Carrier-free functionalized lignin, preparation method and application in preparation of product for preventing and treating osteosarcoma

Abstract

The application discloses a carrier-free functionalized lignin, a preparation method and application thereof in preparing products for preventing and treating osteosarcoma, realizes functionalization of the lignin by regulating the structure of the lignin, has cell recognition ability, is specific to different attribute cells, regulates growth of osteosarcoma cells (MNNG / HOS) and mouse adipocytes (3T3-L1), and shows that the modified lignin can play an anti-cancer cell proliferation role while having little influence on normal cell activity, overcomes side effects and high cost of drugs, and has the advantages of simple preparation method, low cost, small side effects and great potential in the biomedical field.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of lignin functionalization processing and utilization, and particularly relates to application of carrier-free functionalized lignin in preparation of a product for preventing and treating osteosarcoma. BACKGROUND

[0002] Traditional cancer treatment methods, including surgery, chemotherapy and radiotherapy, have been proved to be insufficient to completely eradicate cancer cells. In addition, traditional chemotherapy methods have obvious shortcomings, such as obvious adverse reactions, development of drug resistance and limited selectivity. Natural biopolymers, such as chitosan, gelatin, sodium alginate, albumin and lignin, are widely used in the biomedical field. They have rich advantages, including antibacterial, antioxidant and other biological properties, which provide a potential improvement path for current biomedical technology.

[0003] Among them, lignin, as an aromatic polymer in plants, is cheap and environmentally friendly. It has many advantages, such as sun protection, antioxidant, antibacterial and the like. In addition, the low toxicity and biodegradability of lignin have attracted great attention in the biological field. The conventional methods for developing lignin in the biomedical field include lignin functionalization, nanotechnology and structural modification. Although these methods improve the availability and antitumor efficacy of drugs, lignin can only be used as a passive carrier or active site provider, i.e. it is processed into nanoscale and used in trace amounts, which cannot fundamentally avoid the side effects of drugs, and the advantages of lignin itself are not fully displayed, which limits its potential as an independent antitumor biological material. Therefore, it is crucial to develop functionalized lignin with antitumor activity to fully exploit the potential of lignin as a multifunctional and efficient anticancer biological material. SUMMARY

[0004] The technical problem to be solved by the present application is to provide a carrier-free functionalized lignin and its application in preparation of a product for preventing and treating osteosarcoma, aiming at overcoming the deficiencies of the prior art.

[0005] To achieve the above-mentioned purpose, the technical solutions adopted by the present application are as follows:

[0006] A preparation method of carrier-free functionalized lignin, comprising the following steps:

[0007] (1) dissolving lignin in an alkali solution to obtain a lignin alkali solution;

[0008] (2) adding a formaldehyde aqueous solution and an amination reagent into the lignin alkali solution obtained in step (1) in sequence, and fully reacting;

[0009] (3) transferring the solution obtained in step (2) to a dialysis bag, dialyzing with deionized water until neutral, and freeze-drying to obtain the carrier-free functionalized lignin.

[0010] Specifically, in step (1), the lignin is coniferous kraft lignin with a purity of greater than 90%; the alkali solution has a concentration of 0.5-1 mol / L, preferably 0.8 mol / L of sodium hydroxide aqueous solution.

[0011] Specifically, in step (2), the reaction system has a ratio of alkali, formaldehyde, lignin and amine reagent of (0.6-1.2) mmol:(0.8-1.2) mL:1 g:(9-15) mmol, preferably 0.8 mmol:1 mL:1 g:12 mmol.

[0012] Specifically, in step (2), the reaction is carried out at a temperature of 60-80°C, preferably 70°C, for 2-5 h, preferably 3 h.

[0013] Specifically, in step (3), the dialysis bag has a molecular weight of 1000 Da.

[0014] Further, the carrier-free functionalized lignin prepared by the preparation method of the present application is also within the protection scope of the present application, and the carrier-free functionalized lignin contains a chlorobenzylamine group.

[0015] Further, the carrier-free functionalized lignin prepared has a chlorobenzylamine group concentration of 0.1-1.0 mg / mL, preferably 0.2-0.8 mg / mL.

[0016] Further, the present application also claims protection of the use of the above carrier-free functionalized lignin in the preparation of a product for preventing and treating osteosarcoma.

[0017] Further, the present application also claims protection of a preparation for preventing and treating osteosarcoma, wherein the active ingredient comprises the carrier-free functionalized lignin containing a chlorobenzylamine group.

[0018] Further, the present application also claims protection of the use of the above carrier-free functionalized lignin in the preparation of a product for inhibiting the proliferation of osteosarcoma cells (MNNG / HOS).

