CN112294776B

CN112294776B - Reduction response type carbon dot drug-loaded nanocluster coated with cell membrane and preparation and application thereof

Info

Publication number: CN112294776B
Application number: CN202011016540.0A
Authority: CN
Inventors: 沈明武; 郭云琦; 范钰; 史向阳
Original assignee: Donghua University
Current assignee: Donghua University
Priority date: 2020-09-24
Filing date: 2020-09-24
Publication date: 2021-08-31
Anticipated expiration: 2040-09-24
Also published as: CN112294776A

Abstract

The invention relates to a reduction response type carbon dot drug-loaded nanocluster for coating a cell membrane and preparation and application thereof. The preparation method comprises the following steps: preparing a fluorescent carbon dot, preparing a reduction response type carbon dot nanocluster solution, preparing an adriamycin-loaded carbon dot nanocluster, preparing a B16 cell membrane suspension, and preparing a reduction response type carbon dot drug-loaded nanocluster coating a cell membrane. The method has simple process, simple reaction condition, easy operation and separation and good development prospect; the prepared reduction response type carbon dot drug-loaded nanocluster coated with the cell membrane has a remarkable anti-tumor effect after being injected into a mouse body through tail veins, and has potential clinical application values.

Description

Reduction response type carbon dot drug-loaded nanocluster coated with cell membrane and preparation and application thereof

Technical Field

The invention belongs to the field of responsive nano-drug carriers and preparation and application thereof, and particularly relates to a reductive responsive carbon dot drug-loaded nanocluster for coating a cell membrane and a preparation method and application thereof.

Background

Currently, cancer has become a major threat to human survival and health. Chemotherapy is the most important and effective treatment method for middle and late-stage cancers, but traditional chemotherapy kills cancer cells and causes strong toxic and side effects on normal tissues and organs. Chemotherapeutic drugs used clinically, such as: doxorubicin, paclitaxel, etc., have greatly reduced their therapeutic effects due to their poor water solubility, rapid metabolism, etc. limitations. Therefore, the nano platform constructed by the nano material loaded with the chemotherapeutic drug can deliver the drug to the tumor part in a targeted manner, realize the responsive release of the drug in the tumor microenvironment, reduce the toxic and side effects on normal tissues and improve the curative effect of chemotherapy. Among the numerous nanomaterials, carbon dots are favored due to their fluorescent properties and their ability to serve as drug carriers.

The carbon dots are a novel carbon nano material with the size less than 10nm, and have the characteristics of good biocompatibility, good water solubility, good stability, high fluorescence adjustability and the like, so the carbon dots have good application prospect in the aspect of fluorescence imaging, and the prepared carbon dots are used for accurate fluorescence imaging of brain glioma (Min Qian, et al. ACS appl. mater. interfaces,2018,10, 4031-. Due to the fact that the carbon dots have various functional groups on the surfaces, such as negatively charged carboxyl, hydroxyl and the like, the carbon dots can be combined with chemotherapeutic drugs (such as adriamycin and the like) with positive charges on the surfaces through non-covalent interaction and used as drug carriers for tumor chemotherapy. In a literature report before the subject group (Dan Li, et al.J.Mater.chem.B,2019,7(2),277-285), fifth generation dendrimers are combined with carbon dots loaded with adriamycin to construct a hybrid nano platform for overcoming the research of multi-drug resistance chemotherapy. Most nanoparticles can only stay around blood vessels after entering the body and cannot enter the central area of the tumor, and the permeability is poor, so that the treatment effect of the nano-drug is greatly limited. Monodisperse carbon dots are small in size, can easily diffuse in tumor tissues, can promote loaded drugs to permeate into the tumor, but are small in size, so that the EPR effect is weak, and the loaded drugs are easily and rapidly eliminated through renal metabolism. Therefore, it is very critical to design a cluster-type carbon dot with a proper size, which can respond to dissociation after reaching the tumor microenvironment, and further improve the release amount and permeability of the drug at the tumor site.

In recent years, the bionics technology, especially the nano-carrier with cell membrane biomimetic camouflage can effectively avoid the capture and removal of the mononuclear macrophage system and the reticuloendothelial system to the nano-carrier, increase the in vivo circulation time of the carrier and facilitate the enrichment of the carrier at tumor sites due to the excellent biocompatibility and low immunogenicity. The malignant tumor cell membrane is based on the in-vitro easy culture of tumor cells, is convenient for collecting a large amount of tumor cells, loses carcinogenicity after organelles such as cell DNA and the like are removed, and the collected cancer cell membrane can be used for the bionic camouflage nano carrier. Because the surface of the cancer cell membrane has immune evasion protein and homologous targeting protein, the nano-carrier coated on the basis of the cancer cell membrane can avoid immune clearance and has the capability of actively targeting to a tumor part, thereby realizing the targeted therapy of the tumor. For example, Xie et al prepared silica nanoparticles wrapped by tumor cell membranes for targeted delivery of drugs to tumor sites to achieve better antitumor effect (Wei Xie, et al. ACS Nano,2019,13, 2849-2857).

The research on domestic and foreign documents and patents does not find the preparation of the reduction response type carbon dot drug-loaded nanocluster for coating the cell membrane and the related report of the application of the Doxorubicin (DOX) loaded by the non-covalent action to the tumor chemotherapy in the organism.

Disclosure of Invention

The invention aims to solve the technical problem of providing a reduction response type carbon dot drug-loaded nanocluster for coating a cell membrane and a preparation method and application thereof, so as to overcome the defect of poor tumor treatment effect of carbon dot drug-loaded nanoclusters in the prior art.

The invention provides a reduction response type carbon dot drug-loaded nanocluster for coating a cell membrane, which is obtained by crosslinking a fluorescent carbon dot through a crosslinking agent to form the carbon dot nanocluster, then loading adriamycin and further coating a melanoma cell membrane.

The fluorescent carbon dots are obtained by carrying out hydrothermal reaction on 4-aminosalicylic acid suspension, centrifuging, filtering and freeze-drying.

