CN114949247B - Hybrid nanoparticle capable of stably loading DNA and preparation method and application thereof - Google Patents

Hybrid nanoparticle capable of stably loading DNA and preparation method and application thereof Download PDF

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CN114949247B
CN114949247B CN202210473633.9A CN202210473633A CN114949247B CN 114949247 B CN114949247 B CN 114949247B CN 202210473633 A CN202210473633 A CN 202210473633A CN 114949247 B CN114949247 B CN 114949247B
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CN114949247A (en
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孟英才
黄嘉鑫
周文虎
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Central South University
Xiangya Hospital of Central South University
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Xiangya Hospital of Central South University
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Abstract

The invention provides a hybrid nanoparticle capable of stably loading DNA, a preparation method and application thereof, wherein the hybrid nanoparticle is prepared from Mn 2+ Self-assembling with DNA in sodium hydroxide solution, mn 2+ Oxidizing to form manganese dioxide nanoparticles, forming nanoparticles and nanosheet hybridized nanoparticles by the manganese dioxide nanoparticles under the control of the DNA, wherein the DNA is stably loaded in the hybridized nanoparticles, and one end of the DNA is a Poly T DNA fragment, and the other end of the DNA is a double-block DNA of functional DNA; the preparation method of the hybrid nanoparticle comprises the following steps: mixing manganese chloride solution with a certain concentration with DNA, adding a certain amount of sodium hydroxide solution, immediately mixing, performing ultrasonic treatment for several minutes, and finally centrifuging to collect nanoparticles. The preparation method of the hybrid nanoparticle is simple, and the capacity of the loaded DNA for resisting phosphate or serum replacement is stronger; the method is used for constructing DNA functionalized nano particles, can realize stable modification of the nano particles, can realize targeting of tumors, and can be used for delivering nucleic acid medicines and small molecular medicines.

Description

Hybrid nanoparticle capable of stably loading DNA and preparation method and application thereof
Technical Field
The invention relates to the field of nano materials and nano biological medicines, in particular to a hybrid nanoparticle capable of stably loading DNA, a preparation method and application thereof.
Background
Tumors are a global significant public health problem with high incidence and mortality. Most patients are not treated by conventional methods to achieve good results. Thus, development of new antitumor technologies remains the main subject of current scientific researchThe direction is desired. With the increasing understanding of tumor biology, it is widely accepted that tumor-fast growing cells and incomplete vasculature can induce a microenvironment (TME) that produces hypoxia, trace acids and high levels of hydrogen peroxide. This is also an important factor in reducing the efficacy of the drug and in developing resistance. With the development of nanotechnology, TME response-based nanosystems have demonstrated great potential for tumor therapy. For example, nanomaterials with peroxidase activity can catalyze high levels of H in TME 2 O 2 Generates cytotoxic ROS and realizes the treatment of tumor.
Nanometer manganese dioxide (MnO) 2 ) Is an ideal TME responsive nano material, and can decompose H 2 O 2 Is O 2 Relieving tumor hypoxia, or degrading into Mn by GSH 2+ MRI imaging and chemo-kinetic treatment are achieved. Therefore, the method has great application value in the diagnosis and treatment of tumors. To achieve biomedical applications, nanomaterials often require better biological stability and can be further modified functionally. However, mnO 2 Limited by its nature, lacks chemical reaction sites and is therefore more difficult to modify and modify.
Biomineralization is an important method of building organic/inorganic hybrid materials, such as tooth, bone formation, by depositing inorganic components on an organic substrate. The mineralized nano material of many DNA templates is developed by simulating the biomineralization process, and has the advantages of good biocompatibility, unique bioactivity, structural controllability and the like. Thus, the biomineralization method is used to construct functionalized MnO 2 The nanoparticle has important significance for biomedical application, and explores the stability of functional modification.
Disclosure of Invention
In order to solve the technical problems, the invention provides a hybridized nano-particle capable of stably loading DNA, a preparation method and application thereof, and aims to construct a nano-material capable of stably loading functionalized DNA through a biomineralization method, and control and construct hybridized MnO through the functionalized DNA 2 The nanoparticle is stable in DNA loading, realizes stable modification of the nanoparticle, and can realize swelling of the hybrid nanoparticleTumor targeting and drug delivery, enhancing anti-tumor efficacy.
