CN112546243A - Targeted platinum nanocluster based on hyaluronic acid and preparation method and application thereof - Google Patents
Targeted platinum nanocluster based on hyaluronic acid and preparation method and application thereof Download PDFInfo
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- CN112546243A CN112546243A CN202011509538.7A CN202011509538A CN112546243A CN 112546243 A CN112546243 A CN 112546243A CN 202011509538 A CN202011509538 A CN 202011509538A CN 112546243 A CN112546243 A CN 112546243A
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Abstract
The invention belongs to the field of new materials, and particularly relates to a targeted platinum nanocluster based on hyaluronic acid and a preparation method and application thereof. And the full reaction of the platinum nanocluster solution and the hyaluronic acid solution is realized through EDC/NHS amide, a crude hyaluronic acid platinum nanocluster product is prepared, and the target hyaluronic acid platinum nanocluster is finally obtained after ultracentrifugation and dialysis. The synthesized hyaluronic acid targeted platinum nanocluster can be used for identifying the non-small cell lung cancer in a targeted manner and realizing selective targeted fluorescence imaging on the non-small cell lung cancer, the small cell lung cancer and normal lung cells.
Description
Technical Field
The invention belongs to the field of new materials, and particularly relates to a targeted platinum nanocluster based on hyaluronic acid and a preparation method and application thereof.
Background
The latest global tumor statistics in 2018 show that the incidence rate of the cancer in the global range ranks the first lung cancer, and accounts for 11.6% of the total cancer patients. Among them, non-small cell lung cancer (NSCLC) accounts for about 85% of all newly diagnosed lung cancers, and currently, clinical treatment modes of NSCLC mainly include surgery, radiation, drug therapy and the like. However, due to poor screening programs and late clinical symptoms, most patients are diagnosed with advanced lung cancer, and only 25% to 30% of NSCLC patients have a surgical indication. Therefore, the research on the early identification and diagnosis of the non-small cell lung cancer is of great significance. The platinum nanocluster material consisting of several to hundreds of atoms can realize 'calibration-treatment' multifunctionalization without combining other fluorescent markers (quantum dots, fluorescein and the like) based on the size dependence fluorescence characteristic of the platinum nanocluster material in the process of being used for novel platinum-based antitumor drugs, and has a huge application prospect in clinical cancer diagnosis and treatment. The interaction between the hyaluronic acid molecule and the CD44 can trigger multiple intracellular signal paths, so that the adhesion, migration, invasion and growth of tumor cells are caused, and the hyaluronic acid molecule is used as an early molecular diagnosis index for gastric cancer, head and neck cancer, breast cancer, lymphoma and the like.
The patent CN201910078873.7 discloses a T1-T2 bimodal targeting imaging contrast agent for lung cancer diagnosis and a preparation method thereof, wherein a self-assembly process of a nano carrier is utilized to wrap hydrophobic magnetic nano particles in a hydrophobic inner core of a polymer carrier, the hydrophobic nano particles comprise superparamagnetic bead nano particles and manganese dioxide nano particles, then targeting peptide cNGQGEQc is coupled on a maleimide group at the tail end of a hydrophilic chain, and the targeting peptide is positioned on an outer shell layer of a micelle after the micelle is formed, so that the T1-T2 bimodal targeting contrast agent SPIO-PEG-MnO 2-cNGQc is synthesized, and the contrast agent has the developing effects of lung cancer cell active targeting identification, T1 weighted images and T2 weighted images. Most of the existing fluorescent markers with targeting recognition need to be combined with targeting peptides to realize the targeting function.
