CN104353082A - Functional nano material drug delivery system for identifying, capturing and restraining circulating tumor cells - Google Patents

Abstract

The invention belongs to the application field of nanomaterial coating technology in the recognition, capture and activity regulation of circulating tumor cells, especially for the early warning and prevention of cancer metastasis, and in particular relates to a functional nanometer that specifically recognizes, captures and inhibits circulating tumor cells. Material drug delivery system. The functional nanomaterial drug delivery system is composed of a central nanomaterial carrier, a surface targeting antibody or aptamer, and a drug for anticancer or preventing cancer metastasis. The functional nanomaterial drug-loading system can be used in vitro for specific recognition and capture of microcirculating tumor cells in simulated and clinical patient blood samples, and can be used to regulate the activity of captured circulating tumor cells, so as to play an important role in the early warning and prevention of tumor metastasis. Open up application prospects in prevention.

Description

A functional nanomaterial drug-loaded system that recognizes, captures, and inhibits circulating tumor cells

technical field

  The invention belongs to the application field of nanomaterial coating technology in the recognition, capture and activity regulation of circulating tumor cells, especially for the early warning and prevention of cancer metastasis, and in particular relates to a functional nanometer that specifically recognizes, captures and inhibits circulating tumor cells. Material drug delivery system.

Background technique

  Over the past 50 years, the mortality rate of many major diseases (such as heart disease) has decreased by >60%, but the cancer mortality rate has only decreased by about 5%, and most of them died of tumor re-metastasis after surgery. Traditional "anticancer drugs" mainly target those malignantly proliferating tumor cells rather than dormant circulating tumor cells, so not only cannot selectively kill circulating tumor cells, but also defeat the body's immune system, making "disease - The balance between the human body's own disease resistance" is tilted towards the unfavorable side, and even causes mutagenesis and carcinogenesis. Therefore, the research and development of anticancer drugs follows a slow and stupid route of "fighting cancer after cancer metastasis", with little effect. The current application of nanotechnology in cancer also mainly focuses on the combination of postoperatively transferred chemical drugs and monoantibody-coated nanomaterials, but this method cannot effectively intervene due to its toxicity and non-specificity. The process of cancer metastasis by circulating tumor cells. Therefore, the development of a functionalized nanomaterial drug delivery system that can specifically recognize and capture a small amount of circulating tumor cells in the blood and down-regulate their activity can effectively prevent tumor metastasis induced by the activation of circulating tumor cells, thereby eliminating tumor re-metastasis and recurrence after surgery hidden dangers.

purpose of invention

  The purpose of the present invention is to avoid the non-specific problem of detecting and capturing circulating tumor cells by traditional nanotechnology, and then provide a functional nanomaterial drug-loading system that specifically recognizes, captures and inhibits circulating tumor cells. Because the system can specifically capture and inhibit the captured circulating tumor cells, it is expected to be applied in the early warning and prevention of cancer metastasis.

A functional nanomaterial drug-carrying system of the present invention, the functional nanomaterial drug-carrying system is composed of a central nanomaterial carrier, a surface targeting antibody or aptamer, and a drug for anticancer or preventing cancer metastasis.

The central nanomaterial carrier is PAMAM dendrimers, gold nanoparticles AuNPs, silicon nanoparticles SiNPs, magnetic nanoparticles MNPs or graphene oxide.

The surface-targeting targeting antibody or aptamer is composed of two or more targeting antibodies or aptamers that can specifically recognize and bind circulating tumor cell surface biomarkers, and the biomarker is EpCAM , Sialyl Lewis X, HER2, EGFR, ALDH1, or CD44.

The medicines for anticancer or preventing cancer metastasis include ursolic acid and its derivatives, mifepristone and its derivatives, captopril and its derivatives, emodin and its derivatives.

The central nanomaterial carrier is covalently connected to the surface targeting antibody or aptamer, and the covalent connection method is 1-ethyl-(3-methylaminopropyl)carbodiimide salt salt/N-hydroxysuccinimide method, avidin/biotin method or N-hydroxysuccinimide/maleimide chemistry.

The functional nanomaterial drug-carrying system is prepared by chemically coupling, electrostatically adsorbing, and physically embedding the anti-cancer or cancer metastasis-preventing drug and the central nanomaterial carrier.

The surface of the central nanomaterial carrier has a reactive functional group, and the reactive functional group is -OH, -NH ₂ , -COOH or -SH.

The central nanomaterial carrier and the surface targeting antibody or aptamer are connected by an intermediate linker, and the intermediate linker is carboxylated polyethylene glycol, thiolated polyethylene glycol, hydrazide-polyethylene glycol- dithiol or OPSS-PEG-NHS.

A functional nanomaterial drug-carrying system that specifically recognizes, captures and inhibits circulating tumor cells of the present invention is composed of a central nanomaterial carrier, surface targeting antibodies or aptamers, and drugs for anti-cancer or cancer metastasis prevention, wherein the central Part of it is composed of nanomaterial scaffolds with multivalent complexation effect, good size effect, high loading capacity and rich surface functional groups; the peripheral part is composed of two biomarkers that can specifically recognize and bind to the surface of circulating tumor cells (CTCs). One or more than two targeting antibodies or aptamers; anti-cancer or cancer metastasis prevention drugs are composed of low-toxicity and high-efficiency small-molecular-weight chemical drugs for anti-cancer or cancer metastasis prevention. The nanomaterial drug-carrying system obtained in the present invention not only has a stable structure, but also can maintain the excellent characteristics of nanomaterials and targeting antibodies or aptamers themselves, and can also be used in vitro for the specificity of trace CTCs in simulated and clinical patient blood samples Identification and capture research, and can be used to regulate the activity of captured CTCs, so as to open up application prospects in the early warning and prevention of tumor metastasis.

