthermore, the responsive polypeptide includes any one or more of membrane type metalloproteinase-1 (MT1-MMP) cleavable polypeptide (KRRQLGLPALSβAla, KRRQLGLGLTAβAla, KRRQLGPLGVASβAla, KRRQLASGLTGGβAla, KRRQLGGASGVLLβAla), matrix metalloproteinase 13 (MMP13) cleavable peptide (PGAPPL, QPQGLAK, GPLGMHGK, GPLGLSLGK, CPGAPPG, PLGLAARK), metalloproteinase domain protein 5 (ADAMTS5) cleavable peptide (aggrecan, Fibulin-2), metalloproteinase domain protein 4 (ADAMTS4) cleavable peptide (aggrecan, Fibulin-2), tumor necrosis factor α (TNF-α), interleukin 1 (IL-1), interleukin 2 (IL-2), interleukin 6 (IL-6), etc.

[0012] Theoretically, any chondrocyte-responsive peptide can be used to prepare nanogels. By cross-linking neutral and positively charged small molecules with responsive peptides, the nanogels can be endowed with pathologically responsive release capabilities, enabling the targeted release and activation of growth factors on the surface of chondrocytes in osteoarthritis. This reduces the waste of expensive growth factors in non-active areas and reduces off-target effects, thereby increasing their utilization efficiency.

[0013] In some embodiments, the responsive polypeptide is a responsive polypeptide with a double bond modification.

[0014] The responsive peptide needs to have a double bond because only the responsive peptide with a double bond can cross-link with acrylamide.

[0015] Furthermore, the cationic small molecule with a double bond includes any one or more of methacrylamide (APM-AAM), vinyltrimethylammonium (VTM), acrylic acid (AAc), acryloyl chloride (AACl), phenol formic acid (PMA), fumaric acid (FMA), methyl methacrylate (MMA), vinyltrimethylammonium chloride (ETMAC), butenol (E), dimethyl butadiene diacid (DEGDMA), and ethyl acrylate (EMA).

[0016] Cationic small molecules with double bonds can impart a positive charge to growth factors, enhancing electrostatic interactions with the negatively charged cartilage matrix. This improves penetration into the dense cartilage matrix and enhances retention within the matrix. Cationic small molecules require double bonds because they can crosslink with acrylamide.

[0017] It is understandable that any cationic small molecule with a double bond that carries a positive charge can be used to prepare nanogels that quickly penetrate articular cartilage and responsively release growth factors, and all have the effect of promoting the penetration of growth factors in cartilage.

[0018] In some methods, different cationic small molecules have different effects on promoting the penetration of growth factors in cartilage. It is preferred to use methacrylamide (APM-AAM) as a cationic small molecule, which helps to increase the penetration depth, has less toxicity to chondrocytes, and has higher chondrocyte activity.

[0019] Furthermore, the neutral small molecule with a double bond is acrylamide (AAM).

[0020] Furthermore, the mass fraction of the growth factor is 0.01-1%; the mass fraction of the responsive polypeptide is 0.1-5%, the mass fraction of the acrylamide is 5-40%, and the mass fraction of the cationic small molecule is 5-40%.

[0021] The ratio of each component in the nanogel will directly affect the penetration effect of the growth factor in the cartilage, especially the content of responsive peptides and acrylamide, which will directly affect the penetration depth of the growth factor in the cartilage and also affect the activity of chondrocytes.

[0022] In another aspect, the present invention provides a method for preparing a nanogel that rapidly permeates articular cartilage and responsively releases growth factors, the method comprising the following steps:

[0023] (1) mixing a growth factor, a responsive polypeptide, a neutral small molecule with a double bond, and a cationic small molecule to produce a mixed product;

[0024] (2) Cross-linking the mixed product in situ.

[0025] Furthermore, step (1) includes the following steps:

[0026] (a) The growth factor solution and the responsive polypeptide are dissolved in an alkaline buffer to reach equilibrium, obtaining a completely dissolved macromolecular solution;

[0027] (b) The completely dissolved macromolecular solution is mixed with neutral small molecules with double bonds and cations with double bonds to prepare a nanogel precursor solution.

[0028] Furthermore, in step (a), the growth factor solution reacts with the responsive polypeptide solution using a weakly alkaline buffer solution in an ice bath environment.

[0029] The preparation process of osteoarthritis chondrocyte-responsive growth factor includes two steps: dissolution equilibrium of growth factor and responsive polypeptide, and interaction of growth factor with neutral small molecule and cationic small molecule monomers through hydrophobic interaction or charge interaction to form nanogel precursor.

[0030] Studies have shown that alkaline buffer is very critical in the preparation of osteoarthritis chondrocyte-responsive growth factors. Only in an alkaline environment can the growth factor and the responsive polypeptide undergo cross-linking reaction. Neither acidic nor neutral environments can cause the growth factor and the responsive polypeptide to react.

[0031] In some embodiments, the alkaline buffer used in the preparation of the osteoarthritis chondrocyte-responsive growth factor is a sodium carbonate / sodium bicarbonate buffer.

[0032] In some approaches, a sodium carbonate / sodium bicarbonate buffer is used to solubilize the growth factors.

[0033] The ice bath environment is used to ensure the activity of the growth factors during the reaction.

[0034] In some methods, when a sodium carbonate / sodium bicarbonate buffer is used and the reaction environment is an ice bath environment, the reaction can be promoted in a weakly alkaline environment without affecting the biological activity of the growth factor and the osteoarthritis chondrocyte cleavage peptide. Compared with the existing commonly used room temperature environment reaction, it has a better effect of protecting the biological activity of the growth factor and has a more stable preparation effect.