In patent 201410170073.5, patent 201911041680.0 and patent 201410474210.4, all disclose that hyaluronic acid is used to prepare a substance with a targeting function, but the function of a product prepared by compounding hyaluronic acid and different substances is quite different, and in this subject, a targeting-fluorescence dual-functional anti-tumor platinum nanocluster drug system is constructed by modifying platinum nanoclusters with hyaluronic acid, so that not only different lung cancer cells can be targeted and positioned to realize differentiated imaging, but also a diseased part can be effectively positioned to inhibit the proliferation of specific lung cancer cells, and a new idea can be provided for designing and developing a multifunctional, high-activity and low-toxic-side-effect anti-non-small cell lung cancer chemotherapeutic drug.
Disclosure of Invention
In order to solve the technical problems, the invention provides a targeted platinum nanocluster based on hyaluronic acid and a preparation method and application thereof. The method can not only target and position different lung cancer cells to realize differential imaging, but also effectively position the diseased part to inhibit the proliferation of specific lung cancer cells, thereby providing a new idea for the positioning and identification of non-small cell lung cancer.
The technical scheme of the invention is realized as follows:
the preparation method of the targeted platinum nanocluster based on the hyaluronic acid comprises the following steps: adding a 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) solution into a hyaluronic acid solution, fully mixing, adding an N-hydroxysuccinimide imine (NHS) solution, fully mixing, adding the mixed solution into a platinum nanocluster solution, oscillating and uniformly mixing at 37 ℃ for 1-24h, ultracentrifuging, and finally dialyzing for 1-2 days to obtain the targeted platinum nanocluster.
Preferably, the concentration of the hyaluronic acid solution is 0.5-20g/L, the concentration of the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride solution is 1-50g/L, and the concentration of the N-hydroxysuccinimide imine solution is 1-50 g/L.
Preferably, the volume ratio of the hyaluronic acid solution, the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride solution and the N-hydroxysuccinimide imine solution is (5-10): 3: 2.
Preferably, the mixing temperature at room temperature is 25-37 ℃ and the mixing time is 1-12 h.
Preferably, the dialysis adopts a dialysis bag, and the type of the dialysis bag isM w CO=1000 D。
The targeted platinum nanocluster prepared by the preparation method is formed by wrapping platinum nanoclusters with amine polymer ligands.
The targeted platinum nanocluster is applied to preparation of a fluorescence labeling or fluorescence imaging reagent for targeted recognition of lung cancer cells.
Preferably, the lung cancer cell is human non-small cell lung cancer cell H1299, human small cell lung cancer cell H446 or human embryonic lung fibroblast HFL 1.
The invention has the following beneficial effects:
1. the hyaluronic acid targeting platinum nanocluster synthesized by the application can be used for identifying the non-small cell lung cancer in a targeting manner. Adding the hyaluronic acid targeting platinum nanoclusters into a culture solution containing different lung cells (such as human non-small cell lung cancer cells H1299, human small cell lung cancer cells H446 and human embryonic lung fibroblasts HFL 1) for co-culture, and displaying fluorescence imaging with different intensities under a confocal microscope based on the difference of cell membrane surface CD44 expression.
2. The preparation method of the hyaluronic acid targeting platinum nanocluster has the following characteristics: the cluster size is small (less than 2 nm), the particle size is uniform, and the fluorescence property is excellent; and the full reaction of the platinum nanocluster solution and the hyaluronic acid solution is realized through EDC/NHS amide, a crude hyaluronic acid platinum nanocluster product is prepared, and the target hyaluronic acid platinum nanocluster is finally obtained after ultracentrifugation and dialysis. The preparation method is simple, easy to operate and good in reproducibility, and can realize large-scale production; the novel targeted platinum nanocluster chemotherapeutic drug is constructed, has multiple functions of fluorescence and anti-tumor performance, realizes selective targeted fluorescence imaging on non-small cell lung cancer, small cell lung cancer and normal lung cells, differentially inhibits proliferation of the non-small cell lung cancer, and provides a new idea for early diagnosis and treatment of the non-small cell lung cancer.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a schematic structural diagram of a fluorescent platinum nanocluster in an embodiment of the present invention; wherein: 1-is platinum nanocluster, 2-is amine polymer ligand, and 3-is hyaluronic acid molecule.