In order to achieve the above object, the present invention provides, first, a hybrid nanoparticle stably loaded with DNA, which is composed of Mn 2+ Self-assembly with DNA in sodium hydroxide solution, mn 2+ Oxidizing to form manganese dioxide nanoparticles, wherein the manganese dioxide nanoparticles form nanoparticles and nanosheet hybridized nanoparticles under the control of the DNA, the DNA is stably loaded in the hybridized nanoparticles, and one end of the DNA is a Poly T DNA fragment, and the other end of the DNA is a double-block DNA of functional DNA.
Preferably, the functional DNA is nucleolin receptor targeted AS1411; the double-block DNA composed of AS1411 with one end being poly T DNA and the other end being nucleolin receptor targeting is designed for constructing DNA functional nano-particles, thus realizing the high-efficiency targeting of tumors.
Preferably, the sequence of the DNA is shown in SEQ ID NO. 1.
Preferably, the Mn 2+ The manganese chloride is manganese chloride, and the molar concentration of the manganese chloride is 5-10 mM.
Preferably, the molar concentration of sodium hydroxide is 50 to 100mM.
Preferably, the molar concentration of the DNA is 0.1 to 10. Mu.M.
Based on a general inventive concept, the present invention also provides a method for preparing hybrid nanoparticles capable of stably loading DNA, comprising the steps of:
s1, respectively preparing a manganese chloride solution, a DNA solution and a sodium hydroxide solution;
s2, mixing a manganese chloride solution with the DNA solution, then adding a sodium hydroxide solution, mixing and carrying out ultrasonic treatment, and centrifugally collecting to obtain the hybrid nanoparticle.
Preferably, the ultrasonic power in the step S2 is 100-300W, and the ultrasonic time is 3-10 min.
Based on a general inventive concept, the invention also provides an application of the hybridized nanoparticle capable of stably loading DNA in preparing a nano drug delivery system for tumor targeted therapy.
Preferably, the nano drug delivery system is a hybrid nanoparticle combined photosensitizer Ce6 capable of stably loading DNA, and is applied to preparation of tumor targeted photodynamic therapy drugs.
Compared with the prior art, the invention has the following beneficial effects:
1. the invention provides a hybrid nanoparticle capable of stably loading DNA, which is used for constructing DNA functionalized nanoparticles by designing two-block DNA with one end being a Poly T DNA fragment and the other end being functional DNA, and realizing tumor targeting and drug delivery of the nanoparticles by the functional modified DNA; the hybrid nanoparticle can stably load Poly T DNA, realizes stable modification of the nanoparticle, and has stronger capability of resisting phosphate or serum replacement compared with a loading mode of direct surface adsorption.
2. The hybridization nanoparticle capable of stably loading DNA provided by the invention can mix Mn 2+ Oxidation to form nanoparticles in alkaline environment, DNA control and construction of hybrid MnO 2 Nanoparticles (DNA-MnO) 2 ) MnO whose DNA can prevent formation 2 The nano particles are agglomerated, the formed hybrid nano particles are nano particles with small particle size and grow on a sheet-shaped structure, the structure is stable, and the loading performance is good.
3. The hybridized nano particle capable of stably loading DNA provided by the invention can respond to tumor microenvironment, degrade and release Mn 2+ For use in chemical kinetic therapy; meanwhile, the nanoparticle can be used for delivering nucleic acid medicaments and small molecular medicaments, and can play a synergistic anti-tumor effect by combining the therapeutic effect of the carried medicaments.