The invention also provides a preparation method of the reduction response type carbon dot drug-loaded nanocluster coated with the cell membrane, which comprises the following steps:

(1) dissolving 4-aminosalicylic acid in ultrapure water to form a suspension, carrying out hydrothermal reaction, cooling, centrifuging, filtering, and freeze-drying to obtain the fluorescent carbon dot y-CDs (yellow green), wherein the ratio of the 4-aminosalicylic acid to the ultrapure water is 0.2g:8-15 mL;

(2) dissolving the y-CDs in the step (1) in ultrapure water, adding EDC and NHS for activation (carboxyl group activation), then adding a cross-linking agent, stirring for reaction, and dialyzing to obtain a reduction-responsive carbon-point nanocluster y-CDs NCs solution, wherein the mass ratio of the cross-linking agent to the y-CDs is 1-1.2: 1-1.2;

(3) adding DOX hydrochloride^.Dissolving HCl in a solvent, adding triethylamine, adding the solution into the y-CDs NCs solution obtained in the step (2), stirring the solution in the dark in the open, centrifuging the solution, and freeze-drying the solution to obtain the adriamycin-loaded carbon-point nanoclusters y-CDs NCs/DOX, wherein DOX is DOX^.The ratio of HCl, y-CDs NCs, triethylamine and solvent is 1-1.2 mg: 1-1.2 mg: 2-2.5. mu.L: 100-;

(4) adding the cell lysate into the B16 cell sediment, repeatedly freezing and thawing and crushing, performing gradient centrifugation to extract B16 cell membranes (CCM), and suspending in a PBS solution to obtain a B16 cell membrane suspension;

(5) mixing the y-CDs NCs/DOX in the step (3) with the B16 cell membrane suspension in the step (4), repeatedly extruding, centrifuging to remove the supernatant, and obtaining the reduction response type carbon dot drug-loaded nanocluster y-CDs NCs/DOX @ CCM coated with the cell membrane, wherein the ratio of the y-CDs NCs/DOX to the cell membrane suspension is 200 mu g: 0.4-0.6 mL.

The hydrothermal reaction temperature in the step (1) is 170-190 ℃, and the hydrothermal reaction time is 2-4 h.

And (2) filtering in the step (1) by adopting a microporous filter membrane with a pore size of 220 nm.

The centrifugal speed in the step (1) is 4000rpm, and the centrifugal time is 20 min.

The mass ratio of the y-CDs, the EDC, the NHS and the cross-linking agent in the step (2) is 1-1.2:5.5-6.5:3-4: 1-1.2.

And (3) in the step (2), the cross-linking agent is Cystamine dihydrochloride Cystamine.

And (3) in the step (2), the stirring reaction temperature is room temperature, and the stirring reaction time is 2-4 days.

Adding EDC and NHS in the step (2) for activation: EDC is added firstly and stirred for reaction for 0.5h, NHS is added and stirred for reaction for 3 h.

The dialysis in the step (2) is as follows: selecting a dialysis bag with the molecular weight cutoff of 500, wherein the dialysis solution is an aqueous solution with the volume of 2L, and changing water for 3 times within 24 h.

The solvent in the step (3) is methanol.

And (3) stirring at room temperature for overnight.

The ratio of B16 cells to cell lysate in the step (4) is 1 x 10⁷The method comprises the following steps: 2-4 mL.

The cell lysate in the step (4) contains 1% of PMSF.

The parameters of the repeated freezing-thawing and crushing process in the step (4) are as follows: freezing at-20 deg.C, thawing at 37 deg.C, and repeating for 3 times.

The gradient centrifugation in the step (4) is as follows: at 4 ℃, the solution is centrifuged for 10min at 700g for removing the precipitate, and then centrifuged for 30min at 14000g for removing the supernatant, and the precipitate is resuspended in 1mL of PBS solution.

The repeated extrusion in the step (5) is carried out for 11 times by using an Avanti micro extruder with a filter membrane aperture of 400 nm.

And (3) in the step (5), the centrifugal speed is 10000rpm, and the centrifugal time is 6 min.

The invention also provides application of the reduction response type carbon dot drug-loaded nanocluster coated with the cell membrane in preparation of antitumor drugs.

The invention synthesizes yellow-green fluorescent carbon dot y-CDs by a hydrothermal method, and uses cystamine dihydrochloride as a crosslinking agent to crosslink carbon dot nano-particles into carbon dot nanoclusters y-CDs NCs. By controlling the feeding ratio of the cross-linking agent to the carbon dot nano-particles, the carbon dot nano-cluster with proper size and stable structure can be prepared. The chemotherapy drug adriamycin DOX is loaded in the inner cavity and on the surface of the carbon point nanocluster through non-covalent action, and meanwhile, the B16 cell membrane is coated on the surface of the material in a physical extrusion mode, so that the y-CDs NCs/DOX @ CCM is prepared.

The physical and chemical properties of the prepared carbon dot drug-loaded nanocluster coating the cell membrane are represented by Zeta potential and dynamic light scattering analysis (DLS), infrared spectrum (FT-IR), ultraviolet visible absorption spectrum (UV-vis), steady state/transient state fluorescence spectrum, Transmission Electron Microscope (TEM), SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and other means. And then, evaluating the cytotoxicity of the y-CDs NCs/DOX @ CCM and related control materials by using a CCK-8 method, determining the immune evasion and homologous targeting capability of the y-CDs NCs/DOX @ CCM and the related control materials, simultaneously determining the in vitro cell fluorescence imaging performance of the y-CDs NCs/DOX @ CCM, and detecting the phagocytosis condition of cells to the materials by using flow cytometry. And finally establishing a nude mouse subcutaneous tumor model for an anti-tumor experiment. The specific test results are as follows:

zeta potential and hydrodynamic diameter test results

The potential of y-CDs was-26.9 mV, the potential of y-CDs prepared by crosslinking y-CDs was changed to-4.5 mV, the hydrated particle size was increased to 236.7nm, and the increase in potential and hydrated particle size demonstrated successful synthesis of y-CDs NCs, as shown in Table 1. When the y-CDs NCs are loaded with the chemotherapeutic drug DOX, the potential is changed from negative to positive and is increased to 8.8mV, thus proving the successful loading of DOX. The successful coating of the cell membrane on the surface of the y-CDs NCs/DOX is proved by physically extruding the y-CDs NCs/DOX @ CCM coated with the B16 cell membrane, wherein the potential is-11.7 mV, and the hydrated particle size is increased to 254.8 nm. The hydrodynamic diameter of y-CDs NCs/DOX @ CCM in various solutions (water, physiological saline, 1640 medium) was almost unchanged (see FIG. 2), demonstrating that y-CDs NCs/DOX @ CCM has good colloidal stability.