The present invention is described in detail as follows:

The nanomaterials that can be used in the present invention should have multivalent complexation effect, good size effect, biological stability and high loading capacity, and can be used but not limited to the following substances: dendrimers, gold nanoparticles (AuNPs) ), silicon nanoparticles (SiNPs), magnetic nanoparticles (MNPs), graphene oxide (graphene oxide). The polyamide-amine type dendrimers (PAMAM dendrimers) that are preferentially used in the present invention, in addition to having the common characteristics of ordinary nanomaterials (regular and fine spherical structure on the outside, hydrophobic cavity inside, can be used as a hydrophobic drug And the loading of inorganic probe molecules provides a place to achieve the purpose of controlled release and sustained release of drugs and targeted delivery of inorganic probes), there are a large number of controllable functional groups (amino groups) on the surface, which can be chemically Modification and multivalent complexation targeting antibodies, ligands and drug molecules, etc., to construct safe and effective biological complexes (bioconjugate).

Targeting antibodies that can be used in the present invention are selected for those biomarkers that can be highly expressed on the surface of tumor cells, especially circulating tumor cells, and have low or no expression on normal cells, especially blood cells and leukocytes, but are not limited to The following markers: EpCAM, Sialyl Lewis X, HER2, EGFR, ALDH1, CD44. Antibodies against EpCAM and Sialyl Lewis X highly expressed by epithelial circulating tumor cells are preferentially used in the present invention, wherein EpCAM is also called CD326, TACSTD1, C017-1A, GA733-2 and KSA, etc., and belongs to single transmembrane type I Glycoprotein, with a molecular mass of 30-40 kD, consists of three parts, namely the extracellular domain, the single transmembrane domain and the intracellular domain. The extracellular domain begins with a signal sequence, followed by an epidermal growth factor-like repeat, a human thyroglobulin sequence, and a domain lacking cysteine, the epidermal growth factor-like repeat, and human thyroglobulin The sequence forms a globular structure responsible for the homologous cell-adhesion properties of the EpCAM molecule. The intracellular domain is a short chain of 26 amino acids, containing two a-actinin binding sites, which can bind to actin and interact with the cytoskeleton. Under physiological conditions, EpCAM is expressed in different degrees in all normal epithelium except squamous epithelium, and most of them are located in the tight junction between cells. There is no obvious expression of EpCAM in connective tissue and cells derived from hematopoietic system, brain tissue and vascular endothelial cells. Under pathological conditions, EpCAM is expressed in almost all adenocarcinomas, including colorectal adenocarcinoma, gastric adenocarcinoma, breast cancer, ovarian cancer, lung adenocarcinoma, prostate cancer, pancreatic cancer, hepatocellular carcinoma and retinoblastoma. Therefore, EpCAM is often used as an indicator for early detection and diagnosis of cancer. Another antibody is against the antigen Sialy Lewis X (Slex). Slex, also known as CD15s, is an isomer of the tumor marker monosialoganglioside CA19-9, and one of the more definite ligands recognized by the adhesion molecule selectin (E-selectin, CD62) , composed of galactose (Gal), glucose (Glc), sialic acid (NeuAc), N-acetyl (NAc), fucose (Fuc) combined with five molecules. Cell adhesion molecules, as the molecular basis mediating the mutual recognition and interaction between cells and cells or between cells and matrix, play an important role in the process of tumor invasion and metastasis. The high expression of carbohydrate antigens reflects the changes in the sugar chains of tumor cell membrane carbohydrate antigens. The heterogeneity and distribution of sugar chains produced by chemical modification may mask certain tumor antigens, reduce the immunogenicity of tumor cells, increase the net negative charge of molecules, and weaken the binding and killing effect of lymphocytes and macrophages on tumor cells , to promote immune evasion of tumor cells. Due to the increased repulsion between the cell surfaces, the adsorption and binding capacity is reduced, thus making it easy for tumor cells to fall off and transfer from the tissue. Adhesion molecules play a dual role in the invasion and metastasis of tumor cells. On the one hand, tumor cells must first be down-regulated by cell adhesion molecules and fall off from the primary tumor; The up-regulation of the ligand can adhere to and move with the extracellular matrix or vascular endothelial cells to form metastases. High expression of CD15s and focal dedifferentiation are important signs of tumor metastasis tendency. Therefore, CD15s is also one of the targets of CTCs research.

The medicines that can be used to form the functional nanomaterial drug-carrying system of the present invention usually refer to those chemical medicines that are highly efficient and low-toxic to fight cancer or prevent cancer metastasis, and can adopt but not limited to the following compounds: ursolic acid and its derivatives, rice Fepristone and its derivatives, captopril and its derivatives, emodin and its derivatives. Mifepristone and its derivatives are preferably recommended for use in the present invention. Because they can inhibit the adhesion of circulating tumor cells to the intima of blood vessels and the invasion of peripheral tissues, they can play an important role in the prevention of cancer metastasis.