[0035] Furthermore, the reaction time of step (a) is 5 to 20 minutes; the reaction time of step (b) is 5 to 20 minutes.

[0036] Furthermore, step (2) is: adding a catalyst and a reducing agent to the mixed product to perform in-situ crosslinking, wherein the catalyst is tetramethylethylenediamine (TEMED) and the reducing agent is ammonium persulfate (APS).

[0037] In some embodiments, the catalyst is added in a dosage of 1-5 μl, and the reducing agent is added in a dosage of 10-50 μl.

[0038] In some embodiments, the in situ cross-linking reaction time is 1-2 hours.

[0039] Furthermore, the method further comprises step (3) of purifying the nanogel by ultrafiltration through an ultrafiltration tube. Unreacted monomer molecules are repeatedly ultrafiltered through a 10KDa ultrafiltration tube; or unreacted monomer molecules are filtered through a 10KDa vacuum fiber column using a tangential flow filtration device (TFF). Thus, purified nanogel that rapidly permeates articular cartilage and responsively releases growth factors is obtained.

[0040] On the other hand, the present invention provides a use of a nanogel for preparing a reagent that can quickly penetrate articular cartilage and responsively release growth factors. The nanogel includes growth factors, responsive polypeptides, neutral small molecules with double bonds, and cationic small molecules; the growth factors, responsive polypeptides, neutral small molecules with double bonds, and cationic small molecules are first mixed and reacted, and then cross-linked in situ to obtain the reagent.

[0041] The beneficial effects of the nanogel provided by the present invention that rapidly penetrates into articular cartilage and responsively releases growth factors are:

[0042] 1. A growth factor that responds to osteoarthritis chondrocytes has been invented: by forming a nanogel carrier on the surface of the growth factor, the growth factor is loaded into the nanogel, and by using a peptide cleavable by an enzyme highly expressed on the surface of osteoarthritis chondrocytes as a cross-linker for the nanogel carrier, the nanogel carrier can be disintegrated in a targeted manner on the surface of chondrocytes in the osteoarthritis state, thereby releasing the growth factor encapsulated inside and exerting its effect, reducing the waste of growth factor in non-active areas and achieving higher growth factor utilization efficiency.

[0043] 2. By cross-linking small molecules that can provide cationic groups with polypeptides that respond to osteoarthritis chondrocytes, the nanogel loaded with growth factors has sufficient positive charge, thereby increasing its penetration in the dense cartilage matrix by enhancing the electrostatic interaction force with the negatively charged cartilage matrix and improving its retention effect in the matrix, further improving the penetration and therapeutic effect of growth factors in the cartilage matrix and prolonging the drug utilization rate of growth factors.

[0044] 4. The prepared nanogel has a strong cartilage penetration ability. It takes less than 3 days to completely penetrate 1mm thick cartilage in osteoarthritis cartilage explants. It can be cut by molecules specifically expressed on the surface of osteoarthritis cells, releasing growth factors, which specifically bind to growth factor receptors on the surface of chondrocytes, activate downstream signaling pathways, thereby promoting chondrocyte proliferation and cartilage matrix secretion, thereby achieving the effect of thickening the cartilage matrix and realizing the prevention and treatment of osteoarthritis. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 This is a flow chart for preparing the osteoarthritis chondrocyte-responsive growth factor prepared in Example 1;

[0046] Figure 2 This is a transmission electron microscopy image of the MT1-MMP responsive growth factor nanogel prepared in Example 2;

[0047] Figure 3 This is a graph showing the effect of FGF2 cartilage penetration after 3 days in Example 2;

[0048] Figure 4 This is a diagram showing the 3-day cartilage penetration effect of the MT1-MMP responsive FGF2 nanogel and simple FGF2 in Example 2. DETAILED DESCRIPTION

[0049] The present invention will be described in further detail below in conjunction with the examples. It should be noted that the examples described below are intended to facilitate understanding of the present invention and do not serve to limit the present invention in any way. The reagents not otherwise specified in the present examples are all known products and were obtained by purchasing commercially available products.

[0050] Example 1: Preparation of osteoarthritis chondrocyte-responsive growth factor provided by the present invention

[0051] The preparation flow chart of osteoarthritis chondrocyte responsive growth factor provided in this embodiment is as follows: Figure 1 As shown, the preparation method is as follows:

[0052] (1) Dissolve 1-20 mg of growth factor (in this example, fibroblast growth factor 2 (FGF2), Nanjing Yizhimei Biotechnology Co., Ltd., model: lyophilized powder, the mass of FGF2 is preferably 10 mg) in 1 ml of sodium carbonate / sodium bicarbonate buffer at pH 9.5 to obtain a 10 mg / ml FGF2 solution.

[0053] (2) 0.001-0.02 g of osteoarthritis chondrocyte cleavable peptide (MT1-MMP cleavable peptide was selected in this embodiment and synthesized by Hefei Sener Biotechnology Co., Ltd., including five sequences: KRRQLGLPALSβAla, KRRQLGLGLTAβAla, KRRQLGPLGVASβAla, KRRQLASGLTGGβAla, and KRRQLGGASGVLLβAla (KRRQLGLPALSβAla is preferred in this embodiment). The cleavable peptide is a peptide sequence, and the peptide actually used is a modified peptide with double bonds at both ends of the 5' and 3' ends of the sequence peptide. The modification method is: allylglycine with double bonds is modified at both ends of the peptide by condensation reaction. The molecular formula of KRRQLGLPALSβAla with allylglycine end modification is as follows:

[0054]

[0055] The MT1-MMP cleavable peptide (preferably 0.0065 g) was dissolved in 0.9 ml of sodium carbonate / sodium bicarbonate buffer (pH 9.4-9.6). After complete dissolution, 0.1 ml of 10 mg / ml FGF2 solution was added in an ice bath. The mixture was stirred at 400 rpm for 5-20 minutes (preferably 10 minutes in this embodiment) to prepare MT1-MMP-responsive FGF2.