2. Infrared (FT-IR) test:

the prepared y-CDs and y-CDs NCs are characterized by FT-IR test, as shown in FIG. 3, wherein curve 1 represents y-CDs NCs, and curve 2 represents y-CDs. 3438cm in Curve 2^-1The peak at position corresponds to the O-H stretching vibration absorption peak of carboxyl, 1606cm^-1The peak at position corresponds to the stretching vibration absorption peak of C ═ O, 1495cm^-1The peak at the position corresponds to the stretching vibration absorption peak of C-O, and the fact that the prepared y-CDs have rich carboxyl on the surface is proved. In curve 1, 1631cm^-1And 1535cm^-1The strong absorption peak belongs to an amido bond generated by combining the amino group of cystamine dihydrochloride with the carboxyl group on the surface of y-CDs; 551cm^-1The characteristic peak at (a) is clearly enhanced compared to curve 1, and should be attributed to the S-S bond in cystamine dihydrochloride. The results of the infrared spectrogram demonstrate the successful preparation of the y-CDs NCs nanoparticles.

3. Ultraviolet (UV-vis) test:

the prepared y-CDs, y-CDs NCs and y-CDs NCs/DOX are characterized by UV-vis test, as shown in figure 4, the y-CDs have a characteristic peak at 282nm, and the characteristic peak at 490nm of the y-CDs NCs/DOX proves the successful loading of DOX.

4. Steady state/transient fluorescence spectroscopy test:

the invention characterizes the fluorescence characteristics of the prepared y-CDs, y-CDs NCs/DOX and y-CDs NCs/DOX @ CCM through fluorescence spectra. Fluorescence excitation spectrum and emission spectrum at different excitation wavelengths of y-CDs As shown in FIG. 5(a), the maximum excitation wavelength of y-CDs is 490nm, the maximum emission wavelength of y-CDs is 547nm, and the maximum emission wavelength does not vary with the excitation wavelength. As shown in FIG. 5(b), when nanoclusters are formed, the fluorescence intensity of y-CDs NCs is somewhat reduced due to aggregation of y-CDs as compared with free y-CDs, and the fluorescence intensity is further reduced after cell membranes are coated. And when the y-CDs NCs/DOX @ CCM reacts with the reductive Glutathione (GSH), the fluorescence intensity is recovered to higher intensity, and the dissociation of the prepared carbon dot drug-loaded nanocluster under the reducing condition is proved.

TEM test:

the prepared y-CDs, y-CDs NCs and y-CDs NCs/DOX @ CCM are characterized in size and morphology by TEM test. TEM images and particle size distribution histograms of y-CDs are shown in FIG. 6(a-b), with y-CDs having uniform circles and sizes of 7.2. + -. 0.7 nm. As shown in FIG. 6(c), the prepared y-CDs have NCs with a size of about 150 nm; as shown in FIG. 6(d), the size of the prepared y-CDs NCs/DOX @ CCM was about 200nm, and the thickness of the coated cell membrane was about 10 nm.

SDS-polyacrylamide gel electrophoresis (SDS-PAGE) test:

protein content in the prepared cell membrane suspension and the y-CDs NCs/DOX @ CCM is determined by a BCA protein quantitative kit, and the protein content is adjusted to be 1mg/mL by using a PBS solution. mu.L of protein Marker was added to the first protein lane, and then 15. mu.L of y-CDs NCs/DOX (200. mu.g in 1mL PBS), cell membrane suspension and y-CDs NCs/DOX @ CCM were added to the protein lanes, respectively, with the current set at 100A and the time set at 30 min. As shown in FIG. 7, the y-CDs NCs/DOX group did not run out of the lane with protein streaks, while the y-CDs NCs/DOX @ CCM ran out of a similar lane with protein streaks as the cell membrane suspension (CCM), demonstrating successful coating of the cell membrane on the surface of the y-CDs NCs/DOX.

7. In vitro drug release testing:

buffer solutions having a pH of 7.4, a pH of 7.4(GSH concentration: 10mM), a pH of 5.5, and a pH of 5.5(GSH concentration: 10mM) were prepared, and the prepared y-CDs NCs/DOX @ CCM was dissolved in 1mg/mL of the above different buffer solutions and placed in a dialysis bag, and the dialysis bag was placed in a container containing 9mL of the above different buffer solutions and shaken in a constant temperature shaker at 37 ℃. At different time points, 1mL of the solution outside the dialysis bag was aspirated, 1mL of the corresponding buffer solution was then added to the container, and the absorbance at 480nm of the withdrawn solution was measured. And after the slow release is finished, drawing a drug release curve of the y-CDs NCs/DOX @ CCM under different conditions. As shown in fig. 8, y-CDs NCs/DOX @ CCM released slowly with a drug release rate of 19.13% in a buffer at pH 7.4 (no GSH) and 39.33% at pH 5.5 (no GSH), the latter being significantly higher than the former due to protonation of DOX under acidic conditions and better solubility in water. However, the release rate of DOX reached 62.44% in the buffer at pH 7.4(GSH concentration: 10mM) and the highest release rate of 88% was achieved in the buffer at pH 5.5(GSH concentration: 10mM), indicating that the release of DOX in y-CDs NCs/DOX @ CCM had a significant reduction response. These data demonstrate that the drug release of y-CDs NCs/DOX @ CCM has dual responsiveness of pH and GSH, and also demonstrate that the coated cell membrane does not affect the drug release performance of the carbon dot drug-loaded nanocluster.