The covalent linking method between the central nanomaterial and the surface-targeting antibody or aptamer that can be used in the present invention can be used but not limited to the following methods: 1-ethyl-(3-methylaminopropyl) carbonyl disulfide Amine hydrochloride (EDC) / N-hydroxysuccinimide (NHS) method, avidin (avidin) / biotin (biotin) method and N-hydroxysuccinimide (NHS) / butene Two imide (maleimide) chemical method. The EDC/NHS catalytic method that is preferentially used in the present invention can not only make double antibodies (anti-EpCAM, anti-Slex) covalently linked to the surface of PAMAM dendrimers in turn, but also reduce the physical adsorption (anti-fouling effect) of antibodies on the material surface. ), reduce the toxicity of the nanomaterial itself, and increase the stability of the nanomaterial-antibody complex in the biological environment.

The preparation of the functional nanomaterial drug-loading system that can be used in the present invention can be realized by, but not limited to, the following methods: covalently coupling chemical drugs on the surface of nanomaterials, electrostatically adsorbing chemical drugs on the surface of nanomaterials, encapsulating chemical drugs Buried in nanomaterial cavities. The physical embedding method used in the present invention is preferably recommended, that is, the chemical drug mifepristone and its derivatives are embedded in the 3D cavity structure of PAMAM dendrimers in a certain environment. This method not only avoids the steric hindrance effect caused by the joint connection of chemical drugs and antibodies on the surface of PAMAM dendrimers, but also maximizes the use of the spatial structure of PAMAM dendrimers to increase the drug loading capacity.

The central nanomaterials that can be used in the present invention should have reactive functional groups on the surface to facilitate covalent linkage with targeting antibodies. The following groups can be selected but not limited to: -OH, -NH ₂ , -COOH, -SH.

When the nanomaterials of the present invention are connected with targeting antibodies, intermediates that reduce steric hindrance effects can be used but not limited to the following substances: carboxylated polyethylene glycol, thiolated polyethylene glycol (PEG -SH), hydrazide-polyethylene glycol-dithiol , orthopyridyl-disulfide-polyethyleneglycol-N-hydroxy-succinimide (OPSS-PEG-NHS).

The central nanomaterial and surface targeting antibody that can be used in the present invention, in order to achieve the maximum multivalent complexation effect, the amount of targeting antibody should be much more than the nanomaterial itself, and the specific amount depends on the exposed reactive functional groups on the surface of the nanomaterial.

The tumor cell model of extracorporeal circulation that can be used in the present invention should select those cancer cell lines that are concentrated in research, postoperative metastasis and recurrence, and can be used but not limited to the following cancer cell lines: breast cancer cell lines, prostate cancer cell lines, colon Cancer cell lines, liver cancer cell lines, cervical cancer cell lines, gastric cancer cell lines, lung cancer cell lines. The human colon cancer cell line HT29 is preferentially recommended as the tumor cell model for extracorporeal circulation. Because the colon cancer cell line HT29 has a moderate degree of metastasis and recurrence, and the risk is low; and the biomarkers EpCAM and Slex are highly expressed on the cell surface, so choosing HT29 as an in vitro CTCs model will help the research. successfully launch.

The blood samples containing circulating tumor cells in vitro that can be used in the present invention are taken from those middle and advanced cancer patients with high degree of postoperative metastasis and recurrence in the research concentration, and can be selected but not limited to the following cancer patients: colon cancer patients, breast cancer patients, Liver cancer patients, prostate cancer patients, cervical cancer patients, stomach cancer patients, lung cancer patients.

The physical and chemical properties characterization methods that can be used for the functional nanomaterial drug-carrying system of the present invention can use, but are not limited to, the following methods: nuclear magnetic resonance spectroscopy ( ¹ H NMR), infrared spectroscopy (FTIR), ultraviolet-visible spectroscopy (UV-Vis), Fluorescence spectrum or map, laser particle size (DLS) / Zeta potential, field emission scanning electron microscope (FSEM) image, atomic force microscope (AFM) image, transmission electron microscope image, electrophoresis image.

The methods that can be used for the in vitro CTCs capture research of the functional nanomaterial drug-loaded system of the present invention can be adopted but not limited to the following methods: according to the content of CTCs in the patient's blood (1/10 ³ -10 ⁶ ), first inject a large number of interfering cells ( Add a certain amount of target tumor cells to white blood cells, red blood cells, normal cells that do not express biomarkers, or other tumor cells) or add a certain amount of target tumor cells to whole blood, and then add quantitative nanomaterials-dual/multi-target Perform capture studies to antibody or aptamer complexes. It is highly recommended to use the PAMAM dendriemrs-double antibody complex in the present invention to examine the recognition and capture effect of suspension and adherent HT29 cells alone, or to investigate the presence of a large number of interfering cells (leukemic cells HL-60 or red blood cell RBCs). For the capture of a small amount of HT29 cells, fluorescence photography and flow cytometry analysis were used to evaluate the specific capture effect of the double antibody complex on the CTCs model in simulated blood samples in vitro.

The functional nanomaterial drug-carrying system of the present invention can be used to capture and study CTCs in blood samples of clinical patients. This method can be used but not limited to: obtain blood samples of tumor patients after surgery or chemotherapy, and obtain lymphocyte layer after processing Finally, adding nanomaterials-dual/multiple targeting antibodies or aptamer complexes for co-incubation or directly adding nanomaterials-dual/multiple targeting antibodies or aptamer complexes to whole blood to carry out capture research. It is preferentially recommended to use the postoperative colon cancer blood sample obtained from the tumor hospital in the present invention, add quantitative PAMAM dendrimers-double antibody complex to carry out the capture experiment, after the post-processing of the blood, quantitatively analyze the number of captured CTCs by flow cytometry, confocal laser Analyze its catch.

