CN118045203A - D/N@MSN-CNQDs-FA nano composition and synthetic method and application thereof - Google Patents
D/N@MSN-CNQDs-FA nano composition and synthetic method and application thereof Download PDFInfo
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- CN118045203A CN118045203A CN202410093865.0A CN202410093865A CN118045203A CN 118045203 A CN118045203 A CN 118045203A CN 202410093865 A CN202410093865 A CN 202410093865A CN 118045203 A CN118045203 A CN 118045203A
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Classifications
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- A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
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- A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
- A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
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Abstract
The invention discloses a D/N@MSN-CNQDs-FA nano composition and a synthesis method and application thereof, wherein the synthesis method comprises the following steps: encapsulating the chemotherapeutic drug doxorubicin DOX and the NO donor in MSN at the same time to finish the co-loading of the two anticancer substances; the dicarboxy PEG is combined with FA-CNQDs through amidation reaction and then coupled to the amino end of MSN, so as to synthesize the D/N@M-CN-FA nano-composition. The D/N@MSN-CNQDs-FA nano composition, the synthesis method and the application thereof have high affinity to liver cancer tissues and cancer cells, can penetrate solid tumors to enter the inside of tumors, directly target the liver cancer cells, release loaded antitumor drugs DOX and gas molecules NO in the liver cancer cells to cause apoptosis of the cancer cells, realize selective identification and synergistic killing of the liver cancer tissues and the liver cancer cells, and can be widely applied to preparation of antitumor drugs.
Description
Technical Field
The invention relates to the technical field of nano-compositions, in particular to a D/N@MSN-CNQDs-FA nano-composition and a synthetic method and application thereof.
Background
Cancer is one of the major diseases that threatens human life. Aiming at the problems existing in the current clinical liver cancer detection and treatment, a novel nano diagnosis and treatment compound is developed to improve the identification of tumor cells, enhance the targeting of anti-liver cancer drugs, ensure that the drugs smoothly reach focus positions through blood circulation and enter solid tumors, and is an important means for improving the liver cancer treatment effect. The graphite carbon nitride nano material (GRAPHITIC CARBON NITRIDE NANOMATERIALS, nCN) has wide application prospect in tumor diagnosis and treatment due to the unique structure and optical characteristics. Folate Receptors (FR) are highly expressed on the surface of many tumor cells. Gas therapy is gaining increasing importance in cancer treatment, most commonly in combination therapy. Therefore, the research uses folic acid receptor as a target point, develops various nano diagnosis and treatment complexes based on nCN, realizes the combined treatment of liver cancer, and provides a novel nano targeting composition with conversion possibility for diagnosis and treatment of liver cancer.
Disclosure of Invention
The invention aims to provide a D/N@MSN-CNQDs-FA nano composition, a synthesis method and application thereof, which have high affinity to liver cancer tissues and cancer cells, can penetrate solid tumors to enter the inside of tumors, directly target the liver cancer cells, release loaded antitumor drugs DOX and gas molecules NO in the liver cancer cells to cause apoptosis of the cancer cells, realize selective identification and synergistic killing of the liver cancer tissues and the liver cancer cells, and can be widely applied to preparation of antitumor drugs.
In order to achieve the above purpose, the invention provides a method for synthesizing a D/N@MSN-CNQDs-FA nano composition, which comprises the following steps:
S1, simultaneously encapsulating chemotherapeutic drugs doxorubicin DOX and an NO donor (N-diethylamino-N-oxo-nitrosamine sodium salt DETA NONOate) in mesoporous silica nano-particles MSN to finish the co-loading of two anticancer substances;
S2, the dicarboxyl PEG is combined with folic acid functionalized carbon nitride quantum dots FA-CNQDs through amidation reaction and then coupled to the amino end of MSN, so that the D/N@MSN-CNQDs-FA nano composition is synthesized.
Further, in the step S1, the mass ratio of the chemotherapeutic agent DOX to the NO donor is DOX: NO donor = 1:20.
Further, the preparation process of the FA-CNQDs in the step S2 is as follows: firstly, melamine and cyanuric chloride are polymerized through hot acetonitrile to form a triazine structure, and then, the mixture of the triazine structure and N-hydroxysuccinimide conjugated folic acid FA-NHS is subjected to hydrothermal synthesis of FA-CNQDs under subcritical conditions.