8. Cytotoxicity experiments:

b16 cells were collected at logarithmic growth phase at 1X 10 per well⁴Individual cells were seeded in 3 96-well plates in 5% CO₂And incubating for 12h at 37 ℃. The original medium was discarded, and medium containing Free DOX at different concentrations (DOX concentration 0.1, 0.25, 0.5, 1, 5, 10, 25. mu.g/mL), y-CDs NCs/DOX @ CCM at different concentrations (DOX concentration 0.1, 0.25, 0.5, 1, 5, 10, 25. mu.g/mL) and cells at 5% CO were added to each well plate₂And co-culturing at 37 ℃ for 24 h. Then taking out the pore plate and discarding the originalThe medium was washed three times with PBS, fresh medium containing 10% (v/v) CCK-8 was added and incubation continued in the incubator for 3 h. And finally, testing the light absorption value of each hole at the position with the wavelength of 450nm by using a multifunctional microplate reader, taking the cells treated by the PBS as a blank control, and recording the cell activity as 100%. The results are shown in fig. 9, where the cytotoxicity of each group of materials was gradually increased with increasing concentrations of DOX. Under the same DOX concentration, the y-CDs NCs/DOX @ CCM group has higher cytotoxicity than the y-CDs NCs/DOX group, and proves that the phagocytosis of cells to materials is increased and the effect of inhibiting the proliferation of cancer cells is enhanced due to the homologous targeting of cell membranes (CCM). DOX half maximal Inhibitory Concentration (IC) after 24h of co-incubation of Free DOX, y-CDs NCs/DOX @ CCM with B16 cells₅₀) Respectively 0.3 mug/mL, 11.3 mug/mL and 6.3 mug/mL, and the lethality of the y-CDs NCs/DOX @ CCM to B16 cells is proved to be remarkably higher than that of the y-CDs NCs/DOX under the same experimental operating conditions.

9. Immune evasion and homology targeting ability test:

the immune evasion ability of the y-CDs NCs/DOX @ CCM was evaluated by using RAW 264.7 cells as a cell model, and the homologous targeting ability of the y-CDs NCs/DOX @ CCM was evaluated by using B16 cells as a cell model. RAW 264.7 cells were plated at 2X 10⁵The density of each cell per dish was seeded in 4 confocal cell culture dishes and placed in 5% CO₂And incubating for 12h at 37 ℃. The original medium was discarded, and medium containing y-CDs NCs/DOX (DOX concentration 5. mu.g/mL), y-CDs NCs/DOX @ RBCM (DOX concentration 5. mu.g/mL), y-CDs NCs/DOX @ CCM (DOX concentration 5. mu.g/mL) or PBS was added to each dish separately with the cells in 5% CO₂Co-culture was carried out at 37 ℃ for 6 hours. Similarly, B16 cells were arranged in a 2X 10 format⁵The density of each cell per dish was seeded in 4 confocal cell culture dishes and placed in 5% CO₂And incubating for 12h at 37 ℃. The original medium was discarded, and medium containing y-CDs NCs/DOX (DOX concentration 5. mu.g/mL), y-CDs NCs/DOX @ RBCM (DOX concentration 5. mu.g/mL), y-CDs NCs/DOX @ CCM (DOX concentration 5. mu.g/mL) or PBS was added to each dish separately with the cells in 5% CO₂Co-culture was carried out at 37 ℃ for 6 hours. The medium was discarded, washed three times with PBS, fixed with 2.5% glutaraldehyde, washed three times with PBS, stained with DAPI for 5 minutes, washed three times with PBS after completion, and then washedAnd observing the experimental result by using a laser confocal microscope. As shown in FIG. 10, the cells treated with y-CDs NCs/DOX showed both yellow-green and red fluorescence signals, and the co-localization showed orange fluorescence, demonstrating that the prepared y-CDs NCs/DOX could be phagocytosed by cells and the fluorescence imaging of cells was achieved. As shown in FIG. 10(a), the fluorescence intensity of RAW 264.7 cells co-cultured with y-CDs NCs/DOX @ RBCM and y-CDs NCs/DOX @ CCM was similar, but was significantly lower than that of the y-CDs NCs/DOX group, indicating that y-CDs NCs/DOX @ RBCM and y-CDs NCs/DOX @ CCM were able to evade phagocytosis of RAW 264.7 cells and had immune evasion ability. As shown in FIG. 10(B), the fluorescence intensity of the y-CDs NCs/DOX @ CCM group in B16 cells is significantly higher than that of the y-CDs NCs/DOX @ RBCM and y-CDs NCs/DOX group, indicating that coating B16 cell membrane (CCM) can increase phagocytosis of y-CDs NCs/DOX by B16 cells and endow the cells with homologous targeting ability.

10. Cell phagocytosis assay:

b16 cells were collected at logarithmic growth phase at 1X 10 per well⁴The individual cells were seeded in 12-well plates in 5% CO₂And incubating for 12h at 37 ℃. Discarding the original medium, adding culture medium containing different concentrations of y-CDs NCs/DOX (DOX concentration of 2.5, 5, 10 μ g/mL), y-CDs NCs/DOX @ RBCM (DOX concentration of 2.5, 5, 10 μ g/mL), y-CDs NCs/DOX @ CCM (DOX concentration of 2.5, 5, 10 μ g/mL) and cells in 5% CO₂Co-culture was carried out at 37 ℃ for 6 hours. And then taking out the pore plate, discarding the original culture medium, washing for three times by using PBS (phosphate buffer solution), digesting, centrifuging and collecting the cells in the pore plate, and detecting the fluorescence intensity of the cells by using a flow cytometer. As can be seen from fig. 11 and 12, each group of cells showed increasing fluorescence intensity with increasing DOX concentration. Under the same conditions, B16 cells treated by y-CDs NCs/DOX @ CCM show higher fluorescence intensity than B16 cells treated by y-CDs NCs/DOX @ RBCM and y-CDs NCs/DOX, and the fact that the homologous targeting of B16 cell membranes (CCM) can enhance phagocytosis of materials by cells is proved.

11. In vivo tumor treatment results:

b16 subcutaneous tumor model was constructed in nude mice, 2X 10⁶Right after inoculating a B16 cell to a nude mouseThe tumor volume of the leg reaches 15-20mm³At this time, the nude mice were randomly divided into 5 groups of 8 mice each. The specific grouping is as follows: control group (Saline, 100 μ L); y-CDs NCs (concentration of y-CDs NCs corresponding to a concentration of 5mg/kg DOX in y-CDs NCs/DOX, 100. mu.L); free DOX ([ DOX)]＝5mg/kg,100μL)；y-CDs NCs/DOX([DOX]＝5mg/kg,100μL)；y-CDs NCs/DOX@CCM([DOX]5mg/kg,100 mul), the injection mode is tail vein injection. The day of treatment initiation was taken as day 0, and the body weight and tumor volume of nude mice were recorded every 2 days, and treated every 2 days. As shown in FIG. 13(a), after the treatment was completed, the relative tumor volumes of the y-CDs NCs and Saline groups were slightly different from each other, and the Free DOX group, the y-CDs NCs/DOX group, and the y-CDs NCs/DOX @ CCM group exhibited antitumor effects to different degrees. The in vivo anti-tumor effect of the y-CDs NCs/DOX group is higher than that of the Free DOX group, which probably is because the y-CDs NCs/DOX has EPR effect and can effectively reach tumor sites, and the y-CDs NCs can be used as a good drug carrier. The treatment effect of the y-CDs NCs/DOX @ CCM group is better than that of the y-CDs NCs/DOX group, probably because immune evasion protein and homologous targeting protein on a cell membrane can prolong the blood circulation time of the material, improve the targeting property of the material and enrich at tumor parts. As shown in FIG. 13(b), the weight of the mice in the Free DOX group was significantly reduced, while the weight of the other groups was not significantly changed, thus demonstrating that Free DOX may cause some toxicity to the mice.