[0056] Example 2: Preparation of MT1-MMP responsive growth factor nanogel provided by the present invention

[0057] The preparation method of the MT1-MMP responsive growth factor nanogel provided in this embodiment is as follows:

[0058] (a) MT1-MMP responsive FGF2 was prepared using the method provided in Example 1.

[0059] (b) Acrylamide (AAM, purchased from Sigma, product number A9099) is reacted with MT1-MMP-responsive FGF2 at an AAM concentration of 5%-40% (preferably 20% in this embodiment) in a volume of 10 μl-100 μl (preferably 49.75 μl in this embodiment). The reaction time with MT1-MMP-responsive FGF2 is in an ice bath for 5-20 minutes (preferably 10 minutes in this embodiment) to obtain a first product.

[0060] (c) A cationic small molecule (in this example, methacrylamide (APM-AAM, purchased from Aladdin, product number A112450) is reacted with MT1-MMP-responsive FGF2. The APM-AAM concentration used is 5%-40% (preferably 20% in this example), the added volume is 10 μl-100 μl (preferably 56.78 μl in this example), and the reaction time with the MT1-MMP-responsive growth factor is ice-bathed for 1-10 minutes (preferably 5 minutes in this example) to obtain a mixed product.

[0061] (d) The mixed product is catalyzed by TEMED (available from Aladdin, catalog number T105497) and reduced by APS (available from Aladdin, catalog number N129096) for in situ crosslinking. The dosage of TEMED used is 1-5 μl (preferably 2 μl in this embodiment), the dosage of APS added is 10-50 μl (preferably 20 μl in this embodiment), and the reaction time is 30-120 minutes (preferably 60 minutes in this embodiment).

[0062] (e) The MT1-MMP-responsive FGF2 nanogel was purified by repeated ultrafiltration using a 10 kDa ultrafiltration tube using a HEPES buffer at pH 7.4, and the volume was finally adjusted to 1 ml to obtain 1 mg / ml MT1-MMP-responsive FGF2 nanogel.

[0063] Transmission electron microscopy images of the prepared MT1-MMP responsive growth factor FGF2 nanogels are shown in Figure 2. Figure 2 shown.

[0064] The cartilage penetration depth of pure FGF2 and MT1-MMP responsive FGF2 nanogels was compared after 3 days and recorded by fluorescence microscopy. Figure 3 and Figure 4 As shown. Figure 3 This is the effect of FGF2 cartilage penetration after 3 days.

[0065] Figure 4 This is the effect of MT1-MMP responsive FGF2 nanogel cartilage penetration after 3 days. Figure 3 and Figure 4 It can be seen that after 3 days, FGF2 alone could not penetrate the cartilage, while the MT1-MMP responsive FGF2 nanogel could penetrate the cartilage to more than 1 mm.

[0066] Example 3: Effects of MT1-MMP Responsive FGF2 and Different Cationic Small Molecules on the Preparation of MT1-MMP Responsive FGF2 Nanogels

[0067] In this example, the MT1-MMP-responsive FGF2 prepared in Example 1 was first reacted with acrylamide (AAM) at a concentration of 20% and a volume of 49.75 μl. The reaction time with the MT1-MMP-responsive growth factor was 10 minutes in an ice bath to obtain AAM / MT1-MMP-responsive FGF2. The FGF2 was then reacted with different small molecules that can provide cationic groups (according to the preparation method provided in Example 2). These small molecules include 11 types: methacrylamide (APM-AAM), vinyltrimethylammonium (VTM), acrylic acid (AAc), acryloyl chloride (AACl), phenolic acid (PMA), butenedioic acid (FMA), methyl methacrylate (MMA), vinyltrimethylammonium chloride (ETMAC), butenol (E), dimethyl butadiene diacid (DEGDMA), and ethyl acrylate (EMA). The results were compared with the case where no cationic small molecules were contained or the neutral small molecule aminoacetic acid was used instead. Twelve groups of MT1-MMP-responsive FGF2 nanogels were prepared and tested for particle size and zeta potential, chondrocyte toxicity, and penetration efficiency in osteoarthritis cartilage explants. Particle size and zeta potential were measured using dynamic light scattering (DLS), chondrocyte toxicity was measured using the Cell Counting Kit-8 cell proliferation / cytotoxicity assay, and penetration efficiency in osteoarthritis cartilage explants was measured by observing the penetration depth of the fluorescently labeled nanogels into the cartilage using a fluorescence microscope. The results are shown in Table 1.

[0068] Table 1. Effects of MT1-MMP-responsive FGF2 on the preparation of nanogels by reacting with different cationic small molecules

[0069]

[0070]

[0071] As shown in Table 1, although neutral small molecules alone can produce nanogels, their affinity for cartilage is significantly reduced due to their near-zero surface charge. Consequently, their cartilage penetration is very poor, making it difficult for them to penetrate the dense chondrocytes to function. However, the addition of cationic small molecules significantly increases the penetration depth of the MT1-MMP-responsive FGF2 nanogels. Without either cationic or neutral small molecules, there are no monomers to provide nanocoagulation and cross-linking, preventing the in situ formation of nanogel carriers for the protein, thus rendering them ineffective.