FIG. 2 is a fluorescence image of non-small cell lung carcinoma cell H1299.
FIG. 3 shows fluorescence imaging of human small cell lung carcinoma cell H446.
FIG. 4 is a fluorescence image of HFL1 from human embryonic lung fibroblasts.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without inventive effort based on the embodiments of the present invention, are within the scope of the present invention.
Example 1
The preparation method of the targeted platinum nanocluster based on hyaluronic acid of the embodiment comprises the following steps:
adding 0.01 g hyaluronic acid into 10 mL water, mixing, adding 3 mL 10 g/L EDC solution, mixing for 20 min, adding 2 mL 10 g/L NHS solution, shaking at 37 deg.C for 1h, ultracentrifuging the above crude product, and mixing with typeM w COAnd (4) dialyzing the obtained product for 1D by using a 1000D dialysis bag, and centrifuging twice by using ultrafiltration to obtain the final targeted platinum nanocluster.
Example 2
The preparation method of the targeted platinum nanocluster based on hyaluronic acid of the embodiment comprises the following steps:
adding 0.02 g hyaluronic acid into 10 mL water, mixing well, adding 3 mL 30 g/L EDC solution, mixing thoroughly for 20 min, adding 2 mL 30 g/L NHS solution, shaking at 37 deg.C for 12h, ultracentrifuging the above crude product, and mixing with typeM w COAnd (4) dialyzing the obtained product for 2D by using a 1000D dialysis bag, and centrifuging twice by using ultrafiltration to obtain the final targeted platinum nanocluster.
Example 3
The preparation method of the targeted platinum nanocluster based on hyaluronic acid of the embodiment comprises the following steps:
adding 0.2 g hyaluronic acid into 5 mL water, mixing well, adding 3 mL 50g/L EDC solution, mixing thoroughly for 20 min, adding 2 mL 50g/L NHS solution, shaking at 37 deg.C for 5 hr, ultracentrifuging the above crude product, and mixing with typeM w COAnd (4) dialyzing by using a 1000D dialysis bag, and centrifuging twice by using ultrafiltration to obtain the final targeted platinum nanocluster.
Example 4
The preparation method of the targeted platinum nanocluster based on hyaluronic acid of the embodiment comprises the following steps:
adding 0.005 g hyaluronic acid into 10 mL water, mixing, adding 3 mL 1 g/L EDC solution, mixing for 50 min, adding 2 mL 1 g/L NHS solution, shaking at 30 deg.C for 1h, ultracentrifuging the above crude product, and mixing with typeM w COAnd (4) dialyzing the obtained product for 1D by using a 1000D dialysis bag, and centrifuging twice by using ultrafiltration to obtain the final targeted platinum nanocluster.
Example 5
The preparation method of the targeted platinum nanocluster based on hyaluronic acid of the embodiment comprises the following steps:
the preparation method of the hyaluronic acid targeting platinum nanocluster comprises the following steps: adding 0.01 g hyaluronic acid into 10 mL water, mixing well, adding 3 mL 1 g/L EDC solution, mixing well for 20 min, adding 2 mL 5 g/L NHS solution, shaking at 37 deg.C for 24 hr to obtain the crude extractThe product is ultracentrifuged and has the combined type ofM w COAnd (4) dialyzing the obtained product for 1.5D by using a 1000D dialysis bag, and centrifuging twice by using ultrafiltration to obtain the final targeted platinum nanocluster.