Advantageous effects

(1) The method has the advantages of simple process, simple reaction conditions, easy operation and separation and good development prospect.

(2) The reduction-responsive carbon dot nanocluster prepared by the invention is used as a carrier of chemotherapeutic drugs, has the advantages of good biocompatibility, high drug loading rate and the like, can release the drugs in response and dissociation of a tumor microenvironment, can perform intracellular fluorescence imaging and remarkably inhibit tumor growth in a nude mouse, and has potential application value in the field of tumor chemotherapy.

(3) The y-CDs NCs/DOX @ CCM prepared by the invention has good reduction responsiveness in vitro, carbon points of a cluster structure can be dissociated into single carbon point nanoparticles in a tumor reduction microenvironment and release drugs, the permeability of tumor parts is improved while the EPR effect is maintained, and the tumor inhibition effect is enhanced.

(4) After the y-CDs NCs/DOX @ CCM prepared by the invention enters a mouse body through tail vein injection, the y-CDs NCs/DOX @ CCM has obvious anti-tumor effect and potential clinical application value.

Drawings

FIG. 1 is a schematic diagram of the synthesis and application of the nanomaterial y-CDs NCs/DOX @ CCM in the present invention;

FIG. 2 is a graph of hydrodynamic diameter as a function of time in water, physiological saline, and 1640 medium for the y-CDs NCs/DOX @ CCM prepared in example 1;

FIG. 3 is an infrared spectrum of y-CDs NCs (1) and y-CDs (2) prepared in example 1;

FIG. 4 is a UV spectrum of y-CDs (1), y-CDs NCs (2), y-CDs NCs/DOX (3) prepared in example 1;

FIG. 5 is a fluorescence excitation (1) spectrum and fluorescence emission (2-7) spectra (a) at different excitation wavelengths of the y-CDs prepared in example 1, and fluorescence emission spectra (b) of y-CDs (1), y-CDs NCs (2), y-CDs NCs/DOX (3), y-CDs NCs/DOX @ CCM (4), y-CDs NCs/DOX @ CCM +10mM GSH (5);

FIG. 6 is a TEM image (a) and a particle size distribution histogram (b) of y-CDs prepared in example 1, a TEM image (c) of y-CDs NCs, a TEM image (d) of y-CDs NCs/DOX @ CCM;

FIG. 7 is a SDS-polyacrylamide gel electrophoresis (SDS-PAGE) chart of y-CDs NCs/DOX, cell membrane suspension (CCM) and y-CDs NCs/DOX @ CCM prepared in example 1;

FIG. 8 is a graph of the kinetics of drug release of y-CDs NCs/DOX @ CCM prepared in example 1 under different conditions;

FIG. 9 is a diagram of the cell viability of Free DOX, y-CDs NCs/DOX @ CCM in example 1 after 24h of co-incubation with B16 cells;

FIG. 10 is a confocal laser microscopy image of y-CDs NCs/DOX, y-CDs NCs/DOX @ CCM prepared in example 1 and y-CDs NCs/DOX @ RBCM prepared in comparative example 1 after co-incubation with RAW 264.7(a) and B16(B) cells, respectively, for 6 h;

FIG. 11 is a graph of flow cytometric analysis of y-CDs NCs/DOX, y-CDs NCs/DOX @ CCM prepared in example 1 and y-CDs NCs/DOX @ RBCM prepared in comparative example 1 after incubation with B16 cells for 6 h;

FIG. 12 is flow cytometric data for mean fluorescence of DOX after 6h incubation of y-CDs NCs/DOX, y-CDs NCs/DOX @ CCM prepared in example 1 and y-CDs NCs/DOX @ RBCM prepared in comparative example 1 with B16 cells;

FIG. 13 is a graph showing the change in tumor volume (a) and the change in mouse body weight (b) within 14 days after tail vein injection of Saline, y-CDs NCs, Free DOX, y-CDs NCs/DOX @ CCM in example 12.

Detailed Description

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Further, it should be understood that various changes or modifications of the present invention may be made by those skilled in the art after reading the teaching of the present invention, and such equivalents may fall within the scope of the present invention as defined in the appended claims.

Unless otherwise specified, all chemical reagents were commercially available and used without further purification. 4-Aminosalicylic acid, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS) were purchased from carbofuran technologies, Inc. Cystamine dihydrochloride is purchased from the national pharmaceutical group chemical agents limited (shanghai, china). Doxorubicin HCl (DOX. HCl) was purchased from beijing huafeng technologies ltd (beijing, china). BCA assay kit, PMSF, cell lysate were purchased from Byunnan Biotech, Inc. (Shanghai, China). Cell Counting Kit-8(CCK-8) was purchased from Shanghai seven sea Biotechnology, Inc. (Shanghai, China). B16 cells (mouse melanoma cell line) and RAW 264.7 cells (mouse macrophage line) were from the institute of biochemistry and cell biology, chinese academy of sciences. RPMI-1640 medium, fetal bovine serum, penicillin-streptomycin double antibody and trypsin were purchased from Hangzhou Gino biomedical technologies, Inc. (Hangzhou, China). Nude mice were purchased from shanghai slek experimental animals center (shanghai, china).

Example 1

(1) Dissolving 0.2g of 4-aminosalicylic acid in 10mL of ultrapure water, and then transferring the mixed solution into a polytetrafluoroethylene reaction kettle for hydrothermal reaction at the temperature of 180 ℃ for 3 hours; and after the reaction is finished, naturally cooling to room temperature, centrifuging the product for 20min (4000rpm) to remove particles excessively carbonized, filtering the product through a microporous filter membrane with the pore diameter of 220nm, and finally freeze-drying the product to obtain the carbon dot nano-particles y-CDs.