The functional nanomaterial drug-carrying system that can be used in the present invention can be used to capture CTCs. The following evaluation measures can be used, but not limited to: use MTT, XTT, and LDH methods to test nanomaterial-double/multiple targeting antibody complexes The cytotoxicity of the complex was tested by PI staining method, BrdU incorporation method, etc. to test the distribution of complexes affecting the cell cycle, and Annexin V-FITC/PI, TUNEL, Annexin V-PE/7-AAD, DAPI, AO/EB, Caspase 3/8/9 and other staining methods were used to evaluate the apoptosis status of cells. It is preferred to use MTT method, morphological staining observation method, flow cytometry analysis of cell cycle and cell apoptosis method in this invention to further explore the inhibitory mechanism of PAMAM dendrimers-double antibody complex on the activity of capturing CTCs.

Beneficial effects of the present invention:

(1) The present invention provides a functional nanomaterial complex drug delivery system that specifically recognizes, captures and inhibits circulating tumor cells. The drug delivery system can specifically identify, capture and inhibit circulating tumor cells in blood, It is expected to play an important role in the early warning and prevention of cancer metastasis;

(2) The present invention provides technical information on coupling two or more antibodies or aptamers to the surface of the same nanomaterial. The preparation method is simple, easy to operate, mild in reaction conditions, and requires little equipment, and It has the prospect of industrial implementation.

Description of drawings

Fig. 1 is a schematic diagram of the invention of a functional nanomaterial drug delivery system for identifying, capturing and inhibiting circulating tumor cells. i, the surface of the nanomaterial PAMAM dendrimers is modified by carboxylation; ii, the surface of the modified dendrimers is covalently linked to two antibodies (aEpCAM and aSlex); iii-v, the prepared dendrimers-double antibody drug delivery system is tested in the presence or absence of interfering cells The specific recognition and capture of the target colon cancer cell line HT29; vi, the prepared dendrimers-double antibody drug delivery system inhibits the activity of the captured cancer cells.

Figure 2 is the proton nuclear magnetic spectrum of the corresponding derivative of G6 PAMAM dendrimers (CC G6) and the antibody complex (G6-5aEpCAM-5aSlex) obtained in Examples 1 and 2.

Figure 3 is the infrared spectrum of the corresponding derivative of G6 PAMAM dendrimers (CC G6) and antibody complex (G6-5aEpCAM-5aSlex) obtained in Examples 1 and 2.

Figure 4 is the ultraviolet absorption spectrum of the corresponding derivative of G6 PAMAM dendrimers (CC G6) and antibody complex (G6-5aEpCAM-5aSlex) obtained in Examples 1 and 2.

5 is a scanning electron micrograph of the double antibody complex G6-5aEpCAM-5aSlex obtained in Examples 1 and 2.

Fig. 6 is the fluorescence spectrum of recognition and binding of the fluorescently labeled double antibody complex PE-5aEpCAM-G6-3aSlex-FITC obtained in Example 3 to adherent HT29 cells in vitro.

Fig. 7 is the in vitro recognition and capture fluorescence spectrum of non-adherent HT29 cells by the fluorescently labeled double antibody complex PE-5aEpCAM-G6-3aSlex-FITC obtained in Example 3.

Fig. 8 is a flow cytometric analysis of the capture efficiency of the fluorescently labeled double antibody complex PE-5aEpCAM-G6-3aSlex-FITC obtained in Example 3 on a small amount of target HT29 cells under the interference of a large number of RBCs.

Fig. 9 is the fluorescent spectrum of the fluorescently labeled double antibody complex PE-5aEpCAM-G6-3aSlex-FITC obtained in Example 4 for capturing trace CTCs in the blood samples of postoperative colon cancer patients.

Fig. 10 is the capture flow pattern of trace CTCs in the blood samples of postoperative colon cancer patients by the fluorescence-labeled double antibody complex PE-5aEpCAM-G6-3aSlex-FITC obtained in Example 4.

Figure 11 shows the effects of different concentrations of the double antibody complex G6-5aEpCAM-3aSlex obtained in Example 5 on the cycle distribution of HT29 cells. ** indicates the comparison with the blank control group under the same conditions, p <0.01.

Figure 12 shows the effect of different concentrations of the double antibody complex G6-5aEpCAM-3aSlex obtained in Example 5 on the apoptosis of HT29 cells. * and ** indicate the comparison with the blank control group under the same conditions, ^# and ^## indicate the comparison between different concentrations of the same sample under the same conditions; when it is * or ^# , p <0.05; when it is ** or ^## , p < 0.01.

Detailed ways

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Example 1: Preparation of polyamide-amine dendrimer complex coated with double antibody

Polyamidoamine dendrimer complexes coated with or without fluorescently labeled antibodies can be obtained by covalently linking antibodies to polyamidoamine dendrimers for specific recognition of CTCs in vitro , capture and activity regulation, the specific preparation process is shown in Figure 1.

