CN111920947B - Method for preparing cyanine dye mediated nucleic acid/anticancer drug compound - Google Patents
Method for preparing cyanine dye mediated nucleic acid/anticancer drug compound Download PDFInfo
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- A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
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- A61K49/0034—Indocyanine green, i.e. ICG, cardiogreen
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- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
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Abstract
The invention discloses a method for preparing a cyanine dye mediated nucleic acid/anticancer drug compound, which takes cyanine dye modified functional nucleic acid and anticancer chemotherapeutic drugs as raw materials, and leads the cyanine dye modified functional nucleic acid and the anticancer chemotherapeutic drugs to be self-assembled in aqueous solution through molecular electrostatic attraction, proton transfer and hydrophobic effect to form the nucleic acid/anticancer drug compound. The method for preparing the functional nucleic acid/anticancer drug compound is simple, green, mild and low in cost, wherein the synergistic effect of the anticancer chemotherapeutic drug and the functional nucleic acid can realize the benign combination of chemotherapy and immunotherapy, thereby achieving the purpose of treating cancer. Therefore, the invention is expected to provide theoretical and experimental technical support for preparing efficient anticancer functional nucleic acid medicaments.
Description
Technical Field
The invention belongs to the field of biomedicine, and particularly relates to a preparation method of a cyanine dye-mediated nucleic acid/anticancer drug compound.
Background
Functional nucleic acids, including siRNA, mRNA and antisense nucleic acids, have been widely used in the diagnosis and treatment of cancer. However, single-means nucleic acid therapy is extremely limited by nuclease degradation and low transfection efficiency, severely limiting the use of functional nucleic acids. The combination of functional nucleic acids and other therapeutic modalities (including radiation therapy, chemotherapy, photodynamic therapy, photothermal therapy) can maximize the effectiveness of cancer therapy. Chemotherapy, as a standard means of clinical cancer treatment, exhibits great potential for use in conjunction with functional nucleic acid therapy. However, most of the chemotherapy drugs are small molecules, and due to the large difference between the physicochemical properties of the chemotherapy drugs and the nucleic acid, the direct loading of the nucleic acid and the chemotherapy drugs in the same nanoparticle is difficult to realize. Therefore, the direct realization of the assembly of functional nucleic acids and chemotherapeutic drugs becomes a key means for preparing nucleic acid drugs.
The cyanine dye is used as a common dye molecule, is widely used for nucleic acid modification, and can be further used for quantitative analysis and fluorescence localization of nucleic acid. However, few reports have been made on the physicochemical properties and assembly properties of cyanine dyes. Cyanine dyes are known to belong to the aromatic class of dyes, having a benzene ring structure. Thus, cyanine dyes have a strong pi-conjugated system and hydrophobic interaction. In addition, the cyanine dye has a sulfonic acid group at the side chain end, so that the cyanine dye has stronger electronegativity. Theoretically, cyanine dyes are highly susceptible to binding to positively charged, pi-conjugated drugs. At present, most of anticancer drugs used in clinic belong to aromatic compounds, and have good conjugation property. In addition, many hydrophilic chemotherapeutic drugs exhibit electropositivity, facilitating phagocytosis by cells. Therefore, when the positively charged anticancer drug and the cyanine modified functional nucleic acid are mixed, the positively charged anticancer drug and the cyanine modified functional nucleic acid can easily form nucleic acid/anticancer drug nanoparticles due to the interaction of electrostatic attraction, pi-pi stacking and the like, and the method has important scientific significance for developing and preparing nucleic acid drugs with different functions.
Disclosure of Invention
The invention aims to provide a preparation method of a cyanine dye-mediated nucleic acid/anticancer drug compound, which adopts a simple method to design a compound drug with the synergistic effect of chemotherapy and immunotherapy and can realize effective antitumor therapy.
