Self-curing injectable pectin hydrogel as well as preparation and application thereof

Abstract

The invention discloses a self-curing injectable pectin hydrogel and a preparation method and application thereof.Based on 100 parts by weight of the hydrogel, the hydrogel is prepared from, by weight, 30-80 parts of Pickering emulsion, 0.5-5 parts of pectin and the balance water, based on 100 parts by weight of the Pickering emulsion, 0.5-5 parts of the pectin and the balance water, and based on 100 parts by weight of the Pickering emulsion, 0.5-5 parts of the pectin are added, the Pickering emulsion contains 2 to 20 parts by weight of pluronic F127 and 0.1 to 5 parts by weight of hydroxyapatite, and on the basis of 100 unit volume of the Pickering emulsion, the Pickering emulsion contains 20 to 70 unit volume of ethyl oleate. According to the hydrogel, hydroxyapatite nanoparticles are used as a hydrogel cross-linking agent and also used as a stabilizer for stabilizing a Pickering emulsion, and a biodegradable pectin substrate is combined, so that the effects of promoting bone repair and preventing tumor recurrence are achieved. The system can effectively promote apoptosis of osteosarcoma cells, inhibit migration of the osteosarcoma cells and regulate and control the cycle of the osteosarcoma cells in vitro, and shows a remarkable anti-osteosarcoma effect and an osteogenesis promotion effect in vivo.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of medicine, and in particular, relates to a self-solidifying injectable pectin hydrogel, a preparation method thereof and the use of the hydrogel in the preparation of drugs for preventing the postoperative recurrence of osteosarcoma and bone repair. BACKGROUND

[0002] Osteosarcoma is an aggressive primary bone cancer that poses a serious threat to patients' health. According to statistical data, the incidence of osteosarcoma in children and adolescents is relatively low, but given its treatment difficulty, it has a very serious impact on patients. Early detection and comprehensive treatment are crucial to improving prognosis, but even so, the treatment of osteosarcoma still faces many challenges. The recurrence rate of osteosarcoma is high, about 20% to 40%, and the treatment of osteosarcoma after recurrence is more difficult. Therefore, prevention of recurrence is an important part of treatment strategies.

[0003] Nano-hydroxyapatite (nHAP) as a kind of nano-material with biocompatibility and bioactivity shows potential application value in the treatment of osteosarcoma. It not only can inhibit the growth and metastasis of cancer cells, but also can promote the repair and regeneration of bone tissue. Although the related research is still in the preliminary stage, the application prospect of nano-hydroxyapatite is promising.

[0004] Pickering emulsion, as a kind of emulsion stabilized by solid particles, has attracted widespread attention in the field of drug delivery in recent years. Compared with traditional emulsions, Pickering emulsion uses solid particles as stabilizers, which can be inorganic or organic, and they form a protective layer on the oil-water interface, effectively preventing droplet coagulation, thereby improving the stability of the emulsion. The stability of this emulsion makes it have potential application value in the fields of food science, cosmetics, pharmaceuticals and materials science.

[0005] In the treatment of osteosarcoma, the application prospect of Pickering emulsion is particularly broad. Due to its stability and biocompatibility, Pickering emulsion can be used as a drug delivery system to deliver therapeutic drugs directly to the tumor site, thereby improving treatment efficacy and reducing systemic toxicity. In addition, Pickering emulsion can also achieve multi-drug co-delivery, providing new possibilities for combination therapy. For example, nano-hydroxyapatite as a Pickering emulsion stabilizer not only can improve the stability of the emulsion, but also may promote the repair and regeneration of bone tissue through its bioactivity, while inhibiting the growth and metastasis of tumor cells.

[0006] In summary, Pickering emulsion as a new type of drug delivery system shows great potential in the treatment of osteosarcoma. With further research and development, Pickering emulsion is expected to become an important tool to improve treatment effect and reduce side effects, bringing new hope for osteosarcoma patients. SUMMARY

[0007] According to one aspect of the present application, one object of the present application is to provide a self-curing injectable pectin hydrogel, based on 100 parts by weight of the hydrogel, comprising the following components by weight: 30-80 parts by weight of Pickering emulsion, 0.5-5 parts by weight of pectin and the balance of water, wherein based on 100 parts by weight of the Pickering emulsion, the Pickering emulsion contains 2-20 parts by weight of Pluronic F127 and 0.1-5 parts by weight of hydroxyapatite, and based on 100 unit volumes of the Pickering emulsion, the Pickering emulsion contains 20-70 unit volumes of ethyl oleate.

[0008] Preferably, based on 100 parts by weight of the hydrogel, it comprises the following components by weight: 40-60 parts by weight of Pickering emulsion, 1-3 parts by weight of pectin and the balance of water, wherein based on 100 parts by weight of the Pickering emulsion, the Pickering emulsion contains 5-15 parts by weight of Pluronic F127 and 0.5-3 parts by weight of hydroxyapatite, and based on 100 unit volumes of the Pickering emulsion, the Pickering emulsion contains 25-60 unit volumes of ethyl oleate.

[0009] Preferably, based on 100 parts by weight of the hydrogel, it comprises the following components by weight: 50 parts by weight of Pickering emulsion, 2.5 parts by weight of pectin and the balance of water, wherein based on 100 parts by weight of the Pickering emulsion, the Pickering emulsion contains 10 parts by weight of Pluronic F127 and 1 part by weight of hydroxyapatite, and based on 100 unit volumes of the Pickering emulsion, the Pickering emulsion contains 30-50 unit volumes of ethyl oleate.