(1) Complete carboxylation modification of polyamide-amine dendrimers:

Take 60 mg of the 6th generation polyamide-amine dendrimers (G6 PAMAM dendrimers) dissolved in methanol containing 60 mg of amino-terminated dendrimers. After rotary evaporating the methanol solution, dissolve it completely with 2 ml of DMSO solution, and then add 246 mg of succinyl Anhydrides were reacted under strong magnetic stirring at room temperature for 24 h, and then the reaction product was dialyzed in 500 ml×3 ultrapure water with a cellulose dialysis membrane with a molecular weight cut-off of 8,000-14,000 for 24 h. After complete purification, it was finally frozen Dry to obtain the product CC G6 (G6-(COOH) ₂₅₆ ) of the complete carboxylation of the amino group on the surface of the dendrimer;

(2) Partial acetylation modification of polyamide-amine dendrimers:

Dissolve G6-(COOH) ₂₅₆ in a phosphate buffer solution at pH 7.4 to a concentration of 0.276 μg/ml, take 8 ml of G6-(COOH) ₂₅₆ with a concentration of 0.276 μg/ml, add EDC 302 ng and NHS 181.5 ng, stirred and activated at room temperature for 1 h in the dark, so as to obtain the intermediate product of partial acetylation on the surface of dendrimers;

(3) Diabody (aEpCAM) and (aSlex) modification of polyamide-amine dendrimers

Take 1 ml of the activated intermediate product solution, add 118.6 μl of aEpCAM solution with a concentration of 50 μg/ml and 7.12 μl of aSlex solution with a concentration of 500 μg/ml, stir overnight at room temperature in the dark, then purify by dialysis, and freeze-dry to obtain The G6-5aEpCAM-3aSlex double antibody complex of dendrimer surface-coupled antibodies aEpCAM and aSlex;

(4) Diabody (aEpCAM) and (aSlex) modification of polyamide-amine dendrimers

Take 1 ml of the activated intermediate product solution, add 118.6 μl of aEpCAM solution with a concentration of 50 μg/ml and 11.86 μl of aSlex solution with a concentration of 500 μg/ml, stir overnight at room temperature in the dark, then purify by dialysis, and freeze-dry to obtain The G6-5aEpCAM-5aSlex double antibody complex of dendrimer surface-conjugated antibodies aEpCAM and aSlex.

(5) Diabody (aEpCAM and aSlex) modification of fluorescently labeled polyamidoamine dendrimers

Take 1 ml of the above-mentioned activated product CC G6, first add 3.56 μl of aSlex and 3.56 μl of secondary antibody IgG-FITC solution to react in the dark for 12 h at room temperature, then add 15 μl of aEpCAM-PE solution to continue the reaction for 12 h, and then The product was purified by dialysis for 24 h, and finally freeze-dried to obtain the double fluorescently labeled PE-5aEpCAM-G6-3aSlex-FITC complex.

(6) Diabody (aEpCAM and aSlex) modification of fluorescently labeled polyamidoamine dendrimers

Take 1 ml of the above-mentioned activated product CC G6, add 5.93 μl of aSlex and 5.93 μl of secondary antibody IgG-FITC solution to react in the dark for 12 h at room temperature, then add 15 μl of aEpCAM-PE solution to continue the reaction for 12 h, and then The product was purified by dialysis for 24 h, and finally freeze-dried to obtain double fluorescently labeled PE-5aEpCAM-G6-5aSlex-FITC complex.

Example 2: Characterization of the polyamide-amine dendrimer complex coated with double antibodies

(1) Take a certain amount of newly prepared CC G6 and G6-5aEpCAM-5aSlex substances, dissolve them fully with 700 μl of D ₂ O respectively, place them in NMR tubes, and then perform ¹ H NMR with a 400 MHz superconducting NMR instrument analyze. (2) Take a certain amount of newly prepared CC G6 and G6-5aEpCAM-5aSlex substances, fully dissolve them in PBS (pH 7.4), treat them with ultrasonic waves for 5 min, filter them through a 0.22 μm disposable water-soluble filter, and put them in DLS/Zeta potential instrument was used to measure the respective water-soluble particle size and potential distribution. (3) Take a certain amount of newly prepared CC G6, G6-5aEpCAM-5aSlex substances, mix them with an appropriate amount of KBr powder, press them into tablets, and test their FTIR spectra in an infrared analyzer.

(4) Take a certain amount of newly prepared CC G6, G6-5aEpCAM-5aSlex substances, dissolve them in PBS (pH 7.4) to a certain concentration, and use PBS (pH 7.4) as the benchmark, and use the Q5000 ultramicro spectrophotometer The ultraviolet absorption spectra of these substances at a wavelength of 210-600 nm were respectively measured on a photometer.

(5) Take the newly prepared G6-5aEpCAM-5aSlex complex, first bond the conductive adhesive on the sample holder, and then use a capillary to absorb the 0.1% (W/W) aqueous solution of the complex dispersed by ultrasound, drop by drop Drop it on the conductive glue, put it into the scanning electron microscope to observe after air-drying.

According to the analysis of the characterization results, the difference in the ¹ H NMR spectra of CC G6 and G6-5aEpCAM-5aSlex (as shown in Figure 2) shows the difference in the material structure before and after the antibody is linked, especially the appearance of the ester peak, which shows that the antibody is in G6 PAMAM dendrimers surface presence. In the FTIR spectrum (as shown in Figure 3), when CC G6 is linked to antibodies aEpCAM and aSlex, although the C=O stretching vibration at 1680-1630 cm ^-1 is maintained, the CN at 1420-1400 cm ^-1 The characteristic absorption peak of the stretched amide bond is enhanced, and the difference in the surface groups of the visible substances also reflects the successful coupling of the diabody on the surface of the nanomaterial. Since CC G6 itself has no UV absorption at 220 nm, and antibodies aEpCAM and aSlex both have characteristic UV absorption values at 220 nm, the absorption peak at 220 nm was selected as an indicator for judging whether the antibody is connected to the nanomaterial. The UV spectrum shows (as shown in Figure 4) that although it is a low concentration of G6-5aEpCAM-5aSlex complex, there is still a characteristic absorption peak at 220 nm, which shows that the surface of the nanomaterial has been successfully coated with antibodies. What are the physical and chemical properties of the prepared double-antibody complex G6-5aEpCAM-5aSlex? The particle size and potential measurements of its monodisperse aqueous solution particles show that (see Table 1), when the nanomaterial CC G6 is covalently linked to the double-antibody , although the electronegativity characteristics did not change significantly, but its water-soluble particle size increased sharply, showing that the presence of antibodies would cause a certain aggregation tendency. The scanning electron microscope (as shown in Figure 5) further confirmed the morphology and diameter of the prepared bis-antibody complex. The surface of the complex is approximately spherical, and the horizontal diameter is about 100 nm.

