CN114392354A - DNA nano system for tumor targeting and preparation method and application thereof - Google Patents
DNA nano system for tumor targeting and preparation method and application thereof Download PDFInfo
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- CN114392354A CN114392354A CN202210042758.6A CN202210042758A CN114392354A CN 114392354 A CN114392354 A CN 114392354A CN 202210042758 A CN202210042758 A CN 202210042758A CN 114392354 A CN114392354 A CN 114392354A
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- streptavidin
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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
- A61K47/62—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 the modifying agent being a protein, peptide or polyamino acid
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Abstract
The invention provides a DNA nano system for tumor targeting and a preparation method and application thereof, wherein the DNA nano system for tumor targeting comprises a DNA nano sheet layer, a single-stranded DNA coupled with a low pH insertion peptide, a biotin-modified single-stranded DNA and a streptavidin-functional molecule conjugate; wherein the DNA nanosheet layer, the single-stranded DNA coupled with the low-pH insertion peptide and the biotin-modified single-stranded DNA are combined in a base complementary pairing manner to form a bifunctional DNA nanosheet layer; the streptavidin-functional molecule conjugate is combined with the bifunctional DNA nano-sheet layer through the specific recognition effect of the streptavidin and the biotin to form the DNA nano-system. The DNA nano system provided by the invention has good biological safety and low toxic and side effects, can be used as a drug delivery system, can construct an artificial target on the surface of a tumor cell, achieves the aim of targeting a tumor lacking a specific target, and has potential application value in tumor treatment and diagnosis.
Description
Technical Field
The invention belongs to the field of nano-drugs, relates to a DNA nano-system and a preparation method and application thereof, and particularly relates to a DNA nano-system for tumor targeting and a preparation method and application thereof.
Background
In recent years, malignant tumors (cancers) have become one of the major diseases that endanger human health. At present, the clinical treatment of cancer mainly depends on radiotherapy, chemotherapy and surgical treatment, and the methods relieve the pain of patients to a certain extent, but have certain limitations and low cure rate. With the continuous development of nanotechnology, nanotechnology and modern medicine are continuously fused, and a cross discipline of nanomedicine is formed, and the potential application of the cross discipline relates to aspects of diagnosis, monitoring, treatment and the like of diseases. The nano-carrier is used for loading the chemotherapeutic drug, so that the damage of the chemotherapeutic drug to normal tissues and cells of a human body can be reduced, and the toxic and side effects are reduced. Meanwhile, the long circulation time of the drug molecules in the body is prolonged, and the bioavailability of the drug is improved. Various molecular targets exist in tumor cells, such as transmembrane oncogenic protein MUC1, which plays a central role in malignant transformation of tumors, glutathione overexpressed in cancer cells, and the like. Based on these specific molecules, in combination with the excellent properties of nanosystems, a variety of nanosystems for tumor targeting have been developed.
For example, CN109806402A discloses a tumor-targeted fluorescent nano-chain probe, its preparation method and application. The tumor-targeted fluorescent nano-chain probe comprises a self-assembled nano-chain serving as a carrier, and a fluorescent material and a tumor-targeted ligand which are loaded on the self-assembled nano-chain, wherein the fluorescent material and the tumor-targeted ligand are loaded on the self-assembled nano-chain through chemical crosslinking. The preparation method comprises the following steps: self-assembling the amphiphilic polypeptide to form a self-assembled nano chain; and chemically crosslinking the fluorescent material and the tumor targeting ligand to the self-assembled nano-chain respectively by using a chemical crosslinking mode. The tumor-targeted fluorescent nano-chain probe has the characteristic of near-infrared fluorescent tumor targeting, can realize high-efficiency in-vitro and in-vivo targeting of tumor cells, can overcome the difficulty that the traditional operation is difficult to monitor and guide in real time in the diagnosis of tumors and the operation navigation, and provides a visual and precise navigation tool for the operation treatment of a series of tumors such as abdominal cavity metastasis tumor and the like.