[0072] It can also be seen that the effects of nanogels prepared with different cationic small molecules also vary. Compared with other cationic small molecules such as VTM, AAc, AAC1, PMA, FMA, MMA, ETMAC, E, DEGDMA, and EMA, the nanogels obtained by reacting MT1-MMP-responsive FGF2 with AAM+APM / AAM (methacrylamide) can significantly enhance the penetration of FGF2 in cartilage explants and minimize toxicity to chondrocytes. However, when MT1-MMP-responsive FGF2 reacts with other small molecules, the resulting nanogels may have too large a particle size, affecting the penetration effect, carry too low a positive charge, reducing the cartilage penetration efficiency, or carry too high a positive charge, increasing cytotoxicity. Therefore, compared with other cationic small molecules, MT1-MMP-responsive FGF2 reacts significantly better with AAM+APM / AAM (methacrylamide).

[0073] In addition, this example further examined the cartilage penetration depth of 12 groups of MT1-MMP-responsive FGF2 nanogels after 3 days, and found that the cartilage penetration depth of the nanogel prepared using the cationic small molecule methacrylamide exceeded 1 mm after 3 days, reaching 1.04 mm, which was significantly higher than that of the other cationic small molecule groups.

[0074] Example 4: Effects of different responsive polypeptides on the preparation of responsive growth factor nanogels

[0075] In this example, different responsive peptides were used to prepare responsive FGF2 according to Example 1. All responsive peptides were peptides modified with allylglycine with double bonds at the 5' and 3' ends of the peptide sequence via a condensation reaction. Responsive FGF2 nanogels were then prepared according to Example 2. The different responsive peptides included MT1-MMP (KRRQLGLPALSβAla), MMP13 (PGAPPL, QPQGLAK, GPLGMHGK, GPLGLSLGK, CPGAPPG, and PLGLAARK, with QPQGLAK being preferred in this example), and ADAMTS5 (KY(NO2)SENESRGK(Abz)IYYKKG), and the results were compared with a sample without a responsive peptide. Nine groups of responsive FGF2 nanogels were prepared and tested for particle size and zeta potential, chondrocyte toxicity, and penetration efficiency in osteoarthritis cartilage explants. Particle size and zeta potential were measured using dynamic light scattering (DLS), chondrocyte toxicity was measured using the Cell Counting Kit-8 cell proliferation / cytotoxicity assay, and penetration efficiency in osteoarthritis cartilage explants was measured by observing the penetration depth of the fluorescently labeled nanogels into the cartilage using a fluorescence microscope. The results are shown in Table 2.

[0076] Table 2. Effects of different osteoarthritis chondrocyte-responsive peptides on the preparation of responsive nanogels

[0077]

[0078] As shown in Table 2, nanogels cannot be prepared without responsive peptides. The reason is that the responsive peptides, modified with double bonds at both ends, act as crosslinkers for nanogels, crosslinking the small molecule monomers described above in situ to form nanogels on the protein surface. Without these responsive peptides, crosslinking to form nanogels is impossible. Nanogels constructed with different responsive peptides exhibited distinct effects. Some responsive peptides helped enhance the permeability of nanogels into cartilage explants. This may be due to the responsive peptides' influence on the particle size and charge of the FGF2 nanogels, with the extent of this influence primarily related to the molecular weight of the cleavable peptides themselves and the positive and negative charged groups they carry.

[0079] Comparison of different responsive peptides showed that the FGF2 nanogel constructed with MT1-MMP cleavable peptide had a relatively small particle size and a relatively high positive charge level, and had the strongest permeability to cartilage explants.

[0080] Example 5: Effects of different growth factors on the preparation of MT1-MMP responsive growth factor nanogels

[0081] The present embodiment adopts different growth factors respectively, prepares MT1-MMP responsive growth factor according to embodiment 1, and then prepares MT1-MMP responsive growth factor nanogel according to embodiment 2. Different growth factors include fibroblast growth factor 2 (FGF2), recombinant human transforming growth factor beta (TGF-β), insulin-like growth factor 1 (IGF-1), bone morphogenetic protein 7 (BMP-7), a total of 4 kinds, and are compared with the situation that does not contain growth factor. The 5 groups of MT1-MMP responsive growth factor nanogels prepared were respectively detected their particle size and Zeta potential level, detected their toxic effect on chondrocytes, and detected their penetration efficiency in osteoarthritis cartilage explants, wherein the detection method of particle size and Zeta potential is dynamic light scattering (DLS), the detection method of toxic effect on chondrocytes is Cell Counting Kit-8 cell proliferation / cytotoxicity detection, and the detection method of penetration efficiency in osteoarthritis cartilage explants is fluorescence microscopy to observe the penetration depth of fluorescent-labeled nanogel in cartilage. The test results are shown in Table 3.

[0082] Table 3. Effects of different growth factors on the preparation of MT1-MMP responsive nanogels

[0083]

[0084] As shown in Table 3, MT1-MMP-responsive growth factor nanogels could not be produced without growth factors. This is because nanogels are formed by in situ polymerization on the protein surface. Without the protein core, all small-molecule monomers and crosslinkers would be free in the solution and unable to self-assemble into nanogels. Nanogels containing growth factors can increase the positive charge level and enhance the activity of chondrocytes.