4. After the DNA-stably-loaded hybrid nanoparticle is loaded with Ce6, the functional hybrid nanoparticle can target tumors and exert a strong chemical-photodynamic combined anti-tumor effect, so that the functional hybrid nanoparticle has great application value in the field of tumor treatment.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 shows DNA functionalized MnO in example 1 of the present invention 2 The construction and particle size and morphology of the nanoparticles are characterized, and FIG. 1A shows the relationship between the DNA concentration and the particle size of the nanoparticles; FIG. 1B is a graph showing the relationship between the DNA length and the nanoparticle size; FIG. 1C is a relationship between DNA base type and nanoparticle size; FIG. 1D is a TEM characterization of DNA construct nanoparticles of different base types;
FIG. 2 is a diagram showing the case of the various competing ligand-induced release of DNA in the nanoparticle according to example 2 of the present invention, and FIG. 2A is a diagram showing the case of complementary DNA-MnO 2 The release of DNA in the nanoparticles; FIG. 2B shows the induction of DNA-MnO by different nucleosides 2 The release of DNA in the nanoparticles; FIG. 2C is a phosphate-inducible DNA-MnO 2 The release of DNA in the nanoparticles; FIG. 2D is a serum-induced DNA-MnO 2 The release of DNA in the nanoparticles; FIG. 2E is a phosphate-inducible DNA-MnO 2 Nanoparticles and DNA/MnO 2 The release of DNA; FIG. 2F is a serum-induced DNA-MnO 2 Nanoparticles and DNA/MnO 2 The release of DNA;
FIG. 3 shows construction of DNA functionalized MnO according to example 3 of the present invention 2 Schematic representation of nanoparticles and their characterization, FIG. 3A is a schematic representation of the construction of DNA functionalized MnO 2 Nanoparticles (TA-MnO) 2 Schematic representation of/Ce 6); FIG. 3B is a DNA functionalized MnO 2 Particle size distribution of nanoparticles; FIG. 3C shows the release of DNA from phosphate or serum-induced nanoparticles; FIG. 3D is Ce6, TA-MnO 2 And TA-MnO 2 Ultraviolet absorption spectrum of/Ce 6; FIG. 3E is a SOSG probe detecting the release of ROS from nanoparticles; FIG. 3F shows the degradation of MB by different nanoparticles; FIG. 3G is a graph showing TA-MnO after GSH treatment at various incubation times 2 Conditions in which Ce6 degrades MB;
FIG. 4 is an in vitro characterization of the antitumor Activity of DNA functionalized nanoparticles in example 4 of the present invention, FIG. 4A is TA-MnO 2 And TA/MnO 2 Toxicity to B16 cells; FIG. 4B is a schematic diagram of TA-MnO 2 And TA/MnO 2 IC50 of (a); FIG. 4C is TA-MnO 2 Laser irradiation/Ce 6 plus TA/MnO 2 Toxicity of Ce6 plus laser irradiation to B16 cells; FIG. 4D is TA-MnO 2 Laser irradiation/Ce 6 plus TA/MnO 2 IC50 of Ce6 plus laser irradiation; FIG. 4E shows TA-MnO at various concentrations 2 Imaging characterization of Ce6 and laser irradiation induced apoptosis; FIG. 4F shows TA-MnO at various concentrations 2 Flow characterization of apoptosis induced by Ce6 plus laser irradiation.
FIG. 5 is an in vivo characterization of the anti-tumor activity of DNA functionalized nanoparticles of example 5 of the present invention, FIG. 5A is a graph of tumor growth at various nanoparticle treatments; FIG. 5B is a percent tumor growth inhibition; fig. 5C is a tumor weighing and photographing after treatment; FIG. 5D shows the Ki-67 expression of tumors analyzed by immunohistochemistry; FIG. 5E is an H & E staining analysis of tumor tissue.
FIG. 6 shows the change in body weight of tumor-bearing mice during tumor treatment according to the nanoparticle of example 6 of the present invention.
Detailed Description
In order to make the technical problems, technical solutions and advantages to be solved more apparent, the following detailed description will be given with reference to the accompanying drawings and specific embodiments.
The following examples are illustrative of the invention and are not intended to limit the scope of the invention. Modifications and substitutions to methods, procedures, or conditions of the present invention without departing from the spirit and nature of the invention are intended to be within the scope of the present invention.
The technical means used in the examples are conventional means well known to those skilled in the art unless otherwise indicated; the reagents used in the examples were all commercially available unless otherwise specified.
The percentage "%" referred to in the present invention refers to mass percent unless otherwise specified; however, the percentage of the solution, unless otherwise specified, refers to the grams of solute contained in 100ml of solution.
The parts by weight of the present invention may be those known in the art such as mu g, mg, g, kg, or may be multiples thereof such as 1/10, 1/100, 10 times, 100 times, etc.