CN105288620A discloses a preparation method of gold nanoflowers for tumor targeted therapy, which comprises the steps of preparing gold nanoflowers with different sizes by controlling different reaction conditions, modifying hydrophilic polymers on the surfaces of the gold nanoflowers in situ through sulfydryl, selectively connecting different tumor targeted molecules on the hydrophilic polymers through the reaction of sulfydryl and succinimide, and connecting different numbers of antitumor drugs to the surfaces of the gold nanoflowers through stimulation-responsive acylhydrazone bonds, so that the gold nanoflowers with tumor targeted photothermal therapy and chemotherapy. The gold nanoflowers have the characteristics of near-infrared responsiveness, good stability, good biocompatibility and the like. The obtained product can meet the requirements of clinical application.
However, the existing tumor targeting nano systems provided in the prior art are still very limited, and the problems of low targeting efficiency, poor treatment effect, obvious drug resistance and the like generally exist, and the existing tumor targeting nano systems are not suitable for some tumor restraints lacking specific targets.
Therefore, how to provide a nano system for tumor targeting to improve the tumor specific targeting becomes a problem to be solved in the field.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a DNA nano system and a preparation method and application thereof, and particularly provides a DNA nano system for tumor targeting and a preparation method and application thereof.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the present invention provides a DNA nanosystem for tumor targeting comprising a DNA nanosheet layer, single-stranded DNA coupled to a low pH insertion peptide, biotin-modified single-stranded DNA, and a streptavidin-functional molecule conjugate;
wherein the DNA nanosheet layer, the single-stranded DNA coupled with the low-pH insertion peptide and the biotin-modified single-stranded DNA are combined in a base complementary pairing manner and assembled to form the bifunctional DNA nanosheet layer; the streptavidin-functional molecule conjugate is combined with the bifunctional DNA nano-sheet layer through the specific recognition effect of streptavidin and biotin to form the DNA nano-system for tumor targeting.
The invention creatively constructs a DNA nano system for tumor targeting, and the construction principle is shown in figure 1. The low pH insertion peptide (pHLIP) is a water-soluble polypeptide derived from a bacteriorhodopsin C helix, is a responsive polypeptide with a slightly acidic environment (the tumor environment is slightly acidic, the pH is below 6.5; and the normal physiological environment pH is 7.4), can be spontaneously inserted into a cell membrane through conformation transformation (alpha-helix conformation formation) under the slightly acidic condition, and is not internalized by cells. pHLIP and a DNA single chain can be coupled through covalent interaction, and the DNA single chain can guide the DNA single chain to hybridize at a specific site of a DNA nano plane structure, so that a two-dimensional DNA plane can be embedded into the surface of a tumor cell. The other side of the two-dimensional DNA nano-plane structure is hybridized with a DNA chain of the modified biotin (the DNA chain of the modified biotin is easy to obtain; and the DNA single chain can guide the hybridization at a specific site of the DNA nano-plane structure). Since biotin can be specifically bound with streptavidin, when pHLIP is embedded into a cell membrane under slightly acidic conditions, the biotin sites on the other side of the two-dimensional DNA nano-planar structure are exposed to serve as artificial targets for recruitment of streptavidin-functional molecule conjugates. The functional molecule can be adjusted as required, for example, by modifying a photosensitizer to achieve targeted photothermal therapy, or by modifying a fluorescent dye to achieve tumor imaging. The invention constructs a DNA nano system by taking the nucleic acid polypeptide nano structure as a targeting vector and by virtue of the high affinity of a biotin-streptavidin system, thereby achieving the aim of performing targeted therapy or imaging on tumors lacking specific targets. Compared with other treatment carriers such as inorganic nanoparticles, liposomes, polymeric micelles and the like, the DNA nano system provided by the invention has better biological safety and low toxic and side effects, and thus can be used as a drug delivery system. The system can construct artificial targets on the surface of tumor cells, and the targets can further guide the enrichment of drug molecules on the surface of the tumor cells, so that the system has potential application value in tumor treatment and early diagnosis.