[0010] According to another aspect of the present application, another object of the present application is to provide a preparation method of the self-curing injectable pectin hydrogel, the preparation method comprising the following steps:

[0011] 1) Synthesis of Pickering emulsion

[0012] Pluronic F127 is weighed and dissolved in pure water to form a Pluronic F127 solution with a mass percentage of 2-20% for standby use. Nano-hydroxyapatite is weighed and added to the Pluronic F127 solution and mixed uniformly. Then, ethyl oleate is added in proportion to the mixture, and the mixture is dispersed by a high-speed shearing machine at 12000 rpm for 5 min to obtain a Pickering emulsion containing nano-hydroxyapatite.

[0013] 2) Synthesis of hydrogel

[0014] Pectin is weighed and added to water and stirred to obtain a pectin solution. The Pickering emulsion prepared in step 1) is uniformly mixed with the pectin solution at a volume ratio of 1:1 to form a gel.

[0015] Preferably, the mass percentage of the Pluronic F127 solution in step 1) is preferably 5-15%, more preferably 10%.

[0016] Preferably, the amount of nano-hydroxyapatite added in step 1) is 0.1-5 g, preferably 0.5-3 g, and more preferably 1 g, based on 100 mL of the Pluronic F127 solution.

[0017] Preferably, the amount of ethyl oleate added in step 1) is 25-60 mL, preferably 30-50 mL, and more preferably 40 mL, based on 100 mL of the final Pickering emulsion.

[0018] Preferably, the mass percentage concentration of the pectin solution in step 2) is 1-10%, preferably 2-6%, and more preferably 5%.

[0019] According to another aspect of the present application, another object of the present application is to provide the use of the self-solidifying injectable pectin hydrogel as a carrier in a drug for preventing postoperative recurrence of osteosarcoma and bone repair.

[0020] According to another aspect of the present application, another object of the present application is to provide the use of the self-solidifying injectable pectin hydrogel in the preparation of a drug for preventing postoperative recurrence of osteosarcoma and bone repair.

[0021] Beneficial effects

[0022] The self-solidifying injectable pectin hydrogel according to the present application uses hydroxyapatite (nHAP) nanoparticles as a hydrogel crosslinking agent, and also as a stabilizer for stabilizing Pickering emulsion, in combination with a biodegradable pectin base, to form a self-solidifying injectable hydrogel, achieving the effects of promoting bone repair and preventing tumor recurrence. Experimental results show that the system can effectively promote osteosarcoma cell apoptosis, inhibit osteosarcoma cell migration, and regulate osteosarcoma cell cycle in vitro, and exhibit significant anti-osteosarcoma effect and osteogenesis promotion effect in vivo. BRIEF DESCRIPTION OF DRAWINGS

[0023] In order to more clearly illustrate the technical solutions of the specific embodiments or prior art in the present application, the drawings needed to be used in the description of the specific embodiments or prior art will be briefly introduced as follows. Obviously, the drawings in the following description are some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor on the basis of these drawings.

[0024] Figure 1 and Figure 2 Performance characterization chart of Pickering hydrogel prepared in Example 1.

[0025] Figure 3 Test result chart of Pickering emulsion in vitro promoting osteosarcoma K7M2 cell apoptosis.

[0026] Figure 4 Test result chart of Pickering emulsion in vitro inhibiting migration of osteosarcoma cells K7M2 and regulating cell cycle thereof.

[0027] Figure 5 Test result chart of in vivo anti-osteosarcoma effect of Pickering emulsion.

[0028] Figure 6 In vivo and in vitro effect chart of Pickering emulsion in promoting osteogenesis. DETAILED DESCRIPTION

[0029] Hereinafter, the present application will be described in detail. Before proceeding with the description, it should be understood that the terms used in the specification and the appended claims should not be construed as being limited to the general and dictionary meanings and should be construed as having meanings and concepts corresponding to the technical aspects of the present application based on the principle that the inventor is allowed to define the terms in order to best explain the technical idea of the present application. Therefore, the description presented herein is merely a preferred example for the purpose of illustration and is not intended to limit the scope of the present application, so it should be understood that other equivalent ways or modifications can be derived from the present application without departing from the spirit and scope of the present application.

[0030] As used herein, the terms "comprises," "comprising," "includes," "including," "has," "having," "contains," "containing," or any other similar words, are intended to be open-ended, and to mean including, but not limited to. In other words, use of these terms indicates that the named element is an element that is encompassed by the phrase, but not that the phrase is limited to the named element. For example, a composition or article that comprises a list of elements is not necessarily limited to only those elements but can include other elements not expressly listed or inherent to such composition or article. Further, unless otherwise specified, the use of "or" is to be treated as a disjunctive term that means "and / or," unless otherwise indicated by context. For example, a list of items joined by "or" means any one of the items can be present or each of the items can be present and used together. In addition, unless expressly stated to the contrary, the term "or" as used herein refers to an inclusive "or" and not to an exclusive "or." For example, a condition "A or B" is satisfied by any one of the following: A is true (or present) and B is false (or not present), A is false (or not present) and B is true (or present), and both A and B are true (or present). Also, the use of the terms "comprise," "comprising," "comprises," "include," "including," "includes," "have," "has," "having," or variants thereof are to be construed as open-ended, non-limiting identifiers as to the elements they follow, rather than as recitations of necessary constituent elements. To clarify, recitation of elements using each of these types of