The invention also provides the D/N@MSN-CNQDs-FA nano composition prepared by the synthesis method.
The invention also provides application of the D/N@MSN-CNQDs-FA nano composition in preparation of antitumor drugs.
Further, the tumor includes liver cancer.
The D/N@MSN-CNQDs-FA nano composition and the synthesis method and application thereof have the advantages and positive effects that:
1. The invention develops MSN nanometer combination (D/N@M-CN-FA) which takes CNQDs as pH response switch, and the switch is opened under the acidic condition. The nano composition has high affinity to liver cancer tissues and cancer cells, can penetrate solid tumors to enter the inside of tumors, directly acts on the liver cancer cells in a targeting way, enters the cells through endocytosis, releases the loaded anti-tumor drug DOX and gas molecules NO in the liver cancer cells, causes cancer cell apoptosis, and realizes selective identification and synergistic killing of the liver cancer tissues and the liver cancer cells.
The technical scheme of the invention is further described in detail through the drawings and the embodiments.
Drawings
FIG. 1 is an electron microscope image and Zeta potential measurement of FA-CNQDs in the embodiment of the invention, wherein a is an FA-CNQDs transmission electron microscope, b is the particle size distribution of the FA-CNQDs, and c is the Zeta potential value of the FA-CNQDs and the CNQDs under different pH values;
FIG. 2 shows an electron microscope image of MSN and M-CN-FA in the embodiment of the invention, wherein a and b are M-CN-FA electron microscope images, and c and d are MSN electron microscope images;
FIG. 3 is a graph of D/N@M-CN-FA drug release in an example of the present invention, wherein A is a DOX release profile and B is a DETA NONOate release profile;
FIG. 4 is a CLSM image of HepG2 cells after 2h incubation of D/N@M-CN-FA, D/N@M-CN in the examples of the present invention, DAPI excitation wavelength=405 nm, scale bar=100 μm;
FIG. 5 is a CLSM image showing penetration of D/N@M-CN and D/N@M-CN-FA in HepG2 multicellular spheres in vitro, the surface of which was defined as 0 μm, in examples of the present invention. Excitation wavelength=488 nm, scale bar=50 μm;
FIG. 6 shows the detection results of NO fluorescent probes in the embodiment of the invention, wherein a is a control group, b is a DETA NONOate detection result, c is N@M-CN detection result, and D is a D/N@M-CN-FA detection result;
FIG. 7 shows the cytotoxicity results of CCK-8 assay in accordance with the present invention;
FIG. 8 is a graph showing EdU detection results according to an embodiment of the invention;
FIG. 9 shows the result of a Transwell test in the example of the present invention, wherein a is the control group, b is DETA NONONOate, c is N@M-CN, D is DOX, e is D@M-CN, f is D/N@M-CN, and g is D/N@M-CN-FA;
FIG. 10 shows the result of a scratch test in an embodiment of the present invention, wherein a is a control group, b is DETA NONONOate, c is N@M-CN, D is DOX, e is D@M-CN, f is D/N@M-CN, and g is D/N@M-CN-FA;
FIG. 11 is a graph showing the results of the anti-liver cancer effect test of mice with D/N@M-CN and D/N@M-CN-FA in the examples of the present invention, wherein A is the tumor tissue of the mice, B is the weight change of the mice, A is the control group, B is DETA NONONOate, C is DOX, D is D@M-CN, e is D/N@M-CN, f is D/N@M-CN-FA, and C is the tumor volume change;
FIG. 12 shows the results of H & E staining of major organs after 14 days of treatment with the tumor-bearing mouse nanocomposites according to the examples of the present invention;
FIG. 13 shows apoptosis results of tumor tissue Tunel experiments after 14 days of treatment with the tumor-bearing mouse nano-composition according to the example of the present invention.
Detailed Description
The technical scheme of the invention is further described below through the attached drawings and the embodiments.
Unless defined otherwise, technical or scientific terms used herein should be given the ordinary meaning as understood by one of ordinary skill in the art to which this invention belongs.
All experimental data were analyzed by anova using SPSS26 software. Values are expressed as mean ± Standard Deviation (SD). "p <0.05," p <0.01, "" p <0.001.
In the invention, the D/N@MSN-CNQDs-FA means MSN-CNQDs-FA loaded with DOX and NO donors, which is called D/N@M-CN-FA for short, and M-CN-FA is MSN-CNQDs-FA without loaded with DOX and NO donors, and the preparation steps are the same as those of D/N@M-CN-FA, except that the step of loading DOX and NO donors is not performed.