Preferably, the DNA nanosheet layer is constructed from scaffold strands, staple short strands, upper capture strands and lower capture strands by means of base complementary pairing.
Preferably, the functional molecule comprises a drug molecule or an imaging molecule.
Preferably, the drug molecule is chlorin and/or pheophorbide a.
Preferably, the imaging molecule includes any one of Cy series fluorescent dye, AF series fluorescent dye, or indocyanine green, or a combination of at least two thereof. Examples of the Cy series fluorescent dye include Cy3, Cy5.5, Cy7, etc., and examples of the AF series fluorescent dye include AF488, AF595, AF750, etc.
In a second aspect, the present invention provides a method for preparing a DNA nanosystem for tumor targeting as described in the first aspect, the method comprising the steps of:
(1) mixing the DNA nanosheet layer, the single-stranded DNA coupled with the low-pH insertion peptide and the biotin-modified single-stranded DNA, heating and annealing to obtain the difunctional two-dimensional DNA nanosheet layer;
(2) and mixing the difunctional two-dimensional DNA nano-sheet layer with the streptavidin-functional molecule conjugate to obtain the DNA nano-system for tumor targeting.
Preferably, the preparation method of the DNA nanosheet layer in step (1) includes mixing scaffold chains, stapled short chains, upper capture chains and lower capture chains, heating, and annealing.
Preferably, the heating temperature is 92-98 ℃, such as 92 ℃, 93 ℃, 94 ℃, 95 ℃, 97 ℃, 98 ℃ and the like, and the heating time is 2-8min, such as 2min, 3min, 4min, 5min, 6min, 7min, 8min and the like.
Preferably, the annealing end point temperature is 15-40 ℃, such as 15 ℃, 17 ℃, 20 ℃, 22 ℃, 25 ℃, 27 ℃, 30 ℃, 32 ℃, 35 ℃, 37 ℃, 40 ℃ and the like.
Preferably, the annealing time is 10-16h, such as 10h, 11h, 12h, 13h, 14h, 15h, 16h, and the like.
Preferably, the annealing is further followed by purification.
Preferably, the purification is performed by a centrifugal purification column (molecular weight cut-off is 100 k).
Preferably, the rotational speed of the centrifugation is 3000-8000r/min, such as 3000r/min, 3500r/min, 4000r/min, 4500r/min, 5000r/min, 5500r/min, 6000r/min, 6500r/min, 7000r/min, 7500r/min, 8000r/min, etc.
Preferably, the method for preparing the single-stranded DNA coupled with the low pH insertion peptide in the step (1) comprises the following steps: and mixing the low-pH insertion peptide with the azide-modified DNA single chain to obtain the target.
Preferably, the mixing is carried out under exclusion of light.
Preferably, the mixing temperature is 20-40 ℃, such as 20 ℃, 22 ℃, 25 ℃, 27 ℃, 30 ℃, 32 ℃, 35 ℃, 37 ℃, 40 ℃ and the like, and the mixing time is 12-24h, such as 12h, 14h, 16h, 18h, 20h, 22h, 24h and the like.
Preferably, the mixing further comprises separation and purification.
Preferably, the separation and purification refers to separation and purification by denaturing polyacrylamide gel electrophoresis.
Preferably, the heating temperature in step (1) is 40-50 deg.C, such as 40 deg.C, 41 deg.C, 42 deg.C, 43 deg.C, 44 deg.C, 45 deg.C, 46 deg.C, 47 deg.C, 48 deg.C, 49 deg.C, 50 deg.C, etc., and the heating time is 2-8min, such as 2min, 3min, 4min, 5min, 6min, 7min, 8min, etc.
Preferably, the annealing is performed at an end point temperature of 20 to 30 ℃, for example, 20 ℃, 21 ℃, 22 ℃, 23 ℃, 24 ℃, 25 ℃, 26 ℃, 27 ℃, 28 ℃, 29 ℃, 30 ℃ and the like.