In the following examples, details of the instruments and manufacturers used are shown in Table 1:
table 1 Main Instrument names and manufacturers
In the following examples, the names of the main reagents used and the manufacturers are shown in Table 2:
TABLE 2 Main reagent names and manufacturers
Example 1
DNA functionalized MnO 2 Nanoparticles (DNA-MnO) 2 ) Construction and particle size, morphology characterization:
DNA-MnO of this example 2 The preparation method comprises the following steps:
(1) Preparing sodium hydroxide solution: 80mg of sodium hydroxide is dissolved in 20mL of ultrapure water to obtain 100mmol/L of sodium hydroxide solution for later use.
(2) Preparing a DNA solution: 0.05. Mu. Mol of DNA was dissolved in 500. Mu.L of HEPES buffer (pH 7.4) at 5mmol/L to give 100. Mu. Mol/L of DNA solution for use.
(3) Preparing a manganese chloride solution: 39.6mg of manganese chloride tetrahydrate is dissolved in 20mL of ultrapure water to obtain 10mmol/L of manganese chloride solution for later use.
(4)DNA-MnO 2 Is prepared from the following steps: 36.7. Mu.L of a manganese chloride solution of 10mmol/L and 3.5. Mu.L of a DNA solution of 100. Mu. Mol/L were mixed in 40. Mu.L of water and vortexed for 10s, then 20. Mu.L of a sodium hydroxide solution of 100mmol/L was added,vortex mixing and sonicate for 3min. Finally, the nanoparticles were collected by centrifugation (16000 r/min,15 min) for further use.
Measuring the hydration particle size of the nanoparticles by a Markov particle size meter, wherein the hydration particle size comprises hydration particle sizes of nanoparticles prepared by different DNA concentrations, hydration particle sizes of nanoparticles prepared by different DNA lengths and hydration particle sizes of nanoparticles prepared by different base types of DNA; characterization of MnO by TEM 2 、A 20 -MnO 2 、T 20 -MnO 2 、C 20 -MnO 2 And G 20 -MnO 2 The results are shown in FIG. 1, and FIG. 1A shows the relationship between the DNA concentration and the particle size of the nanoparticles; FIG. 1B is a graph showing the relationship between the DNA length and the nanoparticle size; FIG. 1C is a relationship between DNA base type and nanoparticle size; FIG. 1D is a TEM characterization of DNA construct nanoparticles of different base types.
As can be seen from fig. 1: DNA-MnO prepared by the present invention 2 The hydration particle size of the nanoparticle decreases with increasing DNA concentration, and MnO when DNA is absent 2 Aggregation occurs, and the hydration particle size is obviously increased; the hydration particle size of the nanoparticles gradually decreases along with the increase of the length of the DNA; a is that 20 -MnO 2 、T 20 -MnO 2 And C 20 -MnO 2 The hydration particle size of G is not greatly different 20 -MnO 2 The hydration particle size of (2) is relatively large; TEM results show that: mnO (MnO) 2 In the form of a sheet, A 20 -MnO 2 、T 20 -MnO 2 、C 20 -MnO 2 And G 20 -MnO 2 Not only the sheet structure but also a plurality of small-sized nanoparticles, wherein the small-sized nanoparticles grow on the sheet structure to form a hybrid nanostructure.
The results show that the method provided by the invention can successfully prepare DNA-MnO 2 And (3) hybridized nano particles.
Example 2
The release of DNA in the nanoparticles was examined for various competing ligands.
Hybrid DNA-MnO 2 Prepared by the method of example 1, the DNA used included: FAM-labeled A 20 、T 20 、C 20 And G 20 。
Physically adsorbed DNA/MnO 2 Is prepared from the following steps: adding 40. Mu.L of manganese chloride solution at 10mmol/L and 40. Mu.L of water into an EP tube, vortexing for 10s, then adding 20. Mu.L of sodium hydroxide solution at 100mmol/L, vortexing, mixing, sonicating for 3min, centrifuging (16000 r/min,15 min) and collecting MnO 2 NSs, then dispersed into HEPES buffer. MnO is added to 2 NSs and 3.5. Mu.M FAM-labeled T 20 Incubating at 4deg.C for 1 hr, centrifuging (16000 r/min,15 min), and washing to obtain physical adsorbed T 20 /MnO 2 And (5) standby.