Table 1 shows the water-soluble diameter and Zeta potential of the corresponding derivatives (CC G6) and antibody complexes (G6-5aEpCAM-5aSlex) of G6 PAMAM dendrimers.

Example 3: In vitro recognition and capture analysis of CTCs model (HT29 cells) by polyamide-amine dendrimer complexes coated with double antibodies (1) HT29 cells in logarithmic growth phase were grown at 10 ⁵ / The density of ml was inoculated on a 35 mm laser confocal special dish. After the cells adhered to the wall, they were suspended and sealed with PBS containing 1% BSA for 30 min. After repeated washing, they were mixed with fluorescently labeled double The anti-complex (PE-5aEpCAM-G6-3aSlex-FITC) was co-cultured for 1 h at 37 °C in an incubator with 5% CO ₂ . Finally, unbound complexes or antibodies were washed away, fixed with a fixative, and stained in PBS solution containing nuclear staining agent DAPI for 15 min in the dark. After staining, wash repeatedly, and finally cover with serum-free and phenol-red-free medium and scan under laser confocal microscope DAPI λ _ex 405 nm, λ _em 425-475 nm, FITC λ _ex 488 nm, λ _em 500-535 nm, PE Take pictures and analyze in λ _ex 550 nm, λ _em 570-610 nm modes.

(2) Take HT29 cells in the logarithmic growth phase, trypsinize, take 10 ⁶ HT29 cells per tube, stain with Hoechst 33258 staining solution for 15 minutes to mark the nuclei, wash with PBS, and then use 1% BSA in PBS was blocked for 30 min to remove non-specific binding. Finally, 1 ml of PBS or the fluorescently labeled complex PE-5aEpCAM-G6-3aSlex-FITC at a concentration of 20 μg/ml was added to the control tube and the sample tube, respectively, and incubated in a water bath at 37 °C in the dark for 1 h. After washing to remove unbound complexes, suspend with 100-500 μl of PBS, and use the fluorescence microscope DAPI λ _ex 365 nm, λ _em 445/50 nm, λ _ex 470/40 nm, λ _em 525/50 nm mode Qualitative analysis of the capture situation.

(3) Take HT29 cells in the logarithmic growth phase, trypsinize, take 10 ³ HT29 cells from each tube, and add 10 ⁶ and 10 ⁸ RBCs to simulate blood samples of clinical patients (ie 1 CTCs/ 10 ³ -10 ⁶ blood cells), after treating the cell mixture with 1% BSA in PBS blocking solution for 30 min, directly add 1 ml of fluorescently labeled double antibody complex PE-5aEpCAM-G6-3aSlex at a concentration of 20 μg/ml to each tube -FITC was incubated in a 37°C water bath for 1 h. Two isotype antibodies IgG-PE and IgG-FITC were added to the cell mixture as a negative control. After washing to remove unbound complexes, the cells were suspended with 500 μl of PBS, and the number of cells captured by the complexes was quantitatively analyzed at the 488 nm laser of the flow cytometer.

The prepared fluorescently labeled double antibody complex PE-5aEpCAM-G6-3aSlex-FITC was used in the in vitro recognition and capture experiments of the CTCs model (HT29 cells) (as shown in Figure 1). In the absence of interfering cells, for adherent HT29 cells, confocal laser analysis (as shown in Figure 6), the surface of the cell membrane recognized and bound by the double antibody complex will show superimposed yellow-green fluorescence ( Highlights), while unbound cells, only the nuclei show blue fluorescence (shown in grey). For suspended HT29 cells, the fluorescence microscope atlas (as shown in Figure 7) also showed similar results. The double-antibody complex could specifically recognize and capture HT29 cells within 1 hour, and the surface of the captured HT29 cells had two-color fluorescence (highlighted display). In the presence of a large number of interfering red blood cells, as the number of interfering red blood cells increased from 10 ⁶ to 10 ⁸ , the number of cells captured by the double antibody complex from 1000 target HT29 cells decreased from 39 to 25 (as shown in Figure 8 Shown), it can be seen that the increase in the number of interfering cells will lead to an increase in space and contact resistance, which will further affect the recognition and capture effect of the complex on target cells. But even at the near-clinical CTCs content (ie, 1CTCs/10 ³ ~10 ⁶ hematopoietic cells), the PAMAM dendrimers coated with the double antibody prepared by us can still identify and capture CTCs from a large number of red blood cells with high sensitivity and high specificity, This also illustrates the advantages of the double antibody complex.

Example 4: Recognition and capture analysis of CTCs in blood samples of clinical patients by polyamide-amine dendrimer complexes coated with double antibodies

Take 1 ml of colon cancer patient blood sample, after 1% BSA blocking treatment, directly add 1 ml of fluorescently labeled double-antibody complex PE-5aEpCAM-G6-3aSlex-FITC at a concentration of 20 μg/ml, and incubate for 1 h in a 37 °C water bath Finally, wash with PBS to remove unbound complexes; then use 8-10 ml of erythrocyte lysate to lyse in a 37°C water bath for 5-10 min until the erythrocytes are completely broken, and centrifuge at 1500 rpm to separate the remaining cells; finally use APC-labeled Anti-CD45 was co-incubated for 30 min to label leukocytes. After washing to remove the background color, the number of CTCs captured by the double antibody complex was analyzed on a flow cytometer or the nucleus was further labeled with DAPI, and the double antibody was identified under confocal laser CTCs captured by the complex.