In the invention, the N@MSN-CNQDs mean MSN-CNQDs loaded with NO donors, which are called N@M-CN for short.
In the invention, the D@MSN-CNQDs mean DOX-loaded MSN-CNQDs, which are called D@M-CN for short.
In the invention, the D/N@MSN-CNQDs mean MSN-CNQDs loaded with DOX and NO donors, and are called as D/N@M-CN for short.
The FA-CNQDs in the embodiment of the invention are prepared by adopting a one-pot method, and the specific principle is as follows: firstly, melamine and cyanuric chloride are polymerized through hot acetonitrile to form a triazine structure, and then, the mixture of the triazine structure and N-hydroxysuccinimide conjugated folic acid FA-NHS is subjected to hydrothermal synthesis of FA-CNQDs under subcritical conditions.
Examples
Preparation of MSN.
3.10G of 28% NH 3·H2 O and 10g of ultrapure water were taken and added to 58.5g of 99.7% absolute ethanol. Firstly, stirring and preheating for 30min in a water bath kettle at the temperature of 30 ℃, increasing the stirring speed, dripping 5.6mL of tetraethyl orthosilicate (TEOS) at the uniform speed of 0.2mL/min by using a dropper, and standing for reaction for 1h after the dripping is finished. A mixture of 0.417g TEOS and 0.187g octadecyltrimethoxysilane (C 18 TMDS, pore-forming agent) was added dropwise with vigorous stirring at a rate of 0.2 mL/min. Standing at room temperature for reaction for 1h, adding 600 μl of 3-aminopropyl triethoxysilane, stirring for 1h, washing with water for three times, transferring to a drying oven, and drying at 70deg.C for 12h. Finally, the mixture was put into a muffle furnace, and the temperature was raised to 550℃at 2.3℃per minute, so that the temperature was kept at 550℃for 6 hours. After the muffle furnace was cooled naturally, the material was removed, 4.5632g of ammonium bifluoride (NH 4HF2) was added to the white solid to etch away MSN, and the reaction was maintained on a magnetic stirrer for 12h. After the template is corroded, centrifuging for 5min at 8000 rpm, washing with ultra-pure water for three times, and finally storing in ultra-pure water at 4 ℃ for standby.
2. And (3) synthesizing folic acid functionalized carbon nitride quantum dots (FA-CNQDs).
The synthesis method of the CNQDs and the FA-CNQDs is as follows:
0.125mmol MA and 0.25mmol CC were added to 12.5mL acetonitrile, mixed well, placed in a 100mL Teflon lined autoclave, heated, and held at 180℃for 8h. After the reaction, the autoclave was naturally cooled to room temperature. The resulting product was washed with acetonitrile and distilled water in this order by centrifugation several times until the pH of the solution reached 6.8, and then redispersed in 4mL of distilled water. The suspension was sonicated for 10min and centrifuged at 12000rpm for 5min. The supernatant was collected, filtered through a 0.22 μm filter (Solarbio), then concentrated under reduced pressure to a final concentration of 1.0mg/mL, and stored at 4℃in the absence of light for further use, and the resulting material was CNQDs. FAx-CNQDs were prepared by the same procedure except that the precursor was changed to 0.125mmolMA, 0.25mmol CC and a mixture of different amounts of FA-NHS in 12.5mL acetonitrile. X in FAx-CNQDs represents the molar amount of FA-NHS added.
Synthesis of D/N@M-CN-FA nanocomposites.
10Mg of MSN is weighed and dissolved in 5mL of water, 5mg of medicine (DOX: NO donor=1:20) is added, the mixture is stirred for 24 hours, centrifuged at 12000rpm, washed with water and resuspended in 1mL of ultrapure water; then 1mL of MSN aqueous solution is added into 1mg/mLEDC/NHS activated for 2h of COOH-PEG-COOH to react for 6h, so as to ensure that activated carboxyl on PEG can fully react with inherent amino on the surface of MSN. Centrifuging after stopping the reaction, washing with water, and re-suspending in 5mL of water; then 1mL of 1mg/mL of the solution of FA-CNQDs was added dropwise to the above system, the reaction was continued for 12 hours, centrifuged, washed with water, resuspended in 1mL of ultrapure water, and stored at 4℃in the absence of light.