Release of DNA: mu.L of DNA-MnO was added to the mixture 2 Or DNA/MnO 2 Mix with 150. Mu.L of each ligand and then detect fluorescence recovery in 60min by a microplate reader. The competing ligand included 1. Mu.M DNAs (A 20 ,T 20 ,C 20 And G 20 ) 1mM nucleoside, 1mM phosphate and 10% serum.
The results are shown in FIG. 2, wherein FIG. 2A is a complementary DNA-induced DNA-MnO 2 The release of DNA in the nanoparticles; FIG. 2B shows the induction of DNA-MnO by different nucleosides 2 The release of DNA in the nanoparticles; FIG. 2C is a phosphate-inducible DNA-MnO 2 The release of DNA in the nanoparticles; FIG. 2D is a serum-induced DNA-MnO 2 The release of DNA in the nanoparticles; FIG. 2E is a phosphate-inducible DNA-MnO 2 Nanoparticles and DNA/MnO 2 The release of DNA; FIG. 2F is a serum-induced DNA-MnO 2 Nanoparticles and DNA/MnO 2 In DNA release. As can be seen from fig. 2: hybrid nanoparticle DNA-MnO 2 DNA of four base compositions showed little release, indicating DNA-MnO 2 Can stably load DNA and resist replacement of complementary DNA. Then, DNA substitution experiments were performed with four different bases, and as a result, no significant release of DNA was observed, less than 10%, indicating that DNA was released with MnO 2 Is strong enough to resist displacement of nucleosides. Subsequently, a phosphate displacement experiment was performed, and as a result, four kinds of DNA were released to different extents, and the following relationship was found for the percent release: g 20 >C 20 ≈A 20 >T 20 It is demonstrated that DNA is loaded into nanoparticles primarily through phosphate backbone interactions, where T 20 The loading stability of (2) is the strongest. Next, substitution experiments with serum were performed, and as a result, four kinds of DNA were also released to different extents, and the relationship of the release percentages was: g 20 >C 20 >A 20 >T 20 The results also indicate T 20 The loading stability of (2) is the strongest. Due to T 20 Most of the nanoparticles are loaded and only a small portion is released, so it is presumed that it may be loaded with nanoparticles by surface adsorption and internal encapsulation. Comparative DNA-MnO 2 And DNA/MnO 2 The release of DNA from the nanoparticles in phosphate and serum is known as DNA-MnO 2 DNA release amount ratio of nanoparticles to DNA/MnO formed by physical adsorption 2 The amount of released DNA was much smaller, indicating DNA-MnO 2 The nanoparticle structure can load DNA more stably, so that the targeting delivery of tumor cells can be better realized.
The above indicates T 20 Can be stably loaded in nanoparticles and resist replacement of various competing ligands, and can be used for MnO 2 Is functionally related to the DNA of the subject.
Example 3
Construction of DNA functionalized MnO 2 Schematic representation of nanoparticles and characterization thereof
Based on the research result of the nanoparticle on DNA loading stability, a double-block DNA with one end being Poly T and the other end being AS1411 is designed, and is abbreviated AS TA, and the sequence number is shown AS SEQ ID NO. 1.
TA-MnO was prepared as in example 1 using TA as the functionalized DNA 2 The method comprises the steps of carrying out a first treatment on the surface of the TA-MnO 2 Incubating with 1mmol/L Ce6, and centrifuging to collect nanoparticles to obtain TA-MnO 2 and/Ce 6, for use.
Measuring the hydration particle size of the nanoparticles by using a Markov particle size meter; the release of TA in the presence of phosphate or serum was examined by the method of example 1; and measuring ultraviolet spectra before and after the nano-particles carry Ce 6.
In vitro ROS generation: 160. Mu.L of TA-MnO 2 Ce6 (Ce 6 2. Mu.g/mL) was mixed with 20. Mu.L of 25. Mu.M SOSG solution, then 20. Mu.L of 100mM hydrogen peroxide solution was added and mixed with 635nm,0.1W/cm 2 For 5min, and the fluorescence of SOSG was measured by an enzyme-labeled instrument.The control group is buffer solution, TA-MnO 2 Free Ce6, TA-MnO 2 Laser irradiation was applied to/Ce 6 and free Ce 6.