Blood samples from postoperative colon cancer patients were obtained to further confirm the high specificity of the prepared double-antibody-coated PAMAM dendrimers for capturing CTCs, and to illustrate its clinical application value. The laser confocal microscope atlas shows (as shown in Figure 9) that the bis-antibody complex can capture extremely small amounts of CTCs, and its surface shows specific yellow-green fluorescence (shown by bright spots), which is used to distinguish the CTCs detected by anti-CD45. Labeled leukocytes (red fluorescence on the membrane surface, indicated by dots). Flow cytometry analysis (as shown in Figure 10) shows that, compared with the isotype control group, the double antibody complex can capture CTCs, and the captured CTCs are displayed in the PE ⁺ FITC ⁺ APC ^- area. Statistics show that the prepared double antibody complex PE-5aEpCAM-G6-3aSlex-FITC can capture an average of 11±3 CTCs from 1 ml blood of 8 patients.

Example 5: Analysis of activity regulation of captured HT29 cells by polyamide-amine dendrimer complexes coated with double antibodies

(1) Take HT29 cells in the logarithmic phase of growth ^, digest ^with trypsin, and make cell suspension by pipetting with medium The suspension was inoculated into 96-well plates, 100 μl per well, and then placed in a 37 °C, 5% CO ₂ incubator for 24 h. Next, on a sterile operating bench, first filter the double antibody complex mother solution G6-5aEpCAM-3aSlex with a 0.22 μm filter membrane, then remove the old medium, and add 100 μl of different concentrations of complexed complexes diluted with medium to each well. (0, 1.25, 2.5, 5, 10, 20 μg/ml) co-cultured. A blank control group and a solvent control group were also set up, with 6 replicate wells in each group. After 48 h of action, the medium containing the complex was removed, and 100 μl of serum-free phenol red-free medium containing MTT (500 μg/ml) was added to each well. After continuing to incubate for 4 h, the 96-well plate was removed to stop the culture. Gently remove the supernatant of each well, add 150 μl of DMSO to each well, and shake on the shaker for 10 min. After the blue-purple crystals are completely dissolved, measure the light absorption value of each well at a wavelength of 570 nm on a microplate reader (A _{570 nm} ). The survival rate can be obtained by the formula: survival rate (%)=(experimental group A _{570 nm} -solvent control group A _{570 nm} )/(blank control group A _{570 nm} -solvent control group A _{570 nm} )×100%. (2) Human colon cancer cells HT29 in the logarithmic growth phase were selected and digested with trypsin, and the cell density was adjusted to 5×10 ⁵ cells/ml, seeded in 6-well plates at 1.5 ml/well, and placed at 37 °C, 5 Incubate in an incubator with % CO ₂ for 24 h; filter the above complex mother solution with a 0.22 μM filter membrane to prepare a series of concentration gradients (0, 10, 20 μg/ml), CC G6 is used as a negative control, no complex The treatment group served as the blank control group. Aspirate the old medium, wash the cells with PBS, add 2 ml of the corresponding complex to each well, and continue to incubate in the incubator for 48 h. After co-cultivation, the cells were washed three times with PBS to remove complexes, the cells were digested and collected, transferred to a centrifuge tube, centrifuged at 200 g for 6 min, the supernatant was discarded, and the cells were washed twice with pre-cooled PBS. Aspirate the supernatant carefully, leave about 200 μl of PBS in the centrifuge tube to avoid sucking the cells, then add 1 ml of pre-cooled 70% ethanol to each tube, mix and fix, overnight at -20°C, centrifuge at 200 g for 6 min, discard Remove ethanol, wash once with pre-cooled PBS, absorb the supernatant, leave about 50 μl of PBS, and bounce to disperse the cells. Add 500 μl of the prepared propidium iodide staining solution (staining buffer 500 μl, propidium iodide staining solution (20X) 25 μl, RNase A (50X) 10 μl) into the corresponding blank control tube and sample In the tube, stain at room temperature for 30 min in the dark. Cell cycle distribution was detected by flow cytometry at excitation channel 488 nm.

(3) Take HT29 cells in the logarithmic growth phase, trypsinize to adjust the cell concentration to 5×10 ⁵ cells/ml, inoculate 1.5 ml/well on a six-well culture plate, and culture at 37°C and 5% CO ₂ Cultivated in a box. After the cells adhered to the wall, the old medium was removed, and fresh medium was added to the negative wells, and different concentrations of the above complexes and CC G6 (0, 10, 20 μg/ml) were added to the sample wells. After 48 h of action, the pancreatic After enzyme digestion, the cells were collected by centrifugation, washed three times with PBS, and the remaining 50 μl of PBS was used to resuspend the cells. Prepare double staining solution in the same proportion as the number of samples, that is, 500 μl Binding Buffer + 5 μl Annexin V-FITC + 5 μl PI. Take 500 μl of the above staining mixture and add it to the negative control tube and the sample tube one by one, and react at room temperature for 15 min in the dark. Set up two single-positive compensation tubes, that is, dead cells + 5 μl Annexin V-FITC or dead cells + 5 μl PI, adjust the voltage, and use a flow cytometer to detect cell apoptosis at the excitation channel 488 nm.