Preferably, the annealing is at a rate of 3-8 min/deg.C, such as 3 min/deg.C, 3.5 min/deg.C, 4 min/deg.C, 4.5 min/deg.C, 5 min/deg.C, 5.5 min/deg.C, 6 min/deg.C, 6.5 min/deg.C, 7 min/deg.C, 7.5 min/deg.C, 8 min/deg.C, and the like.
Preferably, the annealing is further followed by purification.
Preferably, the purification is carried out by polyethylene glycol precipitation.
Preferably, the method for preparing the streptavidin-functional molecule conjugate in step (2) comprises: mixing streptavidin with functional molecules to obtain the product.
Preferably, the mixing is performed at 20-40 ℃, such as 20 ℃, 22 ℃, 25 ℃, 27 ℃, 30 ℃, 32 ℃, 35 ℃, 37 ℃, 40 ℃ and the like.
Preferably, the mixing time is 1-3h, such as 1h, 1.2h, 1.5h, 1.7h, 2h, 2.2h, 2.5h, 2.7h, 3h, and the like.
Preferably, the mixing further comprises purification.
Preferably, the purification is performed by passing through a desalting column (molecular weight cut-off is 40 k).
Preferably, the temperature of the mixing in step (2) is 25-40 deg.C, such as 25 deg.C, 26 deg.C, 27 deg.C, 28 deg.C, 29 deg.C, 30 deg.C, 31 deg.C, 32 deg.C, 33 deg.C, 34 deg.C, 35 deg.C, 36 deg.C, 37 deg.C, 38 deg.C, 39 deg.C, 40 deg.C, etc., and the mixing time is 20-120min, such as 20min, 30min, 40min, 50min, 60min, 70min, 80min, 90min, 100min, 110min, 120min, etc.
As a preferred embodiment of the present invention, the method for preparing the DNA nano system for tumor targeting comprises the following steps:
(1) mixing scaffold chains, stapled short chains, upper capture chains and lower capture chains, heating to 92-98 ℃, keeping for 2-8min, annealing to 15-40 ℃, and purifying by a centrifugal purification column to obtain a DNA nanosheet layer;
mixing the low-pH insertion peptide and the azide modified DNA single chain for 12-24h at the temperature of 20-40 ℃ in a dark place, and performing separation and purification by using modified polyacrylamide gel electrophoresis to obtain the single-chain DNA of the coupled low-pH insertion peptide;
mixing streptavidin and functional molecules at 20-40 ℃ for 1-3h, and purifying by a desalting column to obtain a streptavidin-functional molecule conjugate;
(2) mixing the DNA nanosheet layer, the single-stranded DNA coupled with the low-pH insertion peptide and the biotin-modified single-stranded DNA, heating to 40-50 ℃, keeping the temperature for 2-8min, annealing to 20-30 ℃ at the speed of 3-8 min/DEG C, and purifying by using a polyethylene glycol precipitation method to obtain the bifunctional two-dimensional DNA nanosheet layer;
(3) and (3) mixing the difunctional two-dimensional DNA nano-sheet layer with the streptavidin-functional molecule conjugate for 20-120min at 25-40 ℃ to obtain the DNA nano-system for tumor targeting.
In a third aspect, the present invention provides the use of the DNA nanosystem for tumor targeting according to the first aspect or the method for preparing the DNA nanosystem for tumor targeting according to the second aspect for the preparation of a medicament for the treatment of tumors.
The recitation of numerical ranges herein includes not only the above-recited values, but also any values between any of the above-recited numerical ranges not recited, and for brevity and clarity, is not intended to be exhaustive of the specific values encompassed within the range.