MB degradation: 30 μg TA-MnO 2 Ce6 was incubated with 250. Mu.L of 10mM GSH for 5min, then centrifuged and the supernatant was taken and added 475. Mu.L of 25mM NaHCO 3 /5％CO 2 Buffer solution (10. Mu.g/mL MB and 8mM H) 2 O 2 ) Then incubating at 37 ℃ for 0 to 60min, and measuring the ultraviolet absorption spectrum of the solution between 400 and 800 nm. Wherein control group, H 2 O 2 Group and TA-MnO 2 Ce6 plus H 2 O 2 After 60min incubation, the uv absorbance spectrum was determined.
The results are shown in FIG. 3, where FIG. 3A is a schematic diagram of the construction of DNA functionalized MnO 2 Nanoparticles (TA-MnO) 2 Schematic representation of/Ce 6); FIG. 3B is a DNA functionalized MnO 2 Particle size distribution of nanoparticles; FIG. 3C shows the release of DNA from phosphate or serum-induced nanoparticles; FIG. 3D is Ce6, TA-MnO 2 And TA-MnO 2 Ultraviolet absorption spectrum of/Ce 6; FIG. 3E is a SOSG probe detecting the release of ROS from nanoparticles; FIG. 3F is a graph showing the degradation of MB by various nanoparticles; FIG. 3G is a graph showing TA-MnO after GSH treatment at various incubation times 2 Conditions in which Ce6 degrades MB; as can be seen from fig. 3: the invention constructs TA-MnO by TA 2 Ce6, the particles are distributed in nano-scale, and TA can be stably loaded in the nanoparticles and resist the replacement of phosphate or serum; TA-MnO 2 Ce6 exhibits a characteristic absorption peak at 660nm similar to Ce6, and this characteristic peak is found in TA-MnO 2 None of them indicates that Ce6 was successfully loaded in the nanoparticles. Detection of ROS production by nanoparticles in vitro by SOSG, only free Ce6 plus laser irradiation group and TA-MnO 2 the/Ce 6 plus laser irradiation group showed the generation of large amounts of ROS. MB degradation results showed that TA-MnO treated with GSH 2 Ce6 can degrade and fade MB, and after 60min incubation, almost completely fade, while other groups do not cause MB degradation and fade, indicating Mn release after nanoparticle degradation by GSH 2+ MB degradation is discolored by the Fenton reaction.
The above shows that TA-MnO constructed according to the present invention 2 Ce6 loaded TA resistant to phosphorusReplacement of acid salts or serum; ROS can be generated under the irradiation of an in vitro laser; after being degraded by GSH, the chemical kinetics can be exerted; therefore, the nanoparticle can be used for chemical-photodynamic combination treatment of tumors.
Example 4
In vitro characterization of the anti-tumor Activity of DNA functionalized nanoparticles
In vitro cytotoxicity evaluation: b16 cells were seeded in 96-well cell culture plates (5×10 per well) 3 Individual cells), culturing at 37deg.C, and replacing cell culture medium with TA/MnO containing various concentrations after cells are grown completely by adherence 2 、TA-MnO 2 、TA/MnO 2 Ce6 or TA-MnO 2 Culture medium of Ce6, TA/MnO 2 Ce6 and TA-MnO 2 The Ce6 group was incubated for 6h at 0.1W/cm 2 Laser irradiation was performed for 1 min. Then incubating the cells for 48h, removing the culture medium, washing with PBS, adding 100 mu L of the culture medium containing MTT into each well, continuously incubating for 4h, removing the culture medium, dissolving formazan crystals by using DMSO, measuring ultraviolet absorption at 490nm by using a microplate reader, and calculating the cell survival rate.
Apoptosis imaging: inoculating B16 cells into confocal dish, culturing overnight at 37deg.C, and replacing cell culture medium with TA-MnO with different concentration after cells are grown completely 2 Fresh medium of/Ce 6, after incubation for 6h, laser irradiation was performed. Incubation was then continued for 48h, medium was removed, washed twice with PBS, stained with Magic Red and Hoechst 33342, and cells were subsequently imaged with laser confocal.
Flow analysis: inoculating B16 cells into 24-well cell culture plate, culturing overnight at 37deg.C, and replacing cell culture medium with TA-MnO containing different concentrations after cells are grown completely by adherence 2 Fresh medium of/Ce 6, after incubation for 6h, laser irradiation was performed. Then, the incubation was continued for 48 hours, the medium was removed, washed twice with PBS, and the cells were collected and, after incubation with Annexin V-FITC and PI, flow cytometry was performed.