4. And (6) testing the drug loading rate.
The drug loading is the weight percentage of the drug in the nano material, and the drug content is quantified by measuring the absorbance of DOX at 480nm by an ultraviolet-visible spectrophotometer. The freeze-dried drug-loaded MSN is subjected to ultrasonic treatment in dimethyl sulfoxide and then is centrifuged, and the supernatant is taken for absorbance measurement. The drug content was determined from a calibration curve obtained using different concentrations of DOX in dimethyl sulfoxide solution. The DOX solutions with different concentrations are prepared to measure the absorbance at 480nm, a standard curve of DOX concentration and absorbance is made, the drug loading is calculated according to a formula, and the nitric oxide donor (N-diethylamino-N-oxo-nitrosaminide sodium salt) is detected at the wavelength of 540nm by the same method.
The results are shown in Table 1,
TABLE 1
As can be seen from table 1, the simultaneous encapsulation of DETA NONOate and DOX in MSN significantly improves the drug delivery efficiency of MSN, providing greater therapeutic efficacy.
5. Characterization of the material.
(1) And detecting Zeta potential.
Filtering the synthesized nano composition with a filter membrane of 0.22 mu m, removing bubbles from the filtered solution, adding 2mL into a plastic cuvette, and detecting with a dynamic light scattering instrument.
(2) A transmission electron microscope.
The nanomaterial was centrifuged with ultra-pure water at 8000rpm for 5min, washed twice, and resuspended in ultra-pure water. And (3) dripping the mixed solution onto a copper mesh to form spherical water drops, standing for overnight and drying. Analysis was performed by TEM.
(3) Scanning electron microscope.
The nanomaterial was centrifuged with ultra-pure water at 8000rpm for 5min, washed twice, and resuspended in ultra-pure water. And (3) dripping the mixed liquid on a silicon wafer to form spherical water drops, standing for overnight and drying. And then analyzed by SEM.
The particle size and morphology of the FA-CNQDs were analyzed by transmission electron microscopy. As shown in FIG. 1, the FA-CNQDs have good monodispersity (a in FIG. 1) and a particle size of about 6.97.+ -. 0.84nm (b in FIG. 1). In order to explore the amphoteric nature of FA-CNQDs, zeta potentials at different pH values were examined, and the result shows that the isoelectric point of FA-CNQDs is 6.2-6.3 as shown in c of 1. The FA-CNQDs are negatively charged when the pH of the solution environment is higher than the isoelectric point, and the FA-CNQDs are positively charged when the pH of the solution environment is lower than the isoelectric point.
The particle size and morphology of M-CN-FA were analyzed by scanning electron microscopy. As a result, as shown in FIG. 2, MSN and M-CN-FA generally exhibit a round nanosphere structure, and the particle size of a single M-CN-FA is substantially the same, about 220.+ -. 8.96nm (a, c in FIG. 2). In FIG. 2b, the surface of the M-CN-FA material is in a rugged state due to the presence of mesopores.
6. The pH of the nano-drug responds to the slow release of the drug.
The pH responsiveness of the nanocomposites was studied at 37 ℃ under PBS conditions of different pH. 2mL of pH7.2, 1mg/mL of the D/N@M-CN-FA solution and 2mL of the pH6, 1mg/mL of the D/N@M-CN-FA solution were transferred to two dialysis bags (MW=10 kDa), respectively, and then immersed in 4mL of release medium (10 mM pH7.2 PBS and 10mM pH6 PBS), respectively. The release medium is removed for UV-vis analysis and replaced with fresh release medium for a selected time interval. DOX concentration was calculated from absorbance at 480 nm. In the release profile of drug release behavior, the cumulative percent release of drug from MSN is plotted over time. The cumulative release percentage is calculated as equation (2):
Wherein C n and W 0 are the DOX concentration and total DOX amount in MSN for n releases collected. Release experiments were divided into 3 replicates. The same method detects DETA NONOate at a wavelength of 540 nm.
The results are shown in FIG. 3, where A is the DOX release profile of D/N@M-CN-FA at pH7.2 and pH6 at a constant temperature of 37 ℃. At pH7.2, there was about 36% DOX release in 5 hours and about 58.9% DOX release in 24 hours. At pH6, there was about 58% DOX release in 5 hours and about 87% DOX release in 24 hours. The results demonstrate that pH gating can regulate the release of drug from MSN. B is the DETA NONOSAte release profile of D/N@M-CN-FA at pH7.2 and pH 6. At pH6, release of DETA NONONOate was about 56% in 5h and about 83% in 24 h. The NO donor can be stably released.