[0085] Nanogels prepared from TGF-β, IGF-1, BMP-7, and FGF2 can all enhance the Zeta potential level of the growth factors themselves and increase their positive charge levels. Among them, FGF2 nanogel has the highest positive charge level, the strongest active effect on chondrocytes, and the strongest penetration ability in cartilage.

[0086] Example 6: Effects of Responsive and Non-Responsive Peptides on the Preparation of Growth Factor Nanogels

[0087] In this example, the particle sizes and zeta potential levels of MT1-MMP responsive growth factor nanogel and non-responsive growth factor nanogel were compared, and their toxic effects and permeability on chondrocytes were detected.

[0088] The preparation method of MT1-MMP responsive growth factor nanogel is as follows:

[0089] (1) Dissolve 10 mg of FGF2 in 1 ml of sodium carbonate / sodium bicarbonate buffer to obtain a 10 mg / ml FGF2 solution.

[0090] (2) Dissolve 0.001-0.02 g of osteoarthritis chondrocyte-cleavable peptide (MT1-MMP-cleavable peptide is selected in this example, and the mass of MT1-MMP-cleavable peptide is preferably 0.0065 g) in 0.9 ml of sodium carbonate / sodium bicarbonate buffer. After complete dissolution, add 0.1 ml of 10 mg / ml FGF2 solution in an ice bath environment, stir at 400 rpm for 5-20 minutes (preferably 10 minutes in this example) to prepare MT1-MMP-responsive FGF2.

[0091] (3) Acrylamide (AAM) is reacted with the MT1-MMP responsive growth factor, the AAM concentration used is 5%-40% (preferably 20% in this embodiment), the added volume is 10 μl-100 μl (preferably 49.75 μl in this embodiment), and the reaction time with the MT1-MMP responsive growth factor is 5-20 minutes (preferably 10 minutes in this embodiment).

[0092] (4) The cationic small molecule (APM-AAM is selected in this embodiment) is reacted with the MT1-MMP responsive growth factor. The APM-AAM concentration used is 5%-40% (preferably 20% in this embodiment), the added volume is 10 μl-100 μl (preferably 56.78 μl in this embodiment), and the reaction time with the MT1-MMP responsive growth factor is 1-10 minutes (preferably 5 minutes in this embodiment).

[0093] (5) MT1-MMP responsive growth factor is cross-linked in situ using TEMED catalysis and APS reduction. The dosage of TEMED used is 1-5 μl (preferably 2 μl in this embodiment), the dosage of APS added is 10-50 μl (preferably 20 μl in this embodiment), and the reaction time is 30-120 minutes (preferably 60 minutes in this embodiment).

[0094] (6) Using HEPES buffer at pH 7.4, the MT1-MMP responsive FGF2 nanogel was purified by repeated ultrafiltration using a 10 kDa ultrafiltration tube, and the volume was finally adjusted to 1 ml to obtain 1 mg / ml MT1-MMP responsive FGF2 nanogel.

[0095] The preparation method of the non-responsive growth factor nanogel provided in this embodiment is as follows:

[0096] (1) Dissolve 1-20 mg of growth factor (fibroblast growth factor 2 (FGF2) is selected in this example, and the mass of FGF2 is preferably 10 mg) in 1 ml of sodium carbonate / sodium bicarbonate buffer to obtain a 10 mg / ml FGF2 solution.

[0097] (2) Dissolve 0.1 ml of 10 mg / ml FGF2 solution in 0.9 ml of sodium carbonate / sodium bicarbonate buffer and stir at 400 rpm in an ice bath for 5-20 minutes (preferably 10 minutes in this embodiment).

[0098] (3) Acrylamide (AAM) is reacted with the growth factor, the AAM concentration used is 5%-40% (preferably 20% in this embodiment), the added volume is 10 μl-100 μl (preferably 49.75 μl in this embodiment), and the reaction time with the growth factor is 5-20 minutes (preferably 10 minutes in this embodiment).

[0099] (4) The cationic small molecule (APM-AAM is selected in this embodiment) is reacted with the growth factor. The APM-AAM concentration used is 5%-40% (preferably 20% in this embodiment), the added volume is 10μl-100μl (preferably 56.78μl in this embodiment), and the reaction time with the growth factor is 1-10 minutes (preferably 5 minutes in this embodiment).

[0100] (5) Adding a non-osteoarthritis chondrocyte-responsive compound 2-mercaptobenzoic acid for reaction (2-mercaptobenzoic acid can be used to replace the MT1-MMP responsive polypeptide substance, thereby being able to construct a nanogel without responsive properties, otherwise the nanogel cannot be constructed), the concentration of 2-mercaptobenzoic acid used is 1%-10% (preferably 4% in this embodiment), and the added volume is 10 μl-100 μl (preferably 53.95 μl in this embodiment).

[0101] (6) In situ crosslinking is performed using TEMED catalysis and APS reduction of growth factors. The dosage of TEMED used is 1-5 μl (preferably 2 μl in this embodiment), the dosage of APS added is 10-50 μl (preferably 20 μl in this embodiment), and the reaction time is 30-120 minutes (preferably 60 minutes in this embodiment).

[0102] (7) Using HEPES buffer at pH 7.4, the non-osteoarthritis chondrocyte-responsive FGF2 nanogel was purified by repeated ultrafiltration using a 10 kDa ultrafiltration tube, and the volume was finally adjusted to 1 ml to obtain 1 mg / ml non-osteoarthritis chondrocyte-responsive FGF2 nanogel.