[0031] As used herein, all features or conditions of a range or a percentage range are intended to be merely for convenience and brevity in providing specifics. Accordingly, description of a range or a percentage range shall be deemed to have been specifically disclosed and to specifically disclose all possible sub-ranges and individual numerical values within the range, particularly integer values. For example, a range of "1 to 8" shall be deemed to have been specifically disclosed as all sub-ranges, particularly sub-ranges defined by all integer values, such as 1 to 7, 2 to 8, 2 to 6, 3 to 6, 4 to 8, 3 to 8, and the like, and as all individual numerical values within the range, such as 1, 2, 3, 4, 5, 6, 7, 8, and the like. The foregoing interpretation applies to all aspects of the present application, whether broad or narrow, unless otherwise indicated.

[0032] If an amount or other numerical value or parameter is expressed in a range, a preferred range or a series of upper and lower limits, it is to be understood that the range specified in this document is specifically disclosed and can be included in the disclosure even if the range is not expressly recited. In addition, when referring to a range of values, unless otherwise indicated, the range is to include the endpoints and all integers and fractions within the range.

[0033] As used herein, numerical values are to be understood as having the precision of the number of significant figures of the numerical value. For example, the number 40.0 is to be understood as encompassing the range from 39.50 to 40.49.

[0034] The following examples are set forth to illustrate the embodiments of the application and are not intended to limit the scope of the application. Those skilled in the art will readily understand that modifications can be made to the present application without departing from the spirit and scope of the application. Unless otherwise indicated, the reagents and instruments used in the following examples are commercially available.

[0035] Materials and Instruments

[0036] 1. Materials

[0037] Nanohydroxyapatite (Shanghai Aladdin Bio-Chem Technology Co., Ltd., Shanghai); Ethyl oleate (Shanghai Chemical Reagent Co., Ltd., Shanghai); Pluronic F127 (Sigma-Aldrich, USA); Pectin / polygalacturonic acid (Shanghai Macklin Biochemical Technology Co., Ltd., Shanghai); Dextran gel G-100 (GE Healthcare, USA); Methanol (Shanghai Chemical Reagent Co., Ltd., Shanghai); Ethanol (Shanghai Chemical Reagent Co., Ltd., Shanghai); Chloroform (Shanghai Chemical Reagent Co., Ltd., Shanghai); Isopropanol (Shanghai Chemical Reagent Co., Ltd., Shanghai); Coumarin-6 (Bioland Scientific Technology Co., Ltd., Shanghai); Alizarin red dye (Bi Yun Tian Biotechnology Co., Ltd., Shanghai); DMEM cell culture medium (Gibco, USA); a-MEM cell culture medium (Gibco, USA); Australian blood source fetal bovine serum (Gibco, USA); 0.25% trypsin (Bi Yun Tian Biotechnology Co., Ltd., Shanghai); Penicillin-streptomycin double antibody (Bi Yun Tian Biotechnology Co., Ltd., Shanghai); Cell apoptosis kit (Dalian Melon Biotechnology Co., Ltd., Dalian); Cell apoptosis and cycle kit (Dalian Melon Biotechnology Co., Ltd., Dalian); TRIzol RNA separation reagent (Youxian Biotechnology Co., Ltd., Shanghai); DAPI staining solution (Bi Yun Tian Biotechnology Co., Ltd., Shanghai); PageRuler TM Prestained Protein Ladder, 10 to 180 kDa (Cat No. 26616) (Thermo Scientific TM , USA); Cell Counting Kit-8 CCK-8 kit (Bi Yun Tian Biotechnology Co., Ltd., Shanghai); Calcein / PI cell activity and cytotoxicity detection kit (Bi Yun Tian Biotechnology Co., Ltd., Shanghai); Culture medium filter (Nalgene, Thermo Scientific, USA). Isoflurane (1-chloro-2,2,2-trifluoroethyl difluoromethyl ether); Polymethyl alcohol powder (Shanghai Chemical Reagent Co., Ltd., Shanghai); Strong sodium oxide (flaky) (Shanghai Chemical Reagent Co., Ltd., Shanghai); Xylene (Shanghai Chemical Reagent Co., Ltd., Shanghai); BSA bovine serum albumin (Dalian Melon Biotechnology Co., Ltd., Dalian); DEPC water (Bi Yun Tian Biotechnology Co., Ltd., Shanghai); PrimeScriptTM RT reagent Kit (RR037A, TaKaRa, Japan) CellAmp TMDirect TB / Probc RT-qPCR Kit (3735S, TaKaRa, Japan); 5-0 non-absorbable silk thread (Johnson & Johnson Medical Limited, Shanghai); immunohistochemical pen (Cat: H-4000, Vector Labs, Beijing); peroxidase blocker (Cat: 318701, GEPbio, Shanghai); antibody diluent (Cat: Antibody Diluent, GEPbio, Shanghai); DAB developing solution (Cat: 721611, GEPbio, Shanghai); Gill's hematoxylin staining solution (Cat: 721600, GEPbio, Shanghai); ALP detection kit (Biuntian Biotechnology Co., Ltd., Shanghai); bone tissue decalcification solution (Wuhan Bolif Biological Technology Co., Ltd., Wuhan); bone tissue antigen repair kit (Cat: 315810, GEPbio, Shanghai).