[0019] In the present application, the prevention and / or treatment.

[0020] In the present application, the product is a drug or a reagent. When it is a drug, the product is a carrier-free functionalized lignin containing a pharmaceutically effective amount of the carrier-free functionalized lignin and a pharmaceutically acceptable carrier.

[0021] Beneficial effects:

[0022] The carrier-free functionalized lignin realizes functionalization of lignin by regulating the structure of lignin, has cell recognition ability, has specificity for different attribute cells, regulates the growth of osteosarcoma cells (MNNG / HOS) and mouse adipocytes (3T3-L1), and shows that the modified lignin can play an anti-cancer cell proliferation role, while having little effect on normal cell activity, overcomes the side effects and high cost of drugs, and has great potential in the biomedical field. BRIEF DESCRIPTION OF DRAWINGS

[0023] The above and / or other aspects of the present application will become apparent and more readily appreciated from the following description, taken in conjunction with the accompanying drawings, in which:

[0024] Figure 1 The infrared spectrum analysis of the chlorobenzylamine-based lignin (AL) and the original lignin (KL) prepared in the following examples.

[0025] Figure 2 The relative cell activity of the chlorobenzylamine-based lignin (AL) (0.1-0.8 mg / mL) prepared in Example 1 below after being co-cultured with osteosarcoma cells (MNNG / HOS) and normal cells (3T3-L1) for 24 h.

[0026] Figure 3 The relative cell activity of the chlorobenzylamine-based lignin (AL) (0.1-0.8 mg / mL) prepared in Example 2 below after being co-cultured with osteosarcoma cells (MNNG / HOS) and normal cells (3T3-L1) for 48 h.

[0027] Figure 4 The relative cell activity of the chlorobenzylamine-based lignin (AL) (0.1-0.8 mg / mL) prepared in Examples 1-2 below after being co-cultured with osteosarcoma cells (MNNG / HOS) and normal cells (3T3-L1) for 24 h and 48 h.

[0028] Figure 5 The relative cell activity of the unmodified lignin in the comparative example after being co-cultured with osteosarcoma cells (MNNG / HOS) and normal cells (3T3-L1) for 24 h.

[0029] Figure 6 The relative cell activity of the unmodified lignin in the comparative example after being co-cultured with osteosarcoma cells (MNNG / HOS) and normal cells (3T3-L1) for 48 h. DETAILED DESCRIPTION

[0030] The present application can be better understood from the following examples.

[0031] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0032] The chlorobenzylamine-based lignin described in the following examples is prepared as follows:

[0033] S1: 200 mg of lignin is dissolved in 1 mL of 0.8 mol / L sodium hydroxide;

[0034] S2: To the lignin solution in step S1, aqueous formaldehyde and amination reagent are sequentially added, the temperature is adjusted to 70°C, and the reaction time is 3 h;

[0035] S3: The solution in step S2 is removed and transferred to a 1000 Da dialysis bag, and dialysis is performed in deionized water. After dialysis to neutral, the obtained solid is chlorobenzylamine-based lignin by freeze-drying.

[0036] From the infrared spectrum, the changes in lignin absorption peaks of the original lignin (KL) and the chlorobenzylamine-based lignin (AL) can be clearly observed. In KL, for example: the wide peak at 3379 cm -1 belongs to the hydroxyl group in aliphatic and phenolic structures. The peak at 2840 cm -1 comes from the stretching vibration of C-H in the methylene structure, the peaks at 1603 cm -1 and 1512 cm -1 are attributed to the stretching vibration of the aromatic skeleton, and the peak at 1260 cm -1 belongs to the guaiacyl unit, and the peak at 1140 cm -1 comes from the ether bond in the lignin structure, indicating that the skeleton structure of lignin is not destroyed during the Mannich reaction. In the spectrum of AL, the peak at 3379 cm -1 is obviously widened, and the overlapping band moves from 3379 cm -1 in KL to 3523 cm -1 (O-H and N-H stretching) in AL lignin. The intensity of the C-H vibration peak from the lignin aromatic skeleton decreases, such as 1603 cm -1 , 1512 cm -1 , 1451 cm -1 and 855 cm -1 . The intensity of the peak at 2840 cm -1 from the stretching vibration of C-H in the methylene structure increases significantly. In addition, the N-H bending group at 1640 cm -1 produces a peak at 1090 cm -1 due to the stretching vibration of C-N. Figure 1). The results were obtained by subsequent elemental analysis, and the N element content of the chlorobenzylamine-based lignin increased from 0.2% to 3.59% (Table 1). In summary, it is shown that chlorobenzylamine is successfully grafted on lignin, and the reaction principle is as follows:

[0037]

[0038] Table 1

[0039] g％ N(％) C(％) H(％) S(％) KL - 0.2 62.37 5.00 0.77 AL 34.2 3.59 58.61 5.34 1.644

[0040] In the following examples, the relative cell viability % = (experimental group OD value-background value) / (control group OD value average-background value) x 100%; wherein, the OD value is the ultraviolet absorbance, and the background value is the blank control.