7. Selective cellular uptake and targeting capabilities of nanomedicines.
(1) Fluorescence imaging of D/N@M-CN-FA.
HepG2 cells were seeded in 35mm confocal dishes, 1.0X10 5 cells per well were cultured, and the whole medium was used to culture about 70%. The culture broth was removed and incubated with 1.0mL of fresh serum-free medium containing 0.05mg/mL of D/N@M-CN or D/N@M-CN-FA, respectively, for 2h. Cells were stained with DAPI, washed three times with PBS and 1mL of PBS was added according to the instructions provided by the manufacturer. The targeting efficiency of the nanomedicine was directly observed by CLSM with the excitation wavelength fixed at 488nm of the DOX channel and 405nm of the DAPI channel.
The targeting ability of D/N@M-CN-FA and D/N@M-CN was observed with a laser confocal microscope using the fluorescent properties of DOX. The experiment is carried out under the serum-free condition, and the result is shown in fig. 4, only a small amount of DOX in the D/N@M-CN treatment group is endocytosed by liver cancer cells, and a large amount of DOX in the D/N@M-CN-FA targeting vector treatment group is endocytosed by liver cancer cells after co-incubation for 2 hours, so that more nano-drugs can be endocytosed into cancer cells by combining nano-drug surface folic acid with a folic acid receptor on the surface of HepG2 cells.
(2) 3D cell pellet culture.
3D cell spheres were cultured and the penetration capacity of the drug carrier was observed. 10 mL of 1.5w/v% hot agarose solution was added to a 25mL dish and after cooling, the semi-solid agarose coating provided a non-adherent surface to prevent cell adhesion. 1.0X10 6 HepG2 cells were suspended in 15 ml of medium and then transferred to a petri dish and cultured for 4 days until spheroids were produced. After centrifugation of the collected spheres, PBS was washed and resuspended in serum-free medium containing 0.05mg/mL D/N@M-CN-FA for 4h, respectively, after which the spheres were washed 3 times with PBS and observed with a CLSM Z-stack excitation scan, the wavelength was fixed at 488nm. The surface of the 3D cell sphere is defined as (starting point) 0 μm. As can be seen from the CLSM image in FIG. 5, the DN@M-CN nano-drug has good permeability in the 3D cell sphere, and red fluorescence is obviously enhanced after the targeting group FA is added, which indicates that more DN@M-CN-FA enters the inside of the 3D cell sphere.
(3) Intracellular NO level detection.
HepG2 cells were cultured in 35mm confocal dishes with complete medium, 1.0X10 5 cells per dish, and when the cells grew to around 70%, the medium was removed and 1.0mL fresh serum-free medium (containing control, DETA NONOate, 0.1mg/mL N@M-CN or N@M-CN-FA) was added and incubated for 2h, respectively. Cells were washed with PBS, 15. Mu.M DAF-FM DA probe incubated in PBS for 40min, washed 3 times with PBS, and 1mL of PBS was added. The NO-initiated fluorescent signal was directly observed with CLSM under excitation at a fixed wavelength of 495 nm.
DAF-FM DA is a membrane permeable NO fluorescent probe, E x/Em = 495/515mm. DAF-FM DA actively diffuses across the cell membrane into the cell to produce DAF-FM, which increases fluorescence about 160-fold after binding to NO. The detection results are shown in fig. 6, and the DETA NONOate group has a small amount of fluorescence, which indicates that NO carrier is present, and only a small amount of DETA NONOate enters the cancer cell to generate NO; the N@M-CN group fluorescence intensity is higher than that of the DETA NONOate group, which shows that mesoporous silicon plays a good role in transportation, and more NO is generated in cancer cells; the fluorescence intensity of the D/N@M-CN-FA group is higher than that of the DETA NONONOate group and the N@M-CN group, which shows that the targeting of the D/N@M-CN-FA is good, more medicines can be transported into cancer cells, and the medicine release efficiency is good.
D/N@M-CN-FA inhibits cell proliferation, migration and invasion
(1) CCK-8 cytotoxicity assay.