[0038] 2. Instruments

[0039] Electronic balance (BT25S, Sartorius, Germany); magnetic stirrer (85-2A, Shanghai Hengqin Instrument Equipment Co., Ltd.); vortex mixer (R8-1, Beijing Wunuo Technology Co., Ltd.); pure water instrument (Ultrapure plus-12A, Shanghai Hetai Instrument Co., Ltd.); high-speed dispersion machine (IKAT10 basic ULTRA, Germany); ultrasonic constant-temperature cleaning instrument (SB-5200D, Ningbo Xinzhi Biological Technology Co., Ltd.); freeze dryer (Labconco, USA); manual pipette (Eppendorf, Germany); laser particle size analyzer (Zetasizer Nano ZS90, Malvern, UK); fast scanning atomic force microscope (Bruker, Germany); confocal microscope (Olympus SR10, Japan); flow cytometer (ACEA NovoCyte3000, USA); microplate reader (Biotek Synergy4, USA); MALDI-TOF mass spectrometer (MALDI-TOF / TOF 5800analyzer, AB Sciex, Massachusetts, USA); gel imaging system (ChemiDoc MPTM Imaging System, BIO-RAD, California, USA).

[0040] Example 1 Construction of nHAP stabilized Pickering emulsion hydrogel

[0041] Experimental methods

[0042] 1) Synthesis of Pickering emulsion

[0043] 10% Pluronic F127 solution preparation: 10 g Pluronic F127 was weighed into 100 mL of pure water for use, to obtain a 10% Pluronic F127 solution by weight fraction, 1 g of nano-hydroxyapatite was weighed into 6 mL of 10% Pluronic F127 and mixed evenly, then 4 mL of ethyl oleate was added, and the mixed solution was dispersed using a high-speed shearing machine at 12000 rpm for 5 min to obtain a Pickering emulsion.

[0044] Malvern particle size analyzer was used to detect the particle size and zeta potential of the Pickering emulsion; the size was directly observed under a microscope; the morphology of the Pickering emulsion was scanned using a fast scanning atomic force microscope; the stability of the Pickering emulsion: it was placed in a 4°C refrigerator for 0, 3, and 21 days to detect its particle size and zeta potential; the Pickering emulsion prepared using coumarin 6, ethyl oleate, and alizarin red after nHAP synthesis was observed using a confocal microscope.

[0045] 2) Synthesis of Pickering hydrogel

[0046] 5% pectin: 5 g of pectin was weighed into 100 mL of water and stirred evenly to obtain a 50% pectin solution by weight fraction, the Pickering emulsion prepared in step 1) was mixed with the 5% pectin solution at a volume ratio of 1:1 to form a gel.

[0047] FESEM was used to analyze the morphology of the freeze-dried hydrogel. Before analysis, all samples were sputter-coated with gold. Energy dispersive spectroscopy (EDS; Oxford X-Max 20) was coupled to the FESEM.

[0048] The water content of the hydrogel was calculated by the equation:

[0049]

[0050] where W w and W d are the weight of the wet hydrogel, and the dry hydrogel, respectively.

[0051] The hydrogel samples were subjected to rheological shear performance testing. A universal mechanical testing machine (MTS, Instron electric pulse E10000, USA) was used to test the samples at a crosshead speed of 2 mm / min. Five samples (n=5) were tested for each sample.

[0052] The swelling behavior of the hydrogel was tested as follows: The prepared hydrogel was cut into 10 mm length, put into 15 mL centrifuge tube in 10 mL PBS, then the centrifuge tube was kept at 37 °C at different time points (1, 2, 3, 6, 12, 24 h). After that, the hydrogel was taken out from the tube, and the superficial water on the hydrogel was absorbed using Kimwipes. The swelling ratio of the hydrogel was calculated using the following formula (2):

[0053]

[0054] where Wtand W0represent the weight of the swollen hydrogel composite in PBS and the weight of the dried hydrogel composite after swelling, respectively. Four samples were tested in each group (n = 4).

[0055] The degradation profile of the hydrogel was tested as follows: The lyophilized hydrogel (1 mm high, 14 mm in diameter) weighed as W1was immersed in PBS solution (pH = 7.4) for 2 hours, the unabsorbed PBS was discarded, and then immersed in PBS at a mass-volume ratio of 5 mg / mL. The solution was shaken in a constant temperature water bath bed (70 rpm, 37 °C). The extracted liquid was collected and rinsed with PBS at each time point (1, 3, 7, 14, 21, and 28 days). After 4 weeks of degradation, the Ca2+concentration released at each time point and the pH value of the collected solution were measured by inductively coupled plasma mass spectrometry (ICP, Agilent 710, California, USA) and pH meter (Mettler-Toledo, USA), respectively. The content of AM monomer in the degradation solution was determined by spectrophotometry at a wavelength of 205 nm (Nanodrop One, USA). The AM content was calculated according to the standard curve. Four samples were tested in each group. The degraded hydrogel was freeze-dried and weighed as W2(n = 4) at each time point. The weight loss of the hydrogel at different points was calculated using formula (3):

[0056]

[0057] The results are shown in Figure 1 , where A. a schematic diagram of the preparation of Pickering emulsion; B. (a) photographs of Pickering emulsion when Sudan I (left) and Coomassie brilliant blue (right) were just added, (b) after the dye was mixed, the situation in the Pickering emulsion; C. images of Pickering emulsion under atomic force microscope quantitative imaging (QI) mode (a) Height Sentor. (b) Peak Force Error; D. images of Pickering emulsion under laser confocal microscope, green is coumarin 6 labeled ethyl oleate, and red is alizarin red S labeled nano-hydroxyapatite; E. stability test of Pickering emulsion.