[0041] In the following examples, the NEAA non-essential amino acid (100x) is 10 mmol / L, and after 1:100 dilution, the final concentration is 0.1 mmol / L; sodium pyruvate (100x) is 11004 mg / L, and after 1:100 dilution, the final concentration is 110.04 mg / L; glutamine (100x) is 200 mmol / L, and after 1:100 dilution, the final concentration is 2 mmol / L.

[0042] In the following examples, the % is volume percent if not otherwise specified, and the cell viability detection method is MTT method.

[0043] Example 1

[0044] (1) Preparation of cell culture medium

[0045] MNNG / HOS cell culture medium: prepared according to the volume ratio of MEM medium: horse serum of 9:1.

[0046] 3T3-L1 cell culture medium: prepared according to the volume ratio of DMEM-H medium: calf serum of 9:1. Cell complete medium is also required to add NEAA non-essential amino acid, sodium pyruvate and glutamine.

[0047] (2) Adding chlorobenzylamine-based lignin for co-culture with cells

[0048] (i) Co-culture of chlorobenzylamine-based lignin with MNNG / HOS cells:

[0049] Experimental group: MNNG / HOS cells in logarithmic growth phase were taken for cell counting, and the cell concentration was adjusted, all according to 4x10 3Cells were seeded per well into 96-well plates, and 100 μL of chlorbenzylamine lignin (0.1–0.8 mg / mL, filtered and sterilized, in MNNG / HOS cell culture medium) was added. Cells were cultured in a 5% CO2 incubator at 37°C until adherence, and then cultured for 24 h. After 24 h, samples were taken, the culture medium was removed, and each well was washed three times with PBS. 100 μL of medium containing 10% MTT was added to each well, and the wells were incubated at 37°C for 4 h. The supernatant was discarded, and 100 μL of DMSO was added to each well. After gently shaking for 10 min, the absorbance at 570 nm was measured using a microplate reader.

[0050] Control group: The only difference from the experimental group was that chlorbenzylamine lignin was not added, and 100 μL of MNNG / HOS cell culture medium was added directly.

[0051] (ii) Co-culture of chlorbenzylamine lignin with 3T3-L1 cells:

[0052] The only difference from step (i) is that MNNG / HOS cells are replaced with 3T3-L1 cells and the culture medium is replaced with the culture medium required for culturing 3T3-L1 cells.

[0053] Depend on Figure 2 It can be seen that when the concentration is 0.1-0.8 mg / mL, the inhibition rate of osteosarcoma cells increases significantly after 24 h of culture. At 0.4 mg / mL, the inhibition rate of osteosarcoma cells increases to 60.8%, while there is almost no inhibitory effect on normal cells. As the concentration increases to 0.8 mg / mL, the inhibition rate of osteosarcoma cells is 64.3%, while there is almost no inhibitory effect on normal cells. It can be seen that chlorbenzylamine lignin exhibits cell specificity and its toxicity to cancer cells is more than twice that of normal cells.

[0054] Example 2

[0055] (1) Preparation of cell culture medium

[0056] MNNG / HOS cell culture medium: prepared according to a volume ratio of MEM medium: horse serum of 9:1.

[0057] 3T3-L1 cell culture medium: Prepare complete cell culture medium according to the volume ratio of DMEM-H medium to fetal bovine serum of 9:1, and add NEAA non-essential amino acids, sodium pyruvate and glutamine.

[0058] (2) Co-culture of cells with chlorbenzyl lignin

[0059] (i) Co-culture of chlorbenzylamine lignin with MNNG / HOS cells:

[0060] Experimental group: MNNG / HOS cells in logarithmic growth phase were collected, cell counts were performed, and cell concentrations were adjusted to 4 × 10⁻⁶ cells / day. 3 Cells / well were seeded into 96-well plates with 100 μL of chlorbenzylamine lignin (0.1–0.8 mg / mL, filtered and sterilized, in MNNG / HOS cell culture medium) added. Cells were cultured in a 5% CO2 incubator at 37°C until adherence, and then incubated for 48 h. Samples were taken at 48 h, the culture medium was removed, and each well was washed three times with PBS. 100 μL of medium containing 10% MTT was added to each well, and the wells were incubated at 37°C for 4 h. The supernatant was discarded, and 100 μL of DMSO was added to each well. After gently shaking for 10 min, the absorbance at 570 nm was measured using a microplate reader.