Well-grown HepG2 cells were digested, counted with Countstar, diluted with complete medium (DMEM supplemented with 10% fbs and 1% diabody) and the cell suspension adjusted, seeded in 96-well plates with 10 4 cells per well and cultured for 24h at 37 ℃, 5% co 2. The cells were incubated with DMEM medium containing different concentrations of drug carrier (0.1 mg/mL N@M-CN, 0.1mg/mL D@M-CN, 0.1mg/mL D/N@M-CN, 0.1mg/mL D/N@M-CN-FA) for 24h, respectively. Cell viability was determined by CCK-8 assay according to the instructions provided for the kit:
As shown in FIG. 7, after DETA NOnoate, DOX, D/N@M-CN or D/N@M-CN-FA treatment, hepG2 cell growth inhibition rates were 9%, 48%, 66% and 80%, respectively. This demonstrates that DETA NONOate alone had no significant therapeutic effect, but DETA NONOate and DOX were delivered through the nanocarrier into the cells with an increase in inhibition of 66%; the inhibition rate of the D/N@M-CN-FA on the cell growth is up to 80%, because the D/N@M-CN-FA can target HepG2 cells, so that more drugs can target and enter the cells through endocytosis, and the inhibition rate on the cell growth is improved. Therefore, the folic acid targeting and the folic acid receptor mediated endocytosis can increase the concentration of medicines in cells, promote the killing effect of cancer cells, and provide a broad prospect for improving the curative effect and reducing the toxic and side effects of D/N@M-CN-FA in clinical application.
(2) EdU cell proliferation assay.
HepG2 cells were seeded in 12-well plates, 5X 10 5 cells per well, and cultured in complete medium for 24h. The cells were then treated with DMEM medium containing 0.05mg/mL N@M-CN, 0.05mg/mL D@M-CN, 0.05mg/mL D/N@M-CN, or 0.05mg/mLD/N@M-CN-FA nanomedicine, respectively, for 24h. Using BeyoClick TM EdU-594 cell proliferation assay kit, 100. Mu.L of 50. Mu. MEdU medium was added for 2h, PBS was eluted, 4% paraformaldehyde was fixed for 30min, 50. Mu.L of 2mg/mL glycine was added for 5 min, and 100. Mu.L of 0.5% Triton X-100 PBS was added after PBS elution for 10min. 100. Mu.L of 1 XApollo staining reaction solution was added, and the mixture was incubated at room temperature for 30min in the absence of light. 100. Mu.L of 0.5% Triton X-100 PBS was again added to wash 2-3 times for 10min each. Finally, 100 mu L of 1 Xhoechst 33342 reaction solution is added into each hole, incubated at room temperature for 30min in a dark place, sealed, and photographed. The results are shown in FIG. 8, in which the number of living cells gradually decreased after 24h of treatment with different drugs, indicating that the proliferation of HepG2 cells was inhibited by the different drugs. And the minimum number of living cells after the D/N@M-CN-FA treatment shows that the D/N@M-CN-FA has the strongest inhibition effect on the proliferation capacity of HepG2 cells.
(3) Transwell cell migration experiments.
After overnight starvation of the logarithmic growth phase HepG2 cells with serum-free DMEM medium, 0.25% pancreatin was digested. The single cell suspension was then conditioned to a cell concentration of 1X10 5/mL in serum-free DMEM medium. 0.2mL of the culture medium is added into a Transwell cell with the filter membrane aperture of 8 mu m, and DMEM culture mediums of different medicines of 0.05mg/mL N@M-CN, 0.05mg/mL D@M-CN, 0.05mg/mL D/N@M-CN or 0.05mg/mL D/N@M-CN-FA are respectively added into an upper chamber and a lower chamber. After 12h of culture, the filter membrane is taken out, residual cells on the inner surface of the filter membrane in the upper chamber are wiped off by a cotton swab, the cells are fixed for 20min by 90% ethanol, and the cells are stained by 0.1% crystal violet and washed by PBS. The number of cells passing through the membrane was observed under a microscope at 200 x field.
Transwell experiments examined the effect of drugs on HepG2 cell migration invasiveness. The results are shown in FIG. 9, in which the number of HepG2 cells migrating to the lower layer is gradually reduced after the treatment with different drugs, indicating that the drugs have the effect of inhibiting the invasion of HepG2 cells. Compared with the control group, the cell migration quantity of the D/N@M-CN-FA treatment group is obviously reduced, which shows that the D/N@M-CN-FA has the strongest effect of inhibiting the migration capacity of HepG2 cells.