[0058] Prepared Pickering emulsion. Identified as oil-in-water (O / W) type by Sudan I and Coomassie brilliant blue staining detection Figure 1 B) The particle size of the emulsion was about 3 pm, and the surface was uneven by atomic force microscope scanning Figure 1 C) The structure of the Pickering emulsion was clearly visible, with the surface uniformly covered with nano-hydroxyapatite particles and the inner core being ethyl oleate, as shown by laser confocal microscopy Figure 1 D). To determine the stability of the Pickering emulsion Figure 1 E), the emulsion was placed at 4°C, and the particle size and potential were detected on day 0, day 3, and day 21. The results showed that the Pickering emulsion exhibited excellent stability for at least 21 days.

[0059] Figure 2 Pickering hydrogel characterization results, where A. The general appearance of the hydrogel gelation; B. (a) Pickering emulsion fluorescence microscope image; (b) Pickering emulsion image in hydrogel; C. (a) Pickering emulsion hydrogel SEM image after freeze-drying, (b) Pickering emulsion hydrogel SEM image after 28 days in simulated body fluid; D. Pickering emulsion hydrogel can adhere to broken bone; E. Hydrogel rheological shear force detection; F. Swelling detection of hydrogel; G. In vitro degradation rate of hydrogel; H. FIRT results of hydrogel; I. Calcium ion release of hydrogel in vitro for 28 days.

[0060] From Figure 2 A it can be seen that the prepared Pickering emulsion pectin hydrogel has good injectability Figure 2 E), and has certain adhesion Figure 2 D). The Pickering emulsion can not only be uniformly dispersed in the water phase Figure 2 Ba) but also can be uniformly dispersed in the pectin hydrogel Figure 2 Bb). The Pickering hydrogel can deposit salt on its surface after 28 days in the in vitro simulated body fluid environment Figure 2 Cb), which reflects the good in vitro mineralization ability of the Pickering emulsion hydrogel. Swelling behavior is the most important physical property of hydrogel, which enables them to effectively promote material exchange by absorbing nutrients in the surrounding environment. As shown in Figure 2 F, the pickering emulsion hydrogel shows greater water retention capacity than the pectin hydrogel, but there is no significant difference in the in vitro degradation rate Figure 2 G) between the two. FIRT results Figure 2H) The Pickering emulsion hydrogel shows similar bonds with calcium ion cross-linked pectin hydrogel. The calcium ion release Figure 3 I) The results all show the long-acting sustained-release effect of the hydrogel.

[0061] Example 2 Evaluation of the effect of Pickering emulsion hydrogel on osteosarcoma

[0062] 1. Experimental animals and cells:

[0063] Mouse osteosarcoma cell line (K7M2); SPF grade BALB / C female mice were purchased from Shanghai Slek Experimental Animal Co., Ltd. The mice were about 5 weeks old, and the feeding environment was 12h / 12h day and night, constant temperature 25℃, and sterile food and drinking water were added regularly. All animal experiments were conducted in accordance with the specifications and guidelines of the IACUC of the Shanghai Institute of Materia Medica, Chinese Academy of Sciences.

[0064] 2. Experimental method

[0065] 2.1 Cell activity analysis

[0066] (1) Plating: First, the cells were made into a cell suspension (the process is as described above in the cell subculture process, washed with PBS, trypsin digestion, stop digestion, horizontal centrifugation, mix well) K7M2 cells were inoculated in 96-well plates, and the cell density was 8x103 cells / well.

[0067] (2) Drug treatment: the cells were incubated with the Pickering emulsion hydrogel prepared in Example 1 for 48 hours.

[0068] (3) CCK8 incubation: add 10μl of CCK8 to the cells in the 96-well plate and incubate for 1 hour.

[0069] (4) Statistical cell viability: cell viability was measured by measuring the absorbance at 570nm using a microplate reader, and the activity of normal cells was (100%).

[0070] 2.2 Cell uptake experiment

[0071] Intracellular behavior of Pickering emulsion was observed using a laser scanning confocal microscope (LSCM, TCS SP8 STED 3X, Leica, Germany). K7M2 cells were seeded in 6-well plates, with a sterile coverslip at the bottom of each well, at a concentration of 1.5 × 10⁵ cells / well. Cells were cultured in DMEM / F-12 medium at 37°C in a humidified 5% CO₂ incubator. 100 μg / mL Pickering emulsion was added to the medium. After incubation for 2 and 4 hours, the medium was removed and the cells were washed three times with PBS. Cells were fixed in PBS solution with 4% paraformaldehyde for 20 minutes, followed by gentle washing three times with PBS. The fixed cells were stained with Hoechst 33342 for 15 minutes, washed three times with PBS, and then the coverslips were mounted on the microscope slides. Finally, the samples were observed under LSCM.

[0072] 2.3 Apoptosis

[0073] K7M2 was inoculated at a density of 1×10⁻⁶. 5 Cells were cultured in 24-well plates at 100 cells / well for 24 hours. Then, the Pickering emulsion hydrogel prepared in Example 1 was added and the cells were cultured together. After 24 hours of culture, the medium was desorbed, and the cells seeded on the plates were treated with NIR for 5 min and 0 min, as described above. Cells were then collected after 16 hours of incubation, stained with an Annexin V / PI apoptosis kit (Invitrogen, V13241), and analyzed by flow cytometry (BD FACSCanto II, USA), monitoring fluorescence emission at 530 nm and 575 nm using 488 nm excitation according to the manufacturer's protocol (n=3).