In order to achieve the purpose, the invention adopts the following technical scheme:
a cyanine dye mediated nucleic acid/anticancer drug compound preparation method, which is to fully mix cyanine dye modified functional nucleic acid and anticancer chemotherapeutic drugs in aqueous solution, so as to self-assemble into novel functional nucleic acid/anticancer drug compound with uniform size and high loading rate by utilizing proton shift, electrostatic attraction and hydrophilic-hydrophobic change; the method specifically comprises the following steps:
(1) preparing a cyanine dye modified functional nucleic acid stock solution: dissolving the cyanine dye modified functional nucleic acid in sterile water, and blowing and beating uniformly at room temperature in a dark condition to form a uniform cyanine dye modified functional nucleic acid stock solution with the concentration of 100 mu M;
(2) preparing a medicine stock solution: dissolving anticancer chemotherapeutic drug in solvent such as water and dimethyl sulfoxide, and mixing at room temperature in dark condition to obtain drug stock solution with concentration of 10 mM;
(3) preparation of nucleic acid/anticancer drug complexes: uniformly mixing a cyanine dye modified functional nucleic acid stock solution and a drug stock solution, placing the mixture in a metal bath, reacting for 1h at 95 ℃, then cooling to room temperature, centrifuging a product, and washing twice with water to prepare the nucleic acid/anticancer drug compound; the rotation speed of the centrifugation is 8000 rpm, and the time is 5 min.
The cyanine dye is one of any conventionally known cyanine dyes, such as Cy3, Cy3.5, Cy5, Cy5.5, Cy7, Cy7.5, and the like.
The functional nucleic acid is one of any conventionally known functional nucleic acids, such as CPG, siRNA, mRNA, antisense nucleic acid, DNA, etc.
The cyanine dye modified functional nucleic acid is obtained by modifying cyanine dyes at one end of the functional nucleic acid through an amide condensation reaction, and each functional nucleic acid is modified with a cyanine dye molecule.
The anticancer chemotherapeutic drug is zorubicin hydrochloride, doxorubicin hydrochloride or structural analogues and derivatives of doxorubicin hydrochloride.
The mole ratio of the cyanine dye modified functional nucleic acid to the anticancer chemotherapeutic drug is 1: 30.
The invention has the beneficial effects that:
the invention discloses a method for preparing a cyanine dye mediated nucleic acid/anticancer drug compound, which is simple, has easily obtained raw materials and wide universality. The anticancer chemotherapeutic medicine, such as doxorubicin hydrochloride, can effectively inhibit DNA transcription, induce apoptosis, induce immunogenic death of organism, and activate immune response. The functional nucleic acid, such as CPG, can promote the maturation of antigen presenting cells, enhance the antigen presenting ability and further enhance the immune response of the body.
The invention designs a nucleic acid/anticancer drug compound with synergistic effect of chemotherapy and immunotherapy by a simple method, can realize effective antitumor therapy, and can provide good reference for preparation of nucleic acid drugs as a universal method.
Drawings
FIG. 1 is a reaction scheme for example preparation of Cy 5-CPG/DOX.
FIG. 2 shows a transmission electron micrograph (a), a scanning electron micrograph (b), a particle size distribution (c) and a potential change plot (d) of Cy5-CPG/DOX prepared in example.
FIG. 3 is a graph showing the results of performance tests of Cy5-CPG/DOX prepared in examples, wherein (a) is the results of the cycling stability test of Cy5-CPG/DOX, (b) is the graph showing the release of Cy5-CPG/DOX under different environments, (c) is the graph showing the apoptosis induced by Cy5-CPG/DOX and Cy5-CPG, DOX, and (d) is the graph showing the endocytosis rate of Cy5-CPG/DOX and Cy5-CPG, DOX.
FIG. 4 is a graph showing that Cy5-CPG/DOX prepared in the example induces cellular immune response.
Detailed Description
In order to make the present invention more comprehensible, the technical solutions of the present invention are further described below with reference to specific embodiments, but the present invention is not limited thereto.
Examples
Referring to FIG. 1, cyanine dye-mediated nucleic acid/anticancer drug complexes were prepared, using cyanine dye-modified functional nucleic acid Cy5-CPG and doxorubicin hydrochloride (DOX) assembly as an example; the preparation method comprises the following specific steps:
(1) dissolving 10 OD Cy5-CPG (purchased from Shanghai bioengineering Co., Ltd.) in 527 μ L of sterile water, blowing and beating at room temperature in the absence of light to disperse nucleic acid uniformly to form 100 μ M Cy5-CPG stock solution, and storing in the absence of light at-20 deg.C;
(2) dispersing 57.9 mg of doxorubicin hydrochloride (DOX, purchased from sigam) in 10 mL of secondary water at room temperature in a dark place, fully and uniformly dispersing to obtain 10 mM DOX stock solution, and storing in a dark place;
(3) fully mixing 15 mu L of Cy5-CPG stock solution, 5 mu L of DOX stock solution (the molar ratio of Cy5-CPG to DOX is 1: 30) and 300 mu L of secondary water, then placing the mixture in a metal bath, reacting for 60 min at 95 ℃, then cooling to room temperature, taking out a sample, centrifuging for 5 min at 8000 rpm, and washing twice with water to obtain Cy 5-CPG/DOX.