After the prepared double-antibody complex specifically captures the target cancer cells, whether it can further regulate the activity of the captured cancer cells is also a highlight of the present invention. Cell proliferation experiments showed that (see Table 2), compared with CC G6, the double antibody complex G6-5aEpCAM-3aSlex showed a stronger ability to inhibit cell proliferation in a concentration-dependent manner. The complex can further change the distribution of HT29 cells in each phase of the cell cycle. Compared with the blank group, the percentage of cells in the S phase of the complexed cells increased significantly, while the content in the G0/G1 phase decreased (as shown in Figure 11), it can be seen that the double antibody complex can block cell division in S phase, thereby avoiding premature entry into G2/M phase. Quantitative analysis of cell apoptosis by flow cytometry (as shown in Figure 12), the complex can not only change the cell morphology, but also slightly induce the cells to enter the early stage of apoptosis, but the degree of apoptosis does not exceed 20%. The overall analysis shows that the PAMAM dendrimers complex coated with the double antibody can inhibit the activity of the captured cancer cells to a certain extent, but does not produce severe cytotoxicity.

Table 2 shows the inhibitory rate of CC G6 and G6-5aEpCAM-3aSlex obtained in Example 5 with increasing concentration (5, 10, 20 μg/ml) on HT29 cell unit moles by MTT method. ^## indicates that compared with CC G6 under the same conditions, p <0.01.

Example 6: Preparation of antibody-coated magnetic nanospheres

Carboxylated magnetic nanoparticles (MNPs-COOH) were first prepared. Take nanoparticles with amino groups at the end and Fe ₂ O ₃ at the core, add succinic anhydride and react in dimethylformamide (DMF) solution for 2 h, after repeated washing with ethanol and dialysis with ultrapure water, the water-soluble MNPs-COOH.

Second, the Anti-EpCAM antibody was covalently attached to the MNPs-COOH surface. Take 5 mg MNPs-COOH, add 50 mM EDC and 50 mM NHS, and activate in 1 ml 0.01 M PBS (pH 6.8) solution for 30 min. The activated MNPs-COOH was magnetically separated, dissolved in 1 ml of 0.01 M PBS (pH 7.2), and reacted with 50 μg of anti-EpCAM antibody for 4 h under continuous stirring. After repeated washing with PBS (pH 7.2), the antibody-coated magnetic nanospheres were obtained.

Example 7: Preparation of aptamer-coated gold nanospheres

Gold nanoparticles (AuNPs) are first prepared. Take 100 ml of 1mM chloroauric acid (HAuCl4) solution and heat to reflux, then add 10 ml of 38.8 mM sodium citrate and reflux for 20 min to prepare AuNPs.

Second, thiolated DNA aptamer chains were covalently attached to the surface of AuNPs. For example, take 9 μl of 1 mM aptamer, add 1 μl of 500 mM acetic acid solution and 1.5 μl of 10 mM trichloroethyl phosphate (TCEP) to activate for 1 h. After that, 3 ml of the prepared AuNPs were added to react for 16 h. Finally, 30 μl of 500 ml Tris acetic acid and 300 μl of 1M NaCl were added to react for another 24 h. Uncomplexed aptamers were removed by centrifugation at 14,000 rpm for 15 min to prepare aptamer-coated gold nanospheres.

Claims (8)

1. a function nano material drug carrier system, is characterized in that: described function nano material drug carrier system is made up of center nano material carrier, surperficial targeting antibodies or aptamers, medicine that is anticancer or prophylaxis of cancer transfer.

2. function nano material drug carrier system according to claim 1, is characterized in that: described center nano material carrier is PAMAM dendrimers, gold nano grain AuNPs, nano silicon particles SiNPs, magnetic nanoparticle MNPs or graphene oxide graphene oxide.

3. function nano material drug carrier system according to claim 1, it is characterized in that: described surface targets can specific recognition and forming in conjunction with the targeting antibodies of circulating tumor cell surface biomarkers or aptamers by two or more to targeting antibodies or aptamers, and described biomarker is EpCAM, Sialyl Lewis X, HER2, EGFR, ALDH1 or CD44.

4. function nano material drug carrier system according to claim 1, is characterized in that: the medicine of described anticancer or prophylaxis of cancer transfer comprises ursolic acid and derivant, mifepristone and derivant thereof, captopril and derivant, emodin and derivant thereof.

5. function nano material drug carrier system according to claim 1, it is characterized in that: described center nano material carrier and surperficial targeting antibodies or aptamers adopt covalent attachment to carry out, and described covalent attachment is 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate/N-hydroxysuccinimide method, avidin/biotin method or N-hydroxysuccinimide/Maleimide chemistry method.

6. function nano material drug carrier system according to claim 1, is characterized in that: described function nano material drug carrier system by the medicine of described anticancer or prophylaxis of cancer transfer and center nano material carrier are carried out chemical coupling, Electrostatic Absorption, physically trapping mode prepare.

7. function nano material drug carrier system according to claim 2, is characterized in that: described center nano material carrier surface has reactive functionality, and described reactive functionality is-OH ,-NH ₂,-COOH or-SH.

8. function nano material drug carrier system according to claim 5, it is characterized in that: described center nano material carrier adopts intermediate connector to be connected with surperficial targeting antibodies or aptamers, described intermediate connector is carboxylated Polyethylene Glycol, the Polyethylene Glycol of sulfhydrylation, hydrazide-polyethylene glycol-dithiol or OPSS-PEG-NHS.