[0074] 2.4 Cell migration experiment

[0075] 293T-hACE2 cells were seeded at a density of 5000 cells / well in 96-well plates. Once the cells adhered, experiments could begin. Mouse serum samples were incubated at 56°C for 30 minutes to inactivate complement. The serum samples were then diluted a certain factor with serum-free DMEM medium, and the pseudovirus concentrate was also diluted a certain factor. 50 μL of the diluted serum sample and 50 μL of the diluted pseudovirus were incubated together at 37°C for 1.5 h. This mixture was then used to replace the old medium, and polybrene was added to a final concentration of 5 μg / mL. After culturing for 6 h, the medium was changed to fresh serum-containing DMEM medium, and the cells were cultured for another 48 h before being analyzed using a microplate reader.

[0076] 2.5 Western blot

[0077] (1) Preparation of protein samples: Total protein was prepared by organic solvent or water-soluble method.

[0078] (2) Gel preparation: 10-30 kDa antigens were separated by 15% gel; 30-100 kDa antigens were separated by 10% gel; 100-200 kDa antigens were separated by 7.5% gel.

[0079] (3) Running gel: Compressed gel voltage: 80 V (10 mA); Separated gel voltage: 120 V (20 mA); Electrophoresis was stopped when the bromophenol blue indicator reached the bottom of the gel.

[0080] (4) Transferring gel: When the target protein molecule was 80-140 kDa, the gel concentration was 8%, and the transferring time was 1.5-2 hours; when the target protein molecule was 25-80 kDa, the gel concentration was 10%, and the transferring time was 1.5 hours; when the target protein molecule was 15-40 kDa, the gel concentration was 12%, and the transferring time was 0.75 hours; when the target protein molecule was <20 kDa, the gel concentration was 15%, and the transferring time was 0.5 hours.

[0081] (5) Blocking: BSA (2%), Western Blot membrane blocking solution

[0082] (6) Primary antibody hybridization: The antibody (referred to as the primary antibody) was diluted with 2% BSA at a dilution ratio according to the antibody instructions, and was incubated at room temperature for 1 hour, and was washed with TBST for 5 minutes 3-5 times.

[0083] (7) Secondary antibody hybridization: The membrane was incubated with the secondary antibody diluted with 2% BSA at room temperature for 1 hour. TBST was washed for 5 minutes 3-5 times.

[0084] (8) Substrate color development

[0085] 2.6 Construction of osteosarcoma postoperative recurrence model

[0086] Female BALB / c (6 weeks old) were purchased from Shanghai Jiesijie Experimental Animal Company. K7M2 cells were harvested and resuspended in PBS, and the final cell density was 1.0 x 10 7 The mice were anesthetized by injecting 4% chloral hydrate (0.1 mL / 10 g body weight). By using a 25-gauge needle, 50 μL of K7M2 cell suspension was injected subcutaneously at the root of the right thigh of the mouse. After 16 days, the tumor was surgically removed, and 100 μL of hydrogel containing Pickering emulsion was injected. The mouse body weight and tumor volume were measured every other day, and the hydrogel was injected every 5 days. The mice were sacrificed on the 35th day. The heart, liver, spleen, lung, kidney, and tumor were collected. The weights of the organs were measured to calculate the organ absorption, and the tumor weight and volume were measured.

[0087] 2.7 Results and discussion

[0088] Figure 3Figure 1 shows the results of the test on the in vitro promotion of apoptosis of osteosarcoma K7M2 cells by Pickering emulsion. A. K7M2 cells take up coumarin 6-labeled Pickering emulsion (a) 2 hours, (b) 4 hours; B. Effects of each component of Pickering emulsion on K7M2 cell activity; C. Pickering emulsion promotes K7M2 cell apoptosis; D. Killing effect of Pickering emulsion on K7M2 cells.

[0089] To verify the cytotoxic effect of Pickering emulsion on K7M2 cells, the uptake of the emulsion by the K7M2 cell line was first examined. It was found that after 2 hours of addition, the uptake of the emulsion by K7M2 cells was less than 10%. Figure 3 Aa), with an intake rate of approximately 50% after 4 hours. Figure 3 Ab). The effects of each component of Pickering emulsion on K7M2 cell viability were observed after co-incubation with K7M2 cells for 24 hours. Figure 3 B showed that nHAP inhibited the cell viability of K7M2 cells. After co-incubating the formulation with K7M2 cells for 48 hours, the formulation showed a pro-apoptotic effect on K7M2 cells. Figure 4 C and 3D).

[0090] Figure 4 The image shows the results of tests conducted using Pickering emulsion to inhibit the migration of osteosarcoma K7M2 cells and regulate their cell cycle. A. Scratch assay; B. Migration assay; C. (a) Effect of the drug on the cell cycle, (b) Statistical analysis of the cell cycle; D. Western blotting to detect changes in the expression of key cell cycle proteins; E. qPCR showing changes in the expression levels of apoptosis- and cell cycle-related mRNAs in K7M2 cells after drug administration.