FIG. 2 shows a transmission electron microscope (a), a scanning electron microscope (b), a particle size distribution chart (c) and a potential change chart (d) of the prepared Cy 5-CPG/DOX. As can be observed from the figure, the nanoparticles formed by compounding Cy5-CPG and doxorubicin hydrochloride have uniform size, the particle size is intensively distributed at 200 nm, and the surface charge of the nanoparticles is-32.3 mV.
Performance test
1. A Cy5-CPG/DOX sample of 500 mu g/mL is taken and respectively mixed with PBS of 1 mL, serum (FBS) of 5% and cell lysate, the mixture is incubated at 37 ℃ for different times, reaction liquid is taken at regular intervals, 100 mu l of supernatant is taken after centrifugation at 8000 rpm for 3 min, and the fluorescence peak at 593 nm is measured by a microplate reader.
2. The breast cancer cell line 4T1 was used as a validation model. 4T1 cells were seeded in 6-well plates, incubated for 24 h, and washed three times with PBS. The cells were lysed and the supernatants were centrifuged to 1 mL each. Then using PBS buffer and H 2 O 2 The pH was adjusted to give a mixed solution of cell lysate with pH 7.4 and 5.0. Respectively adding 500 mu g/mL Cy5-CPG/DOX, incubating for different times, taking reaction liquid at regular intervals, centrifuging at 8000 rpm for 3 min, taking 100 mu L supernatant, and measuring the fluorescence peak at 593 nm by using an enzyme-linked immunosorbent assay.
3. The breast cancer cell line 4T1 was used as a validation model. 4T1 cells were seeded in 96-well plates, incubated for 24 h, and washed three times with PBS. 100. mu.L of medium containing different concentrations (0, 50, 100, 150, 200, 250, 300. mu.g/ml) of each compound (Cy 5-CPG, DOX, Cy 5-CPG/DOX) was added and incubated for 24 h. Then, the supernatant was washed three times with PBS, 100. mu.L of a medium containing CCK-8 (Cell Counting Kit-8, Byunyan day) was added thereto, the mixture was incubated for 1 hour, and the absorbance at 450 nm was measured with a microplate reader.
4. The breast cancer cell line 4T1 was used as a validation model. 4T1 cells were seeded in 6-well plates, incubated for 24 h, and washed three times with PBS. 1 mL of a medium containing 300. mu.g/mL of each compound (Cy 5-CPG, DOX, Cy 5-CPG/DOX) was added thereto, and the mixture was incubated for 12 hours. The cells were then washed with PBS and harvested by centrifugation. Cells were lysed and the supernatant was centrifuged to determine the peak fluorescence at 593 nm.
5. The breast cancer cell line 4T1 was used as a validation model. 4T1 cells were seeded in a fluorescent plate, incubated for 24 h, and washed three times with PBS. Then, 1 mL of a medium containing 300. mu.g/mL Cy5-CPG/DOX was added thereto, and incubated for 6 hours. Cells were washed three times with PBS, and then fixed for 10 min by adding 4% tissue fixative. Then, antibodies containing calreticulin were added and incubated for 30 min, and further, FITC-labeled IgG was incubated for 60 min. Expression of calreticulin exposed by immunogenic death of cells was determined by confocal microscopy.
FIG. 3 is a graph showing the results of performance tests, wherein (a) shows the results of the in vitro cycling stability of Cy5-CPG/DOX, which shows that Cy5-CPG/DOX can be preserved in PBS for a longer time due to its better binding power; in the serum, although Cy5-CPG/DOX shows certain DOX release performance, the retention time is longer, so that the blood circulation stability is better; in the cell lysate mixed solution, Cy5-CPG/DOX releases more DOX, which shows that the nanoparticles can be released in cells, and the released medicine can better induce apoptosis.