(2) 30mg of y-CDs were dissolved in 15mL of ultrapure water, and stirred at room temperature, EDC (174mg, 1mL of ultrapure water) was added dropwise to the solution, and after 30min, NHS (95mg, 1mL of ultrapure water) was added dropwise to the solution, and stirred for 3h to activate the carboxyl groups on the y-CDs. Subsequently, a solution of cystamine dihydrochloride (30mg, 3mL of ultrapure water) was added to the solution, the reaction was stirred for 3 days, and then dialyzed for 3 days in ultrapure water using a dialysis bag having a molecular weight cut-off of 500 to obtain a carbon dot nanocluster y-CDs NCs solution.

(3) Dissolving 5mg of doxorubicin hydrochloride in 600 mu L of methanol solution, adding 10 mu L of triethylamine for neutralization, then dropwise adding the solution into 3mL of y-CDs NCs solution, stirring overnight in the open in the dark, centrifuging at 8000rpm for 20min, and taking the supernatant for freeze drying to obtain the doxorubicin-loaded carbon-point nanoclusters y-CDs NCs/DOX. Meanwhile, the absorbance of the precipitate at 480nm was measured by ultraviolet to calculate the drug loading rate to be 40.5% and the encapsulation rate to be 81%.

(4) Taking B16 cells in logarithmic growth phase at 1X 10⁷And centrifuging at 1000rpm for 5min to obtain cell precipitate, adding 3mL of hypotonic cell lysis solution (containing 1% PMSF) into the cell precipitate, ice-bathing for 15min, and repeatedly freezing and thawing for 3 times by freezing and thawing method (-20 deg.C freezing and 37 deg.C thawing). Setting the centrifugal temperature at 4 ℃, centrifuging for 10min at 700g of centrifugal force, and removing precipitates; centrifuging at 14000g centrifugal force for 30min, removing supernatant, and suspending the precipitate in 1mL PBS solution to obtain B16 cell membrane suspension.

(5) 200 μ g y-CDs NCs/DOX was dissolved in 0.5mL of B16 cell membrane suspension, the solution was extruded 11 times using an Avanti micro-extruder, centrifuged at 10000rpm for 6min to remove excess cell membranes, and y-CDs NCs/DOX @ CCM was prepared.

Example 2

As a result of taking 1mg each of the y-CDs, y-CDs NCs/DOX, and y-CDs NCs/DOX @ CCM synthesized in example 1, diluting it to 50. mu.g/mL with ultrapure water, and simultaneously taking 50. mu.L of y-CDs NCs dissolved in 950. mu.L of ultrapure water for measuring the surface potential, hydrodynamic diameter and dispersion coefficient, as shown in Table 1, the potential of y-CDs was-26.9 mV, the potential of y-CDs prepared by crosslinking y-CDs was changed to-4.5 mV, the hydrated particle size was increased to 236.7nm, and the increase in potential and hydrated particle size confirmed the successful synthesis of y-CDs. When the y-CDs NCs are loaded with the chemotherapeutic drug DOX, the potential is changed from negative to positive and is increased to 8.8mV, thus proving the successful loading of DOX. The successful coating of the cell membrane on the surface of the y-CDs NCs/DOX is proved by physically extruding the y-CDs NCs/DOX @ CCM coated with the B16 cell membrane, wherein the potential is-11.7 mV, and the hydrated particle size is increased to 254.8 nm. The hydrodynamic diameter of y-CDs NCs/DOX @ CCM in various solutions (water, physiological saline, 1640 medium) was almost unchanged (see FIG. 2), demonstrating that y-CDs NCs/DOX @ CCM has good colloidal stability.

TABLE 1

Example 3

FT-IR characterization was performed on the y-CDs and y-CDs NCs prepared in example 1, as shown in FIG. 3, where curve 1 represents the y-CDs NCs and curve 2 represents the y-CDs. 3438cm in Curve 2^-1The peak at position corresponds to the O-H stretching vibration absorption peak of carboxyl, 1606cm^-1The peak at position corresponds to the stretching vibration absorption peak of C ═ O, 1495cm^-1The peak at the position corresponds to the stretching vibration absorption peak of C-O, and the fact that the prepared y-CDs have rich carboxyl on the surface is proved. In curve 1, 1631cm^-1And 1535cm^-1The strong absorption peak belongs to an amido bond generated by combining the amino group of cystamine dihydrochloride with the carboxyl group on the surface of y-CDs; 551cm^-1The characteristic peak at (a) is clearly enhanced compared to curve 1, and should be attributed to the S-S bond in cystamine dihydrochloride. The results of the infrared spectrogram demonstrate the successful preparation of the y-CDs NCs nanoparticles.

Example 4

The y-CDs, y-CDs NCs and y-CDs NCs/DOX prepared in example 1 were taken for UV-vis characterization, as shown in FIG. 4, the characteristic peak of y-CDs at 282nm, and the characteristic peak of y-CDs NCs/DOX at 490nm proved the successful loading of DOX.

Example 5

The fluorescence characteristics were characterized by taking y-CDs, y-CDs NCs/DOX @ CCM prepared in example 1. Fluorescence excitation spectrum and emission spectrum at different excitation wavelengths of y-CDs As shown in FIG. 5(a), the maximum excitation wavelength of y-CDs is 490nm, the maximum emission wavelength of y-CDs is 547nm, and the maximum emission wavelength does not vary with the excitation wavelength. As shown in FIG. 5(b), when nanoclusters are formed, the fluorescence intensity of y-CDs NCs is somewhat reduced due to aggregation of y-CDs as compared with free y-CDs, and the fluorescence intensity is further reduced after cell membranes are coated. And when the y-CDs NCs/DOX @ CCM reacts with the reductive Glutathione (GSH), the fluorescence intensity is recovered to higher intensity, and the dissociation of the prepared carbon dot drug-loaded nanocluster under the reducing condition is proved.