[0091] Experimental results showed that the formulation inhibited K7M2 in the horizontal direction ( Figure 4 A) and vertical ( Figure 4 B) migration ability. Further to verify that the formulation primarily works by inhibiting tumor proliferation, we examined cell cycle changes in K7M2 cells after administration, and the results showed ( Figure 4 After administration of the Cb formulation, K7M2 cells were primarily arrested in the S phase, thereby inhibiting cell proliferation. Western Blot ( Figure 4 D) and qPCR Figure 5 The results for E also showed cell cycle-related proteins (c-myc, cyclin D1) and mRNAs (myc, ccnd1).

[0092] Figure 5The graph shows the in vivo anti-osteosarcoma effect of Pickering emulsion. A. Animal experiment timeline (blue dots indicate injection treatment time points); B. Mouse body weight change; C. Organ coefficients of each group of mice at the experimental endpoint after administration; D. Gross visual size of subcutaneous tumors in mice at the experimental endpoint; E. Growth curve of subcutaneous tumors in mice after surgical resection; F. Subcutaneous tumor weight in mice at the experimental endpoint; G. Expression of Ki67 within the tumor.

[0093] BALB / c mice were subcutaneously implanted with K7M2 osteosarcoma cells and weighed the following day. The subcutaneous osteosarcoma tumor was removed when it reached approximately 1000 cm³ in volume. Immediately after surgery, the mice were given medication, followed by administration every 5 days. Tumor markers were observed after three administrations. Figure 5 A). No abnormal fluctuations in mouse body weight occurred throughout the entire animal experiment. Figure 5 B), at the experimental endpoint, subcutaneous tumors were collected, and the PBS group showed the largest tumor volume and weight. The nHAP and the hydrogel group containing Pickering emulsion also showed significant effects in inhibiting tumor recurrence. Furthermore, the administration did not affect the organ coefficients of the mice (…). Figure 6 F) can indirectly prove that the preparation has no obvious toxic effects on the body's organs.

[0094] Example 3: Evaluation of the osteogenic effect of Pickering emulsion hydrogel

[0095] 1. Experimental cells and animals

[0096] Mouse osteoblast cell line (MC3T3-E1); 6-8 week old female BALB / c mice (SPF grade) were purchased from Shanghai Silex Laboratory Animal Co., Ltd. They were housed at 25°C room temperature, with a 12-hour day / night cycle, and water and sterile food were provided regularly, with periodic cage changes. All animal experimental procedures followed the standards and guidelines of the IACUC of the Shanghai Institute of Materia Medica, Chinese Academy of Sciences.

[0097] 2. Experimental Methods

[0098] 2.1 ALP detection

[0099] ALP staining was performed using an ALP histochemical diagnostic kit (Beyotime, China). In short, 1×10⁻⁶ ppm was used. 5 MC3T3-E1 cells were co-cultured with the Pickering emulsion hydrogel prepared in Example 1 in 24-well plates in medium (Sigma-Aldrich, USA) for 7 days. Normal medium and osteogenic differentiation medium served as the normal and control groups, respectively. Cells were washed with deionized water and analyzed under a microscope.

[0100] 2.2 Bone defect model

[0101] The hydrogel was placed in the bone defect area for repair using a mouse calvarial defect model. This study used 12 BALB / C mice (20-25 grams, 6 weeks, male) and divided them into three groups (control group, nHAP group, hydrogel containing Pickering emulsion group). The control group had a skull defect, but no material was placed. In the nHAP group, only nHAP (hydroxyapatite powder mixed with F127 solution only) was attached to the mouse calvarial defect. In the hydrogel containing Pickering emulsion group, the hydrogel containing Pickering emulsion prepared according to Example 1 was attached to the mouse calvarial defect. After anesthesia, the mouse's head hair was shaved and sterilized. A 1 cm incision was made along the sagittal suture of the skull. Blunt dissection was performed on the subcutaneous tissue. Then a 3.5 mm diameter bone defect was made on both sides of the parietal bone with a hole drill. A disc-shaped hydrogel with a diameter of 3.5 mm and a depth of 0.5 mm was inserted into the bone defect. The skin incision was sutured with 5-0 silk thread. At 6 weeks after the operation, the animals were euthanized, the skull samples were obtained and placed in 10% formalin for fixation and immersion for 24 hours.

[0102] The fixed skull sample was placed in decalcification solution for 1 month. After the sample was completely decalcified, it was dehydrated in 70% ethanol solution, 80% ethanol solution, 95% ethanol solution, and 100% ethanol solution for half an hour each time, and then placed in xylene for transparency for 30 minutes. Finally, it was embedded with paraffin. The bone defect was cut according to the coronal plane to obtain sample sections, which were dried and then subjected to HE staining, Masson staining, and immunohistochemical staining.

[0103]

[0104] 2.3 Results and discussion

[0105] Figure 6 Figure showing the in vivo and in vitro effects of Pickering emulsion on promoting osteogenesis, wherein A. the effect of the drug on the expression of alkaline phosphatase (ALP) in MC3T3-E1 cells after administration; B. the effect of the drug on the repair of mouse calvarial bone defects (drug placed in the left side of the bone defect, no drug placed in the right side of the bone defect); C. hematoxylin and eosin (H&E) staining and Masson staining of mouse bone defects; D. bone growth rate, green: calcein 0w, red: alizarin red 6w; E. expression of OCN in bone tissue.