Examples of the effects of the invention
Differential targeting of lung cells: 10 μ L of the finally obtained target platinum nanoclusters of example 1 were added to sterile RPMI-1640 medium (containing 10% serum) containing different lung cells and sterilized air (5% CO) at 37 deg.C2) After 4h of co-culture under the conditions of (1), the suspension cells were fixed with 4% paraformaldehyde. After washing cells with PBS, glycerol (50%, v/v) was added dropwise and then cell imaging detection was performed by confocal microscopy. The imaging result is shown in fig. 2-4, fig. 2 is a confocal microscope image of H1299 cells incubated in FBS-containing medium for 4H, the nuclei emit blue fluorescence after DAPI staining, the hyaluronic acid-based targeted platinum nanoclusters emit green fluorescence, and the scale bar is 20 μm; FIG. 3 is a confocal microscope image of H446 cells incubated in FBS-containing medium for 4H, wherein nuclei emit blue fluorescence after DAPI staining, targeted platinum nanoclusters based on hyaluronic acid are green fluorescence, and the scale bar is 20 μm; fig. 4 is a confocal microscope image of HFL1 cells incubated in FBS-containing medium for 4h, the nuclei emitted blue fluorescence after DAPI staining, and the hyaluronic acid-based targeted platinum nanoclusters exhibited green fluorescence with a scale bar of 20 μm.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (8)
1. The preparation method of the targeted platinum nanocluster based on the hyaluronic acid is characterized by comprising the following steps: adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride solution into hyaluronic acid solution, fully mixing, then adding N-hydroxysuccinimide imine solution, mixing at room temperature, ultracentrifuging, and finally dialyzing for 1-2 days to obtain the targeted platinum nanocluster.
2. The method for preparing hyaluronic acid-based targeted platinum nanoclusters according to claim 1, wherein: the concentration of the hyaluronic acid solution is 0.5-20g/L, the concentration of the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride solution is 1-50g/L, and the concentration of the N-hydroxysuccinimide imine solution is 1-50 g/L.
3. The method for preparing hyaluronic acid-based targeted platinum nanoclusters according to claim 2, wherein: the volume ratio of the hyaluronic acid solution, the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride solution and the N-hydroxysuccinimide imine solution is (5-10) to 3: 2.
4. The method for preparing hyaluronic acid-based targeted platinum nanoclusters according to claim 2, wherein: the mixing temperature at room temperature is 25-37 deg.C, and the mixing time is 1-24 h.
5. The method for preparing hyaluronic acid-based targeted platinum nanoclusters according to claim 2, wherein: the dialysis adopts a dialysis bag, and the model of the dialysis bag isM w CO=1000 D。
6. The targeted platinum nanoclusters prepared by the preparation method of any one of claims 1 to 5, characterized in that: the targeted platinum nanocluster is platinum nanocluster wrapped by amine polymer ligand.
7. Use of the targeted platinum nanoclusters of claim 6 in preparation of fluorescent labels or fluorescent imaging reagents for targeted recognition of lung cancer cells.
8. Use according to claim 7, characterized in that: the lung cancer cell is human non-small cell lung cancer cell H1299, human small cell lung cancer cell H446 or human embryonic lung fibroblast HFL 1.
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Citations (2)
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CN108841385A (en) * | 2018-04-27 | 2018-11-20 | 中原工学院 | A kind of preparation method and application of the fluorescence platinum nanoclusters of hematological system suspension cell optical imagery |
CN110787146A (en) * | 2019-09-25 | 2020-02-14 | 中国人民解放军第四军医大学 | Preparation method and application of redox-responsive tumor-targeted cisplatin nano drug delivery system |
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CN108841385A (en) * | 2018-04-27 | 2018-11-20 | 中原工学院 | A kind of preparation method and application of the fluorescence platinum nanoclusters of hematological system suspension cell optical imagery |
CN110787146A (en) * | 2019-09-25 | 2020-02-14 | 中国人民解放军第四军医大学 | Preparation method and application of redox-responsive tumor-targeted cisplatin nano drug delivery system |
Non-Patent Citations (1)
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XIANG LING ET AL: "Synthesis and characterization of hyaluronic acid-platinum(IV) nanoconjugate with enhanced antitumor response and reduced adverse effects", 《RSC ADV.》 * |
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