[0103] The particle size and zeta potential of the two groups of growth factor nanogels were tested, and their cytotoxic effects on chondrocytes were tested using the same testing method as in Example 3. The test results are shown in Table 4.

[0104] Table 4. Effects of responsive and non-responsive compounds on the preparation of nanogels

[0105]

[0106] As shown in Table 4, there were no significant differences in particle size, zeta potential, and one-day cartilage penetration depth between the non-osteoarthritis chondrocyte-responsive nanogels and the MT1-MMP-responsive nanogels. However, the non-osteoarthritis chondrocyte-responsive nanogels significantly weakened their chondrocyte activity-promoting effect compared to the MT1-MMP-responsive nanogels. This is because the MT1-MMP-responsive nanogels can be cleaved and decomposed by chondrocytes, releasing the encapsulated FGF2 growth factor, thereby better exerting the growth factor's effect and improving its utilization efficiency. In contrast, the FGF2 in the non-osteoarthritis chondrocyte-responsive nanogels was encapsulated within the nanogels, preventing their full cell activity-promoting effect.

[0107] Example 7: Effect of different MT1-MMP cleavable peptide sequences on the preparation of MT1-MMP responsive growth factor nanogels

[0108] The preparation method of the MT1-MMP responsive growth factor nanogel provided in this embodiment is as follows:

[0109] (1) Dissolve 10 mg of FGF2 in 1 ml of sodium carbonate / sodium bicarbonate buffer to obtain a 10 mg / ml FGF2 solution.

[0110] (2) 0.0065 g of MT1-MMP cleavable peptides of different sequences (KRRQLGLPALSβAla, KRRQLGLGLTAβAla, KRRQLGPLGVASβAla, KRRQLASGLTGGβAla, KRRQLGGASGVLLβAla) were dissolved in 0.9 ml of sodium carbonate / sodium bicarbonate buffer. After complete dissolution, 0.1 ml of 10 mg / ml FGF2 solution was added in an ice bath environment, and the reaction was stirred at 400 rpm for 5-20 minutes (preferably 10 minutes in this embodiment) to prepare MT1-MMP-responsive FGF2.

[0111] (3) Acrylamide (AAM) is reacted with the MT1-MMP responsive growth factor, the AAM concentration used is 5%-40% (preferably 20% in this embodiment), the added volume is 10 μl-100 μl (preferably 49.75 μl in this embodiment), and the reaction time with the MT1-MMP responsive growth factor is 5-20 minutes (preferably 10 minutes in this embodiment).

[0112] (4) The cationic small molecule (APM-AAM is selected in this embodiment) is reacted with the MT1-MMP responsive growth factor. The APM-AAM concentration used is 5%-40% (preferably 20% in this embodiment), the added volume is 10 μl-100 μl (preferably 56.78 μl in this embodiment), and the reaction time with the MT1-MMP responsive growth factor is 1-10 minutes (preferably 5 minutes in this embodiment).

[0113] (5) MT1-MMP responsive growth factor is cross-linked in situ using TEMED catalysis and APS reduction. The dosage of TEMED used is 1-5 μl (preferably 2 μl in this embodiment), the dosage of APS added is 10-50 μl (preferably 20 μl in this embodiment), and the reaction time is 30-120 minutes (preferably 60 minutes in this embodiment).

[0114] (6) Using HEPES buffer at pH 7.4, the MT1-MMP responsive FGF2 nanogel was purified by repeated ultrafiltration using a 10 kDa ultrafiltration tube, and the volume was finally adjusted to 1 ml to obtain 1 mg / ml MT1-MMP responsive FGF2 nanogel.

[0115] The particle size and zeta potential of the five prepared MT1-MMP responsive FGF2 nanogels were tested, and their cytotoxic effects on chondrocytes were tested using the same method as described in Example 3. The test results are shown in Table 5.

[0116] Table 5. Effects of different MT1-MMP cleavable peptide sequences on the preparation of MT1-MMP responsive growth factor nanogels

[0117]

[0118] As shown in Table 5 , the nanogel prepared from the KRRQLGLPALSβAla cleavable peptide sequence has a smaller particle size and a higher zeta potential level, thereby being able to penetrate deeper into the cartilage within 1 day and having a more effective cartilage penetration ability.

[0119] Example 8: Effects of different doses of AAM on the preparation of MT1-MMP responsive growth factor nanogels

[0120] In this example, MT1-MMP responsive FGF2 was prepared according to the method provided in Example 1, and 19.75 μl, 29.75 μl, 39.75 μl, 49.75 μl, 59.75 μl, 69.75 μl, 79.75 μl, and 89.75 μl of 20% AAM were added, respectively, and the reaction was carried out in an ice bath for 10 minutes. Then, 56.78 μl of 20% APM-AAM was added, and the reaction was carried out for 5 minutes. TEMED 2 μl and APS 20 μl were added, and the reaction was carried out for 60 minutes. Different MT1-MMP responsive FGF2 nanogels were obtained after ultrafiltration and purification through a 10 kDa ultrafiltration tube. The particle size and Zeta potential levels of MT1-MMP-responsive FGF2 nanogels with different AAM doses were detected, their toxic effects on chondrocytes were detected, and their penetration efficiency in osteoarthritis cartilage explants was detected. The particle size and Zeta potential were detected by dynamic light scattering (DLS), the toxic effect on chondrocytes was detected by Cell Counting Kit-8 cell proliferation / cytotoxicity assay, and the penetration efficiency in osteoarthritis cartilage explants was detected by fluorescence microscopy to observe the penetration depth of fluorescent-labeled nanogels in cartilage. The test results are shown in Table 6.