[0106] ​Because osteosarcoma often occurs near the bone, when the tumor is large, it often erodes the bone, in order to ensure the principle of "tumor-free" surgery, the tumor near the bone will be partially resected. Then not only need to prevent postoperative recurrence, but also need to repair the bone. In order to detect the bone repair ability of the prepared hydrogel, first in vitro detection of the material on the early osteogenic differentiation index in MC3T3-E1 osteoblast cell line. The experimental results show that the drug and material indeed promote the expression of ALP in MC3T3-E1 cells( Figure 6 A). Subsequently, two 3.5mm size circular bone defects were prepared on the top of the skull of BALB / C mice. The left side of the skull defect was not placed in any material (corresponding to the right side of the picture defect), and the right side of the skull defect was placed in the material (corresponding to the left side of the picture defect) to eliminate the individual differences between animals. Self-control, the mice were euthanized after 6 weeks, and the skull was collected to detect the skull bone repair, the micro-CT results( Figure 6 B) show that Pickering emulsion hydrogel significantly promotes the repair of skull bone defects after 6 weeks of material placement. The H&E staining and Masson staining results of the decalcified skull specimen( Figure 6 C) show the increase in bone defect thickness, and the preparation can be seen to significantly increase the bone thickness. Fluorescently labeled bone mineralization growth rate can be seen that the preparation promotes the growth of bone tissue( Figure 6 D), and the expression of osteogenesis-related protein OCN in the tissue also increases( ​ E), which proves that the preparation effectively promotes bone tissue regeneration.

[0107] The above merely provides a specific implementation of the present application, but the protection scope of the present application is not limited thereto, any skilled person in the art can easily think of changes or replacements within the technical range disclosed by the present application, which should be covered within the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the protection scope of the claims.

Claims

1. A self-solidifying injectable pectin hydrogel, comprising the following components by weight based on 100 parts by weight of the hydrogel: 30-80 parts by weight of a Pickering emulsion, 0.5-5 parts by weight of pectin, and the balance water, wherein the Pickering emulsion contains 2-20 parts by weight of Pluronic F127 and 0.1-5 parts by weight of hydroxyapatite based on 100 parts by weight of the Pickering emulsion, and the Pickering emulsion contains 20-70 unit volumes of ethyl oleate based on 100 unit volumes of the Pickering emulsion.

2. The self-solidifying injectable pectin hydrogel according to claim 1, characterized in that, The self-solidifying injectable pectin hydrogel comprises the following components by weight based on 100 parts by weight of the hydrogel: 40-60 parts by weight of a Pickering emulsion, 1-3 parts by weight of pectin, and the balance water, wherein the Pickering emulsion contains 5-15 parts by weight of Pluronic F127 and 0.5-3 parts by weight of hydroxyapatite based on 100 parts by weight of the Pickering emulsion, and the Pickering emulsion contains 25-60 unit volumes of ethyl oleate based on 100 unit volumes of the Pickering emulsion.

3. The self-solidifying injectable pectin hydrogel according to claim 1, wherein, The self-solidifying injectable pectin hydrogel comprises the following components by weight based on 100 parts by weight of the hydrogel: 50 parts by weight of a Pickering emulsion, 2.5 parts by weight of pectin, and the balance water, wherein the Pickering emulsion contains 10 parts by weight of Pluronic F127 and 1 part by weight of hydroxyapatite based on 100 parts by weight of the Pickering emulsion, and the Pickering emulsion contains 30-50 unit volumes of ethyl oleate based on 100 unit volumes of the Pickering emulsion.

4. A method for preparing the self-solidifying injectable pectin hydrogel according to any one of claims 1 to 3, comprising the following steps: 1) Synthesis of a Pickering emulsion Pluronic F127 is weighed and dissolved in pure water to form a Pluronic F127 solution with a mass percentage of 2-20% for standby, nano-hydroxyapatite is weighed and added to the Pluronic F127 solution and mixed uniformly, then ethyl oleate is added to the mixture in proportion, and the mixture is dispersed with a high-speed shearing machine at 12000 rpm for 5 min to obtain a Pickering emulsion containing nano-hydroxyapatite; 2) Synthesis of a hydrogel Pectin is weighed and added to water and stirred to obtain a pectin solution, the Pickering emulsion prepared in step 1) is uniformly mixed with the pectin solution in a volume ratio of 1:1 to form a gel.

5. The preparation method according to claim 4, characterized in that, The mass percentage of the Pluronic F127 solution in step 1) is preferably 5-15%, and more preferably 10%.

6. The preparation method according to claim 4, characterized in that, In step 1), the amount of nano-hydroxyapatite added based on 100 mL of the Pluronic F127 solution is 0.1-5 g, preferably 0.5-3 g, and more preferably 1 g.

7. The preparation method according to claim 4, characterized in that, The amount of ethyl oleate added in step 1) is 25-60 mL, preferably 30-50 mL, more preferably 40 mL, based on 100 mL of the final Pickering emulsion.

8. The preparation method according to claim 4, characterized in that, The mass percentage concentration of the pectin solution in step 2) is 1-10%, preferably 2-6%, more preferably 5%.

9. Use of the self-solidifying injectable pectin hydrogel according to any one of claims 1 to 3 as a carrier in a medicament for the prevention of post-surgical recurrence of osteosarcoma and bone repair.

10. Use of the self-solidifying injectable pectin hydrogel according to any one of claims 1 to 3 for the preparation of a medicament for the prevention of post-surgical recurrence of osteosarcoma and bone repair.

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