(4) Cell scratch experiments.
Marking the back of a clean 6-hole plate by using a Mark pen, inoculating a proper amount of cells in each hole for culturing until the cells are completely adhered and the confluence degree reaches about 80%, using a clean 10 mu L gun head, and carrying out a scratching operation by using a gun tip which is perpendicular to the drawn transverse line compared with a ruler. After streaking, the wells were washed 2 times with PBS and 2mL of 10% FBS in DMEM medium was added to each well. Immediately after wrapping the sealing film around the edge of the 6-well plate, a photograph was taken using a microscope. After photographing, discarding the sealing film, adding nano-drugs (0.05 mg/mL N@M-CN, 0.05mg/mL D@M-CN, 0.05mg/mL D/N@M-CN and 0.05mg/mL D/N@M-CN-FA) respectively, culturing for 48h in an incubator, and continuously photographing.
In the cell scratch experiments, hepG2 cells were treated with different drugs and observed with a microscope and photographed at 0h, 48h, respectively. As shown in fig. 10, a1-g1 is a 0h photograph recording result, a2-g2 is a 48h photograph recording result, and as can be seen from fig. 10, b2 is a weak inhibition effect of the NO donor group on HepG2 cells compared with the blank group after 48h addition of the NO donor; c2 is group N@M-CN, again, with weak inhibition of cell migration; d2 is a chemotherapeutic DOX and has good inhibition effect on tumor cell migration; e2 is D@M-CN group, and the inhibition to the tumor growth is higher than that of single-drug DOX; D/N@M-CN is carried on a nano-drug carrier together with a DOX and a NO donor, and the cell migration ability of the D/N@M-CN-FA treatment group is obviously inhibited due to the targeting of FA to cells, and the 48h photographing result is basically indistinguishable from 0h, so that the D/N@M-CN-FA has the strongest inhibition effect on the HepG2 migration ability.
9.D/N@M-CN-FA can inhibit liver cancer tumor growth of mice.
(1) And (6) establishing a tumor model.
The anti-liver cancer activity of mice is measured by in vitro screening of nano drug-carrying system with high anti-cancer activity. Ethical wholesale of all animal experiments were obtained from the animal ethical committee of the university of fuqian. 100 μL of liver cancer hep1-6 cells (5×10 6) were subcutaneously injected on the dorsum (male, 6-8 weeks) of healthy C57BL/6 mice. Tumor volume was calculated as: tumor volume = length x width 2/2 calculation. After a few days, mice are randomly grouped and start intravenous injection of the medicine after tumors grow to 80-120 mm 3. The length and diameter of the tumor were measured every 2 days with a vernier card, and the tumor volume was calculated.
As shown in fig. 11, panel C shows the change in tumor volume during different drug treatments, with insignificant change in tumor growth compared to the control group by DETA NONOate (NO precursor) alone, the D/N@M-CN group (drug co-loaded with NO donor) enhanced the inhibition of tumor growth by drug, and the D/N@M-CN-FA group had the greatest inhibition of tumor growth by drug delivery to tumor cells due to folate targeting. Panel A shows that the D/N@M-CN-FA group significantly inhibited tumor growth and minimal tumor size after 14 days of treatment, demonstrating significant therapeutic effects of D/N@M-CN-FA. As can be seen from the change in weight of the mice in panel B, there was no significant change in weight of the mice during the treatment.