[0121] Table 6. Effects of different AAM doses on the preparation of MT1-MMP responsive FGF2 nanogels

[0122]

[0123]

[0124] Table 6 shows that the AAM dose significantly affects the particle size, zeta potential, chondrocyte toxicity, and cartilage penetration of the MT1-MMP-responsive FGF2 nanogels. When the AAM dose was 49.75 μl, the particle size of the prepared MT1-MMP-responsive FGF2 nanogels remained small, the zeta potential was high, and the cartilage penetration was strongest, with strong chondrocyte proliferation activity. When the AAM dose was reduced, although the particle size decreased, the zeta potential also decreased, the cartilage penetration ability weakened, and no significant enhancement in chondrocyte proliferation was observed. This indicates that zeta potential also plays a significant role in cartilage penetration, and that below a certain level, zeta potential has no significant toxic effect on chondrocytes. When the AAM dose was increased, although the zeta potential increased, the particle size also increased significantly, the cartilage penetration efficiency decreased significantly, and the toxicity to chondrocytes increased. This indicates that particle size is also a key factor limiting the cartilage penetration efficiency of the nanogels, and that increased zeta potential also enhances cell toxicity. Only when the particle size and Zeta potential are maintained at a reasonable level can the cartilage penetration efficiency of the nanogel be maximized and the toxicity to chondrocytes be minimized. The most preferred AAM dose is 39.75 μl.

[0125] Example 9: Effect of different AAM reaction times on the preparation of MT1-MMP responsive FGF2 nanogels

[0126] In this example, MT1-MMP-responsive FGF2 was prepared according to the method provided in Example 1. 49.75 μl of 20% AAM was then added. The AAM reaction was performed on ice for 1, 5, 10, 15, and 20 minutes, respectively. 56.78 μl of 20% APM-AAM was then added for 5 minutes. 2 μl of TEMED and 20 μl of APS were then added for 60 minutes. After ultrafiltration through a 10 kDa ultrafiltration tube, different MT1-MMP-responsive FGF2 nanogels were obtained. The MT1-MMP-responsive FGF2 nanogels, prepared with different AAM reaction times, were tested for particle size and zeta potential, chondrocyte toxicity, and penetration efficiency in osteoarthritis cartilage explants. The testing methods are as described in Example 3. The test results are shown in Table 7.

[0127] Table 7. Effect of different AAM reaction times on the preparation of MT1-MMP responsive FGF2 nanogels

[0128]

[0129] As can be seen from Table 7, the AAM reaction time also directly affects the particle diameter, zeta potential, and cartilage penetration effect of the MT1-MMP responsiveness FGF2 nanogel, but has no significant effect on the toxic effects of chondrocytes. Because the reaction time is relatively short, AAM is less cross-linked with the MT1-MMP responsiveness FGF2, and the particle diameter and the Zeta potential of the FGF2 nanogel of the MT1-MMP response formed are all lower, thereby affecting its penetration efficiency in cartilage. After the reaction time exceeds 10 minutes, AAM and the MT1-MMP responsiveness FGF2 reaction are substantially complete, and increasing the reaction time has no significant effect on the particle diameter, the Zeta potential of the MT1-MMP responsiveness FGF2 nanogel, thereby has no significant effect on the cartilage penetration efficiency. The optimal AAM reaction time selected in the present embodiment is 10 minutes.

[0130] Example 10: Effect of different doses of APM-AAM on the preparation of MT1-MMP responsive growth factor nanogels

[0131] In this example, MT1-MMP-responsive FGF2 was prepared according to the method provided in Example 1. 49.75 μl of 20% AAM was added and reacted on ice for 10 minutes. 36.78 μl, 46.78 μl, 56.78 μl, 66.78 μl, and 76.78 μl of 20% APM-AAM were then added, reacted for 5 minutes, 2 μl of TEMED and 20 μl of APS were added, reacted for 60 minutes, and purified by ultrafiltration through a 10 kDa ultrafiltration tube to obtain different MT1-MMP-responsive FGF2 nanogels. The particle size and zeta potential of the MT1-MMP-responsive FGF2 nanogels with different APM-AAM doses were tested, their cytotoxic effects on chondrocytes were tested, and their penetration efficiency in osteoarthritis cartilage explants was tested. The testing methods are as described in Example 3. The test results are shown in Table 8.

[0132] Table 8. Effects of different APM-AAM doses on the preparation of MT1-MMP responsive FGF2 nanogels

[0133]

[0134] Table 8 shows that the APM-AAM dose significantly affects the particle size, zeta potential, chondrocyte toxicity, and cartilage penetration of the MT1-MMP-responsive FGF2 nanogels. When the APM-AAM dose was 56.78 μl, the prepared MT1-MMP-responsive FGF2 nanogels maintained a small particle size and a high zeta potential, exhibiting the strongest cartilage penetration and enhancing chondrocyte proliferation. Decreasing the APM-AAM dose reduced the particle size but also the zeta potential, weakening the cartilage penetration ability without affecting chondrocyte proliferation. Increasing the APM-AAM dose, while increasing the zeta potential, significantly increased the particle size, significantly reduced the cartilage penetration efficiency, and enhanced the chondrocyte toxicity. Only when the particle size and zeta potential were maintained at appropriate levels could the nanogels achieve the highest cartilage penetration efficiency and minimize chondrocyte toxicity. The most preferred dose of APM-AAM is 56.78 μl.