Example 6

The y-CDs, y-CDs NCs/DOX @ CCM prepared in example 1 were taken for characterization of size and morphology. TEM images and particle size distribution histograms of y-CDs are shown in FIG. 6(a-b), with y-CDs having uniform circles and sizes of 7.2. + -. 0.7 nm. As shown in FIG. 6(c), the prepared y-CDs have NCs with a size of about 150 nm; as shown in FIG. 6(d), the size of the prepared y-CDs NCs/DOX @ CCM was about 200nm, and the thickness of the coated cell membrane was about 10 nm.

Example 7

The protein content in the cell membrane suspension and y-CDs NCs/DOX @ CCM prepared in example 1 was determined by BCA protein quantification kit, and the protein content was adjusted to 1mg/mL with PBS solution. mu.L of protein Marker was added to the first protein lane, and then 15. mu.L of y-CDs NCs/DOX (200. mu.g in 1mL PBS), cell membrane suspension and y-CDs NCs/DOX @ CCM were added to the protein lanes, respectively, with the current set at 100A and the time set at 30 min. As shown in FIG. 7, the y-CDs NCs/DOX group did not run out of the lane with protein streaks, while the y-CDs NCs/DOX @ CCM ran out of a similar lane with protein streaks as the cell membrane suspension (CCM), demonstrating successful coating of the cell membrane on the surface of the y-CDs NCs/DOX.

Example 8

Example 9

B16 cells were collected at logarithmic growth phase at 1X 10 per well⁴Individual cells were seeded in 3 96-well plates in 5% CO₂And incubating for 12h at 37 ℃. The original medium was discarded, and medium containing Free DOX at different concentrations (DOX concentration 0.1, 0.25, 0.5, 1, 5, 10, 25. mu.g/mL), y-CDs NCs/DOX @ CCM at different concentrations (DOX concentration 0.1, 0.25, 0.5, 1, 5, 10, 25. mu.g/mL) and cells at 5% CO were added to each well plate₂And co-culturing at 37 ℃ for 24 h. The plates were then removed, the original medium discarded, washed three times with PBS, fresh medium containing 10% (v/v) CCK-8 was added and incubation continued in the incubator for 3 h. Finally, the multifunctional microplate reader is used for testing at the position with the wavelength of 450nmThe absorbance of each well was recorded as 100% for PBS-treated cells as a blank. The results are shown in fig. 9, where the cytotoxicity of each group of materials was gradually increased with increasing concentrations of DOX. Under the same DOX concentration, the y-CDs NCs/DOX @ CCM group has higher cytotoxicity than the y-CDs NCs/DOX group, and proves that the phagocytosis of cells to materials is increased and the effect of inhibiting the proliferation of cancer cells is enhanced due to the homologous targeting of cell membranes (CCM). DOX half maximal Inhibitory Concentration (IC) after 24h of co-incubation of Free DOX, y-CDs NCs/DOX @ CCM with B16 cells₅₀) Respectively 0.3 mug/mL, 11.3 mug/mL and 6.3 mug/mL, and the killing power of the y-CDs NCs/DOX @ CCM on B16 cells is proved to be remarkably stronger than that of the y-CDs NCs/DOX under the same experimental operating conditions.

Example 10

The immune evasion ability of the y-CDs NCs/DOX @ CCM was evaluated by using RAW 264.7 cells as a cell model, and the homologous targeting ability of the y-CDs NCs/DOX @ CCM was evaluated by using B16 cells as a cell model. RAW 264.7 cells were plated at 2X 10⁵The density of each cell per dish was seeded in 4 confocal cell culture dishes and placed in 5% CO₂And incubating for 12h at 37 ℃. The original medium was discarded, and medium containing y-CDs NCs/DOX (DOX concentration 5. mu.g/mL), y-CDs NCs/DOX @ RBCM (DOX concentration 5. mu.g/mL), y-CDs NCs/DOX @ CCM (DOX concentration 5. mu.g/mL) or PBS was added to each dish separately with the cells in 5% CO₂Co-culture was carried out at 37 ℃ for 6 hours. Similarly, B16 cells were arranged in a 2X 10 format⁵The density of each cell per dish was seeded in 4 confocal cell culture dishes and placed in 5% CO₂And incubating for 12h at 37 ℃. The original medium was discarded, and medium containing y-CDs NCs/DOX (DOX concentration 5. mu.g/mL), y-CDs NCs/DOX @ RBCM (DOX concentration 5. mu.g/mL), y-CDs NCs/DOX @ CCM (DOX concentration 5. mu.g/mL) or PBS was added to each dish separately with the cells in 5% CO₂Co-culture was carried out at 37 ℃ for 6 hours. The medium was discarded, washed three times with PBS, fixed with 2.5% glutaraldehyde, washed three times with PBS, stained with DAPI for 5 minutes, washed three times with PBS after completion, and then the experimental results were observed with a confocal laser microscope. As shown in FIG. 10, the y-CDs NCs/DOX-treated cells showed both yellow-green and red fluorescence signals, andthe prepared y-CDs NCs/DOX was shown to be phagocytosed by cells and to enable cellular fluorescence imaging by co-localization showing orange fluorescence. As shown in FIG. 10(a), the fluorescence intensity of RAW 264.7 cells co-cultured with y-CDs NCs/DOX @ RBCM and y-CDs NCs/DOX @ CCM was similar, but was significantly lower than that of the y-CDs NCs/DOX group, indicating that y-CDs NCs/DOX @ RBCM and y-CDs NCs/DOX @ CCM were able to evade phagocytosis of RAW 264.7 cells and had immune evasion ability. As shown in FIG. 10(B), the fluorescence intensity of the y-CDs NCs/DOX @ CCM group in B16 cells is significantly higher than that of the y-CDs NCs/DOX @ RBCM and y-CDs NCs/DOX group, indicating that coating B16 cell membrane (CCM) can increase phagocytosis of y-CDs NCs/DOX by B16 cells and endow the cells with homologous targeting ability.