The results are shown in FIG. 4, wherein FIG. 4A is TA-MnO 2 And TA/MnO 2 Toxicity to B16 cells; FIG. 4B is a schematic diagram of TA-MnO 2 And TA/MnO 2 Is of (2) 50 The method comprises the steps of carrying out a first treatment on the surface of the FIG. 4C is TA-MnO 2 Laser irradiation/Ce 6 plus TA/MnO 2 Toxicity of Ce6 plus laser irradiation to B16 cells; FIG. 4D is TA-MnO 2 Laser irradiation/Ce 6 plus TA/MnO 2 IC50 of Ce6 plus laser irradiation; FIG. 4E shows TA-MnO at various concentrations 2 Imaging characterization of Ce6 and laser irradiation induced apoptosis; FIG. 4F shows TA-MnO at various concentrations 2 Flow characterization of apoptosis induced by Ce6 plus laser irradiation. As can be seen from fig. 4: with increasing nanoparticle concentration, the survival rate of B16 cells gradually decreases, and TA-MnO 2 Cell viability of the group was significantly lower than TA/MnO 2 Cell viability in the group, resulting in lower IC50 values, probably due to TA stable modification of TA-MnO 2 Endows the nucleolin-mediated tumor active targeting function. In contrast, TA-MnO loaded with Ce6 and given laser irradiation 2 Cell viability was lower in the/Ce 6 group while TA-MnO 2 Cell viability with/Ce 6 plus laser irradiation was also significantly lower than TA/MnO 2 Cell viability of/Ce 6 plus laser irradiation also resulted in lower IC50 values. Thus, TA-MnO 2 The Ce6 plus laser irradiation has a strong effect of inhibiting the growth of tumor cells, which can be attributed to the combined effect of nucleolin receptor-mediated tumor targeted delivery and chemo-photodynamic therapy. Through Magic Red staining and imaging and flow apoptosis analysis, it can be seen that the TA-MnO is followed 2 Increasing concentration of/Ce 6, apoptotic cells also gradually increased.
The above shows that the TA-MnO provided by the invention 2 The Ce6 has strong effect of inhibiting the growth of tumor cells and can be used for the chemical-photodynamic combined treatment of tumors.
Example 5
In vivo characterization of the anti-tumor Activity of DNA functionalized nanoparticles
Subcutaneously inoculating B16 cells into C57BL/6 mice until tumor grows to about 100mm 3 At the time, tumor-bearing mice were randomly divided into 5 groups, and PBS and TA/MnO were injected into the tail vein respectively 2 、TA-MnO 2 、TA/MnO 2 Ce6 and TA-MnO 2 Ce6, injections every other day, for a total of 4 injections. Wherein TA/MnO 2 Ce6 and TA-MnO 2 Ce6 advance 24h after injectionLine laser irradiation (0.1W/cm) 2 6 min). Tumor volume was measured for 10 days of treatment, tumor growth curves were drawn and tumor growth inhibition was calculated for day 10. Mice were euthanized after 10 days and tumor tissue was removed, weighed, photographed, and subjected to Ki-67 immunohistochemical analysis and H&E staining analysis.
The results are shown in fig. 5, where fig. 5A is a graph of tumor growth at various nanoparticle treatments; FIG. 5B is a percent tumor growth inhibition; fig. 5C is a tumor weighing and photographing after treatment; FIG. 5D shows the Ki-67 expression of tumors analyzed by immunohistochemistry; FIG. 5E is H of tumor tissue&E staining analysis. As can be seen from fig. 5: tumor growth in mice injected with nanoparticles by tail vein is significantly inhibited. Wherein, TA-MnO 2 Is greater than TA/MnO 2 Shows that TA stably modifies TA-MnO 2 Endows the nucleolin-mediated in vivo tumor active targeting function. TA/MnO laser irradiation 2 Ce6 and TA-MnO 2 The tumor growth inhibition rate of/Ce 6 is further enhanced, and TA-MnO 2 The tumor growth inhibition rate of/Ce 6 plus laser irradiation is obviously higher than that of TA/MnO 2 Tumor growth inhibition ratio of/Ce 6 plus laser irradiation, shows TA-MnO 2 The Ce6 plus laser irradiation has a strong in vivo anti-tumor effect, which can be attributed to nucleolin receptor-mediated in vivo tumor targeted delivery and combined effect of chemo-photodynamic therapy. The tumor treatment can be visually compared by tumor weighing and photographing. In addition, ki-67 immunohistochemical analysis and H&E-staining analysis results all showed TA-MnO 2 The Ce6 and laser irradiation has strong anti-tumor effect.