[0135] Example 10: Effect of different APM-AAM reaction times on the preparation of MT1-MMP responsive FGF2 nanogels

[0136] In this example, MT1-MMP-responsive FGF2 was prepared according to the method provided in Example 1. 49.75 μl of 20% AAM was then added, and the mixture was reacted on ice for 10 minutes. Then, 56.78 μl of 20% APM-AAM was added, with the APM-AAM reaction times being 1 minute, 3 minutes, 5 minutes, 7 minutes, and 9 minutes, respectively. 2 μl of TEMED and 20 μl of APS were then added for 60 minutes. After ultrafiltration and purification through a 10 kDa ultrafiltration tube, different MT1-MMP-responsive FGF2 nanogels were obtained. The particle size and zeta potential of the MT1-MMP-responsive FGF2 nanogels with different APM-AAM reaction times were measured, as were their cytotoxic effects on chondrocytes and their penetration efficiency in osteoarthritis cartilage explants. The testing methods were as described in Example 3. The test results are shown in Table 9.

[0137] Table 9. Effect of different APM-AAM reaction times on the preparation of MT1-MMP responsive FGF2 nanogels

[0138]

[0139] As shown in Table 9, the APM-AAM reaction time directly affects the particle size, zeta potential, and cartilage penetration of the MT1-MMP-responsive FGF2 nanogels, but has no significant effect on chondrocyte toxicity. Short reaction times result in fewer crosslinks between the MT1-MMP-responsive FGF2 nanogels after crosslinking between APM-AAM and AAM, resulting in lower particle size and zeta potential of the resulting MT1-MMP-responsive FGF2 nanogels, which in turn affects their cartilage penetration efficiency. When the reaction time exceeds 5 minutes, the MT1-MMP-responsive FGF2 nanogels react almost completely after crosslinking between APM-AAM and AAM. Increasing the reaction time has no significant effect on the particle size or zeta potential of the MT1-MMP-responsive FGF2 nanogels, and thus on their cartilage penetration efficiency. The optimal APM-AAM reaction time selected in this example was 5 minutes.

[0140] Although the present invention is disclosed as above, the present invention is not limited thereto. Any person skilled in the art can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the scope defined by the claims.

Claims

1. A nanogel that rapidly penetrates articular cartilage and responsively releases growth factors, characterized in that: The invention comprises growth factors, responsive polypeptides, neutral small molecules with double bonds and cationic small molecules with double bonds; the growth factors, responsive polypeptides, neutral small molecules with double bonds and cationic small molecules with double bonds are firstly reacted together, and then cross-linked in situ to obtain the obtained product.

2. The nanogel according to claim 1, wherein The growth factors include any one or more of fibroblast growth factor 2, recombinant human transforming growth factor β, insulin-like growth factor 1, and bone morphogenetic protein 7.

3. The nanogel according to claim 2, wherein The responsive polypeptide includes any one or more of membrane metalloproteinase-1 cleavable polypeptide, matrix metalloproteinase 13 cleavable peptide, metalloproteinase domain protein 5 cleavable peptide, metalloproteinase domain protein 4 cleavable peptide, tumor necrosis factor α, interleukin 1, interleukin 2, and interleukin 6.

4. The nanogel according to claim 3, wherein The cationic small molecule with a double bond includes any one or more of methacrylamide, vinyltrimethylammonium, acrylic acid, acryloyl chloride, phenolic acid, butenedioic acid, methyl methacrylate, vinyltrimethylammonium chloride, butenol, dimethyl butadienedioate, and ethyl acrylate; The neutral small molecule with double bonds is acrylamide.

5. The nanogel according to claim 4, wherein The mass fraction of the growth factor is 0.01-1%; the mass fraction of the responsive polypeptide is 0.1-5%, the mass fraction of the acrylamide is 5-40%, and the mass fraction of the cationic small molecule is 5-40%.

6. A method for preparing a nanogel that rapidly penetrates articular cartilage and responsively releases growth factors, characterized in that: The following steps are involved: (1) mixing a growth factor, a responsive polypeptide, a neutral small molecule with a double bond, and a cationic small molecule with a double bond to produce a mixed product; (2) Cross-linking the mixed product in situ.

7. The preparation method according to claim 6, wherein Step (1) includes the following steps: (a) The growth factor solution and the responsive polypeptide are dissolved in an alkaline buffer to reach equilibrium, obtaining a completely dissolved macromolecular solution; (b) The completely dissolved macromolecular solution is mixed with neutral small molecules with double bonds and cations with double bonds to prepare a nanogel precursor solution.

8. The preparation method according to claim 7, wherein In step (a), the growth factor solution and the responsive polypeptide solution are dissolved in equilibrium, the buffer used is a weakly alkaline buffer, and the reaction environment is an ice bath environment.

9. The preparation method according to claim 8, wherein The reaction time of step (a) is 5 to 20 minutes; the reaction time of step (b) is 5 to 20 minutes; and step (2) is: adding a catalyst and a reducing agent to the nanogel precursor solution to perform in-situ crosslinking, wherein the catalyst is tetramethylethylenediamine, the reducing agent is ammonium persulfate, the reaction environment is an ice bath, and the reaction time is 1 to 2 hours.

10. A use of a nanogel for preparing a reagent capable of rapidly penetrating articular cartilage and responsively releasing growth factors, characterized in that: The nanogel comprises growth factors, responsive polypeptides, neutral small molecules with double bonds and cationic small molecules with double bonds; the nanogel is prepared by firstly mixing the growth factors, responsive polypeptides, neutral small molecules with double bonds and cationic small molecules and then cross-linking them in situ.

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