(b) Is a graph of the release of Cy5-CPG/DOX in different intracellular environments. As can be seen from the figure, under the tumor microenvironment condition of pH =5, Cy5-CPG/DOX can be better released, showing a certain weak acid responsiveness.
(c) And (d) Cy5-CPG/DOX and Cy5-CPG, DOX, respectively, induced apoptosis and endocytosis. As can be observed in the figure, the nanoparticles formed by Cy5-CPG/DOX have better endocytosis and show the optimal cell killing effect.
FIG. 4 is a graph showing that Cy5-CPG/DOX induces cellular immune response. It can be observed that DOX can induce apoptosis, causing immunogenic death and thus leakage of calreticulin.
The experiments show that the prepared Cy5-CPG/DOX can be well endocytosed by cells, DOX is released in a tumor cell microenvironment to induce apoptosis of the cells, and the apoptotic cells can be used as antibodies to cause immunogenic death. CPG is used as an adjuvant for stimulating antigen presenting cells, can activate the antigen presenting cells to enhance antigen presentation, further synergistically enhance the immune response of an organism, and show stronger antitumor activity.
In a word, the invention provides a preparation method of a cyanine dye-mediated nucleic acid/adriamycin composite drug, which introduces cyanine fluorescent molecules into a self-assembly process of nucleic acid for the first time, and has the advantages of simple method and high drug loading rate. Meanwhile, the prepared compound medicine has good blood circulation stability and weak acid responsiveness, and the GSH and H are over-expressed in a tumor region in vivo 2 O 2 Is easy to degrade under the condition of (1), and can slowly release DOX and CPG. More importantly, DOX can induce cell immunogenic death, and combines with immune adjuvant CPG to wake up the immune response of the organism, thereby showing higher anti-tumor activity.
The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.
Claims (6)
1. A method for preparing cyanine dye mediated nucleic acid/anticancer drug compound is characterized in that: self-assembling the cyanine dye modified functional nucleic acid and the anticancer chemotherapeutic drug into a novel functional nucleic acid/anticancer drug compound with uniform size and high loading rate in an aqueous solution; the method specifically comprises the following steps:
(1) preparing a cyanine dye modified functional nucleic acid stock solution: dissolving the cyanine dye modified functional nucleic acid in sterile water, and blowing and beating the solution uniformly at room temperature in a dark place to form a uniform cyanine dye modified functional nucleic acid stock solution;
(2) preparing a drug stock solution: dissolving the anticancer chemotherapeutic drug in a solvent, and uniformly mixing at room temperature in a dark condition to obtain a drug stock solution;
(3) preparation of nucleic acid/anticancer drug complexes: uniformly mixing a cyanine dye modified functional nucleic acid stock solution and a drug stock solution, placing the mixture in a metal bath, reacting for 1h at 95 ℃, then cooling to room temperature, centrifuging a product, and washing twice with water to prepare the nucleic acid/anticancer drug compound;
the anticancer chemotherapeutic drug is zorubicin hydrochloride, doxorubicin hydrochloride or structural analogues and derivatives of doxorubicin hydrochloride.
2. The method of claim 1, wherein the nucleic acid/anticancer drug complex is prepared by the following steps: the concentration of the cyanine dye modified functional nucleic acid stock solution obtained in the step (1) is 100 mu M.
3. The method for preparing a cyanine dye-mediated nucleic acid/anticancer drug complex according to claim 1, which comprises: the solvent in the step (2) comprises any one of water and dimethyl sulfoxide.
4. The method of claim 1, wherein the nucleic acid/anticancer drug complex is prepared by the following steps: the concentration of the drug stock solution obtained in the step (2) is 10 mM.
5. The method of claim 1, wherein the nucleic acid/anticancer drug complex is prepared by the following steps: the mole ratio of the cyanine dye modified functional nucleic acid to the anticancer chemotherapeutic drug is 1: 30.
6. The method of claim 1, wherein the nucleic acid/anticancer drug complex is prepared by the following steps: the rotation speed of the centrifugation in the step (3) is 8000 rpm, and the time is 5 min.
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