The above shows that the TA-MnO provided by the invention 2 The Ce6 has strong in vivo anti-tumor effect and can realize the chemical-photodynamic combined treatment of in vivo tumor.
Example 6
Weight change of tumor-bearing mice during tumor treatment of nanoparticles
Tumor-bearing mice in example 5 were weighed daily for evaluation of acute toxicity of nanoparticles during treatment. The results are shown in FIG. 6: TA/MnO during treatment 2 Group, TA-MnO 2 Group, TA/MnO 2 Ce6 plus laser irradiation group and TA-MnO 2 There was no significant change in body weight of the/Ce 6 plus laser irradiated mice, indicating no significant acute toxicity of the nanoparticles during treatment.
In summary, the present invention provides a nanoparticle capable of stably loading DNA by designing a two-block DNA with one end being Poly T and the other end being AS1411 and constructing DNA functionalized MnO 2 Nanoparticles, the DNA can be stably loaded against phosphate or serum replacement, thus MnO can be realized 2 Is modified stably. Meanwhile, after Ce6 is loaded, the functionalized nanoparticle can target tumors and exert a strong chemical-photodynamic combined anti-tumor effect, so that the functionalized nanoparticle has great application value in the field of disease treatment.
The above embodiments are only preferred embodiments of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be able to apply equivalents and modifications according to the technical solution and the concept of the present invention within the scope of the present invention.
Sequence listing
<110> university of south-middle school
<120> a hybrid nanoparticle capable of stably loading DNA, and preparation method and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 1
tttttttttt tttttttttt ggtggtggtg gttgtggtgg tggtgg 46

Claims (9)

1. A hybrid nanoparticle capable of stably loading DNA, characterized in that the hybrid nanoparticle is composed of Mn 2+ Self-assembly with DNA in sodium hydroxide solution, mn 2+ Oxidizing to form manganese dioxide nanoparticles, the manganese dioxide nanoparticlesThe DNA is stably loaded in the hybridized nano particles, one end of the DNA is a Poly T DNA fragment, the other end of the DNA is a double-block DNA of functional DNA, and the functional DNA is a nucleolin receptor targeted AS1411.
2. The hybrid nanoparticle for stably loading DNA according to claim 1, wherein the DNA has a sequence shown in SEQ ID No. 1.
3. The stably loadable DNA-hybrid nanoparticle of claim 1, wherein the Mn 2+ The manganese chloride is manganese chloride, and the molar concentration of the manganese chloride is 5-10 mM.
4. The DNA stably loadable hybrid nanoparticle of claim 1, wherein the molar concentration of sodium hydroxide is 50-100 mM.
5. The hybrid nanoparticle for stably loading DNA according to claim 1, wherein the molar concentration of the DNA is 0.1 to 10. Mu.M.
6. The method for preparing the hybridized nanoparticle capable of stably loading DNA according to any one of claims 1 to 5, comprising the following steps:
s1, respectively preparing a manganese chloride solution, a DNA solution and a sodium hydroxide solution;
s2, mixing a manganese chloride solution with the DNA solution, then adding a sodium hydroxide solution, mixing and carrying out ultrasonic treatment, and centrifugally collecting to obtain the hybrid nanoparticle.
7. The preparation method according to claim 6, wherein the ultrasonic power in the step S2 is 100-300W, and the ultrasonic time is 3-10 min.
8. Use of a hybrid nanoparticle capable of stably loading DNA according to any one of claims 1 to 5 or a hybrid nanoparticle capable of stably loading DNA prepared according to any one of claims 6 to 7 in the preparation of a nanodelivery system for targeted tumor therapy.
9. The use according to claim 8, wherein the nano-drug delivery system is a hybrid nanoparticle-conjugated photosensitizer Ce6 that can stably load DNA, and the nano-drug delivery system is used in the preparation of tumor-targeted chemo-photodynamic therapeutic drugs.
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