C57BL/6 mice were randomly divided into 6 treatment groups (5 per group): control 、DETA NONOate(16mg DETA NONOate/kg)、DOX(0.8mg DOX/kg)、D@M-CN(0.8mg DOX/kg)、D/N@M-CN(0.8mg DOX/kg+16mg DETA NONOate/kg)、D/N@M-CN-FA(0.8mg DOX/kg+16mg DETA NONOate/kg), mice were injected every two days with 0.9% NaCl (100. Mu.L) or 100. Mu.L of the nanotargeting material, respectively. In 14 days of observation of antitumor effect, body weight was weighed every 2 days and tumor size was measured. At the end of the experiment, mice were euthanized and major organs including heart, liver, spleen, lung were collected, kidneys, tumors were taken, and paraffin sections H & E stained to evaluate cell and tissue morphology. The results are shown in FIG. 12, where the alveolar epithelial cells were intact in structure and the pulmonary tissue was free of congestion edema and inflammatory cell infiltration. The experimental group and the control group have no obvious difference. The spleen tissue structure is compact, and no obvious pathological change exists. The experimental group and the control group have no obvious difference. The glomeruli of the control group have clear outlines, the renal small capsule cavity is not subjected to stenosis or adhesion, and the epithelial cells of the renal tubular are complete in structure. Experimental group: the kidney tissue is engorged with blood, the tubular epithelial cells are slightly swollen, and a small amount of inflammatory cells are scattered and infiltrated. The control group has regular myocardial fiber arrangement, clear structure, normal myocardial cell morphology, clear and complete nucleus, multiple cell number and slightly alkaline cytoplasm. The cell number of the experimental group is obviously reduced, and the cytoplasm is eosinophilic. For tumor tissue, the experimental group cells were slightly reduced, and the number density was decreased.
(2) Tissue Tunel staining.
Tunel assay was performed following the procedure of the in situ apoptosis assay kit. The slices were dewaxed and rehydrated with xylene and graded alcohol. Proteinase K was added to enhance tissue penetration. After recovery in citrate buffer and 80kPa for 3min, the sections were incubated with the labeling and enzyme solution mixture in the dark at 37℃for 60min. The sections were observed with a fluorescence microscope. Fig. 13 shows that Tunel detected apoptosis of tumor tissue 14 days after tail vein injection of different drugs. It can be seen that the D/N@M-CN-FA treatment group tumor tissue has a large number of apoptotic cells, which indicates that the D/N@M-CN-FA drug induces apoptosis of tumor cells, thereby achieving the treatment effect.
Therefore, the MSN-CNQDs-FA nano composition, the synthesis method and the application thereof have high affinity to liver cancer tissues and cancer cells, can penetrate solid tumors to enter the inside of tumors, directly target the liver cancer cells, release the loaded antitumor drugs DOX and gas molecules NO in the liver cancer cells to cause apoptosis of the cancer cells, realize selective identification and synergistic killing of the liver cancer tissues and the liver cancer cells, and can be widely applied to preparation of antitumor drugs.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention and not for limiting it, and although the present invention has been described in detail with reference to the preferred embodiments, it will be understood by those skilled in the art that: the technical scheme of the invention can be modified or replaced by the same, and the modified technical scheme cannot deviate from the spirit and scope of the technical scheme of the invention.
Claims (6)
- The synthesis method of the D/N@MSN-CNQDs-FA nano composition is characterized by comprising the following steps:S1, simultaneously encapsulating chemotherapeutic drugs doxorubicin DOX and an NO donor (N-diethylamino-N-oxo-nitrosamine sodium salt DETA NONOate) in mesoporous silica nano-particles MSN to finish the co-loading of two anticancer substances;S2, the dicarboxyl PEG is combined with folic acid functionalized carbon nitride quantum dots FA-CNQDs through amidation reaction and then coupled to the amino end of MSN, so that the D/N@MSN-CNQDs-FA nano composition is synthesized.
- 2. The method according to claim 1, wherein the mass ratio of the chemotherapeutic agent DOX to the NO donor in step S1 is DOX: NO donor = 1:20.
- 3. The synthesis method according to claim 1, wherein the FA-CNQDs in step S2 is prepared as follows: firstly, melamine and cyanuric chloride are polymerized through hot acetonitrile to form a triazine structure, and then, the mixture of the triazine structure and N-hydroxysuccinimide conjugated folic acid FA-NHS is subjected to hydrothermal synthesis of FA-CNQDs under subcritical conditions.
- 4. The D/N@MSN-CNQDs-FA nano-composition prepared by the synthesis method according to any one of claims 1-3.
- 5. The use of the D/N@MSN-CNQDs-FA nano composition according to claim 4 in preparing an anti-tumor drug.
- 6. The use according to claim 5, characterized in that: tumors include liver cancer.
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| CN111621030A (en) * | 2020-06-02 | 2020-09-04 | 华侨大学 | Preparation method of hierarchical porous metal organic framework material for drug loading sustained release |
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| CN111621030A (en) * | 2020-06-02 | 2020-09-04 | 华侨大学 | Preparation method of hierarchical porous metal organic framework material for drug loading sustained release |
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