[0061] Control group: The only difference from the experimental group was that chlorbenzylamine lignin was not added, and 100 μL of MNNG / HOS cell culture medium was added directly.

[0062] (ii) Co-culture of phenol-sulfonated lignin with 3T3-L1 cells:

[0063] The only difference from step (i) is that 3T3-L1 cells are replaced with MNNG / HOS cells and the culture medium is replaced with the culture medium required for culturing 3T3-L1 cells.

[0064] Depend on Figure 3 It can be seen that when the concentration is 0.1-0.8 mg / mL, the inhibition rate of osteosarcoma cells increases significantly after 24 hours of culture. At 0.4 mg / mL, the inhibition rate of osteosarcoma cells increases to 59%, while there is almost no inhibitory effect on normal cells. As the concentration increases to 0.8 mg / mL, the inhibition rate of osteosarcoma cells is 58%, while there is almost no inhibitory effect on normal cells. It can be seen that chlorbenzylamine lignin exhibits cell specificity and its toxicity to cancer cells is more than twice that of normal cells.

[0065] Comparative Example

[0066] According to Examples 1-2 above, only the chlorobenzylamine lignin is replaced with the original lignin (excluding step S1, where the lignin in step S2 is replaced with unmodified lignin).

[0067] Depend on Figure 5 It can be seen that when unmodified lignin is co-cultured with cells, as the lignin concentration increases from 0.2 to 0.8 mg / mL, the inhibition rate of cancer cells reaches the highest level of 38% at 24 h.

[0068] Depend on Figure 6It can be seen that the unmodified lignin is co-cultured with cells, and with the increase of lignin concentration from 0.2 to 0.8 mg / mL, the inhibition rate of cancer cells is up to 35% at 48 h, and the normal cells also show a certain toxicity, which is 21.2%, and does not show good cell specificity. The effect is much lower than that of chlorobenzylamine lignin. Figure 4

[0069] The above experiments show that the modified lignin can play an anti-cancer cell proliferation role after the functional treatment of the lignin by modification without using exogenous drugs, and has little effect on normal cell activity, overcomes the side effects and high cost of drugs, and has great potential in the application in the biomedical field.

[0070] The application provides a carrier-free functionalized lignin, a preparation method, and a thought and method for application in preparation of products for preventing and treating osteosarcoma, and there are many methods and approaches for specifically realizing the technical scheme, and the above description is only a preferred embodiment of the application, and it should be pointed out that, for ordinary skilled persons in the technical field, some improvements and refinements can be made without departing from the principle of the application, and these improvements and refinements should also be regarded as the protection scope of the application. The components not explicitly described in the embodiment can be realized by using the prior art.​

Claims

1. A method for preparing a carrier-free functionalized lignin, characterized in that, It comprises the following steps: (1) dissolving lignin in an alkali solution to obtain a lignin alkali solution; (2) adding formaldehyde aqueous solution and aminating reagent into the lignin alkali solution obtained in step (1) in sequence and fully reacting; (3) transferring the solution obtained in step (2) into a dialysis bag, dialyzing with deionized water until neutral, and freeze-drying to obtain the product; In step (1), the lignin is softwood kraft lignin with a purity of more than 90%; and the alkali solution has a concentration of 0.5-1 mol / L; In step (2), the amount ratio of alkali, formaldehyde, lignin and aminating reagent in the reaction system is (0.6-1.2) mmol:(0.8-1.2) mL:1 g:(9-15) mmol; and the aminating reagent is chlorobenzylamine; In step (2), the reaction temperature is 60-80 ℃, and the reaction time is 2-5 h; In step (3), the dialysis bag has a molecular weight of 1000 Da.

2. The vector-free functionalized lignin produced by the method of claim 1, characterized in that, The carrier-free functionalized lignin contains chlorobenzylamine groups.

3. The carrier-free functionalized lignin of claim 2, wherein, The concentration of chlorobenzylamine groups in the prepared carrier-free functionalized lignin is 0.1-1.0 mg / mL.

4. Use of the carrier-free functionalized lignin according to claim 2 or 3 in the preparation of a product for preventing and treating osteosarcoma.

5. A preparation for the prevention and treatment of osteosarcoma, characterized in that, The active ingredient in the preparation comprises the carrier-free functionalized lignin according to claim 2 or 3.

6. Use of the carrier-free functionalized lignin according to claim 2 or 3 in the preparation of a product for inhibiting the proliferation of osteosarcoma cells.

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