Example 11

Example 12

B16 subcutaneous tumor model was constructed in nude mice, 2X 10⁶Inoculating B16 cells to the right hind leg of nude mouse until the tumor volume reaches 15-20mm³At this time, the nude mice were randomly divided into 5 groups of 8 mice each. The specific grouping is as follows: control group (Saline, 100 μ L); y-CDs NCs (concentration of y-CDs NCs/DOX corresponding to DOX 5mg/kg，100μL)；Free DOX([DOX]＝5mg/kg,100μL)；y-CDs NCs/DOX([DOX]＝5mg/kg,100μL)；y-CDs NCs/DOX@CCM([DOX]5mg/kg,100 mul), the injection mode is tail vein injection. The day of treatment initiation was taken as day 0, and the body weight and tumor volume of nude mice were recorded every 2 days, and treated every 2 days. As shown in FIG. 13(a), after the treatment was completed, the relative tumor volumes of the y-CDs NCs and Saline groups were slightly different from each other, and the Free DOX group, the y-CDs NCs/DOX group, and the y-CDs NCs/DOX @ CCM group exhibited antitumor effects to different degrees. The in vivo anti-tumor effect of the y-CDs NCs/DOX group is higher than that of the Free DOX group, which probably is because the y-CDs NCs/DOX has EPR effect and can effectively reach tumor sites, and the y-CDs NCs can be used as a good drug carrier. The treatment effect of the y-CDs NCs/DOX @ CCM group is better than that of the y-CDs NCs/DOX group, probably because immune evasion protein and homologous targeting protein on a cell membrane can prolong the blood circulation time of the material, improve the targeting property of the material and enrich at tumor parts. As shown in FIG. 13(b), the weight of the mice in the Free DOX group was significantly reduced, while the weight of the other groups was not significantly changed, thus demonstrating that Free DOX may cause some toxicity to the mice.

Comparative example 1

(1) Whole mouse blood was centrifuged at 3000rpm for 5min, washed three times with PBS to remove serum, and the erythrocyte pellet was resuspended in 0.2mM EDTA solution to induce membrane disruption. Centrifuging at 12000rpm for 15min at 4 deg.C, removing supernatant, and suspending the precipitate in 1mL PBS to obtain erythrocyte membrane (RBCM) suspension.

(2) Mu.g of y-CDs NCs/DOX prepared in example 1 was dissolved in 0.5mL of the erythrocyte membrane suspension, and the solution was extruded 11 times using an Avanti micro-extruder, centrifuged at 10000rpm for 6min to remove excess cell membranes, to prepare y-CDs NCs/DOX @ RBCM.

Claims

1. The reduction response type carbon dot drug-loaded nanocluster coated with cell membranes is characterized in that the drug-loaded nanocluster is obtained by crosslinking fluorescent carbon dots through a crosslinking agent to form the carbon dot nanocluster, then loading adriamycin and further coating melanoma cell membranes, wherein the crosslinking agent is Cystamine dihydrochloride Cystamine;

the preparation method of the reduction response type carbon dot drug-loaded nanocluster coated with the cell membrane comprises the following steps:

(1) dissolving 4-aminosalicylic acid in ultrapure water to form a suspension, carrying out hydrothermal reaction, cooling, centrifuging, filtering, and freeze-drying to obtain the fluorescent carbon dot y-CDs, wherein the ratio of the 4-aminosalicylic acid to the ultrapure water is 0.2g:8-15 mL;

(2) dissolving the y-CDs in the step (1) in ultrapure water, adding EDC and NHS for activation, then adding a cross-linking agent, stirring for reaction, and dialyzing to obtain a reduction-responsive carbon-point nanocluster y-CDs NCs solution, wherein the mass ratio of the cross-linking agent to the y-CDs is 1-1.2: 1-1.2;

(3) dissolving DOX & HCl in a solvent, adding triethylamine, adding the solution into the y-CDs NCs solution obtained in the step (2), stirring the solution in the dark place, centrifuging the solution, and freeze-drying the solution to obtain the adriamycin-loaded carbon-point nanoclusters y-CDs NCs/DOX, wherein the ratio of DOX & HCl, y-CDs NCs, triethylamine and the solvent is 1-1.2 mg: 1-1.2 mg: 2-2.5. mu.L: 100-;

(4) adding the cell lysate into the B16 cell sediment, repeatedly freezing and thawing and crushing, performing gradient centrifugation to extract B16 cell membranes, and suspending in a PBS solution to obtain a B16 cell membrane suspension;

(5) mixing the y-CDs NCs/DOX in the step (3) with the cell membrane suspension B16 in the step (4), repeatedly extruding and centrifuging to obtain the reduction response type carbon dot drug-loaded nanoclusters y-CDs NCs/DOX @ CCM coating the cell membranes, wherein the ratio of the y-CDs NCs/DOX to the cell membrane suspension is 200 mu g: 0.4-0.6 mL.

2. A preparation method of a reduction response type carbon dot drug-loaded nanocluster for coating a cell membrane comprises the following steps:

(2) dissolving the y-CDs in the step (1) in ultrapure water, adding EDC and NHS for activation, then adding a cross-linking agent, stirring for reaction, and dialyzing to obtain a reduction-responsive carbon-point nanocluster y-CDs NCs solution, wherein the mass ratio of the cross-linking agent to the y-CDs is 1-1.2: 1-1.2, wherein the cross-linking agent is Cystamine dihydrochloride Cystamine;

3. The method as claimed in claim 2, wherein the hydrothermal reaction temperature in step (1) is 170 ℃ and 190 ℃ and the hydrothermal reaction time is 2-4 h.

4. The method of claim 2, wherein the mass ratio of y-CDs, EDC, NHS and the crosslinking agent in step (2) is 1-1.2:5.5-6.5:3-4: 1-1.2; the stirring reaction temperature is room temperature, and the stirring reaction time is 2-4 days.

5. The method according to claim 2, wherein the solvent in the step (3) is methanol; the stirring temperature was room temperature and the stirring time was overnight.

6. The method according to claim 2, wherein the ratio of B16 cells to cell lysate in step (4) is 1X 10⁷The method comprises the following steps: 2-4 mL; the cell lysate contained 1% phenylmethylsulfonyl chloride PMSF.

7. The method according to claim 2, wherein the parameters of the repeated freeze-thaw disruption process in the step (4) are as follows: freezing at-20 deg.C, thawing at 37 deg.C, and repeating for 3 times; gradient centrifugation is as follows: at 4 ℃, the solution is centrifuged for 10min at 700g for removing the precipitate, and then centrifuged for 30min at 14000g for removing the supernatant, and the precipitate is resuspended in 1mL of PBS solution.

8. The method as claimed in claim 2, wherein the repeated extrusion in the step (5) is 11 times of repeated extrusion using an Avanti micro extruder having a filter pore size of 400 nm.

9. Use of nanoclusters according to claim 1 for the preparation of antitumor drugs.