Compared with the prior art, the invention has the following beneficial effects:
the invention creatively constructs a DNA nano system for tumor targeting, the construction principle of which is shown in figure 1, and the system can be divided into two stages, namely a first stage (S1): firstly, a two-dimensional DNA nano plane is constructed by utilizing a DNA paper folding technology, the safety of living body application is ensured by the DNA material, the tumor passive targeting is provided to a certain degree by the nano size, and the accurate positioning and quantitative modification of two different functional components can be realized by the two-dimensional form and the positioning modification property of the DNA material. Then, accurately positioning and quantifying low pH insertion peptide (pHLIP) and biotin on two side surfaces of the two-dimensional DNA nano plane respectively to obtain a dual-functional two-dimensional DNA nano plane, thereby realizing tumor microenvironment positioning (pHLIP) and artificial target point construction (biotin). Second stage (S2): the coupling design of the streptavidin-functional molecules ensures that the streptavidin-functional molecules have smaller nano sizes and are easier to permeate into tumor tissues, the functional molecules can be adjusted according to requirements, for example, the coupling of drug molecules or imaging elements can be realized by using a chemical modification method (carboxyl-amino coupling reaction), so that various different functions are realized, and the same S1 can be adapted to different S2, so that the application range of the DNA nano system is greatly expanded. Finally, S1 and S2 are assembled based on the interaction between biotin and streptavidin (which has extremely high affinity, rapid and specific binding, and can tolerate a wide range of pH and temperature changes), S2 is based on the receptor-ligand specific recognition of streptavidin and biotin for S1 in vivo, which is similar to the recruitment amplification process of biological signals in vivo, and the amplification effect of artificial targets can be regulated and controlled according to the adjustment of the number and density of biotin sites on the surface of S1, the concentration, frequency, interval time and other parameters of S1 and S2, so that the targeting performance of the living body is optimized. According to the invention, the DNA nano system is constructed by adopting the design mode of S1+ S2, and the preparation method is higher in yield and simpler and more convenient compared with a single system formed by positioning and assembling all functional elements in multiple levels, avoids complicated material design and preparation steps, improves the pharmacokinetics of streptavidin-functional components, greatly improves the effect of the functional components reaching/staying tumor tissues, and enhances the corresponding functions of drug molecules/imaging molecules. The invention is based on a DNA two-dimensional nano plane, realizes the positioning of a tumor micro-acid environment by using pHLIP, artificially constructs a biotin target, and realizes targeted delivery at the level of cells and living bodies by using streptavidin-functional molecules, thereby bringing specific molecular targets for tumors, greatly improving the targeting function of the functional molecules, having good targeting effect on the tumors, and having important application value in the fields of tumor imaging, treatment and the like.
Drawings
FIG. 1 is a schematic diagram of the construction of the DNA nanosystem of the present invention.
FIG. 2 is a graph showing the morphology of the DNA origami structure template obtained in step (1) of example 1.
FIG. 3 is a morphological characterization diagram of the bifunctional two-dimensional DNA nanosheets obtained in step (3) of example 1.
FIG. 4 is a graph showing the morphological characteristics of the DNA nanosystem obtained in step (5) of example 1.
FIG. 5 is an SDS-PAGE pattern of the coupling product of SA and SA-Ce 6.
FIG. 6 is a graph showing the comparison of fluorescence intensities of the treatment groups.
Fig. 7 is a graph comparing the survival rates of cancer cells in the respective treatment groups.
FIG. 8 is an image of tumor formation in mice of experimental and control groups.
FIG. 9 is a graph showing the change of the fluorescence intensity of the tumor region with time after the treatment of the experimental group and the control group.
FIG. 10 is a graph of tumor volume versus time for mice in each treatment group.
Detailed Description
The technical solution of the present invention is further explained by the following embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.
Example 1
The present embodiment provides a DNA nano system for tumor targeting, which is prepared by the following steps:
(1) preparation of two-dimensional DNA nanosheet layer
Mixing a long scaffold chain with a pre-designed staple short chain, an upper layer capturing chain and a lower layer capturing chain, cooling by a PCR program, and assembling according to the base complementary pairing principle to obtain the two-dimensional DNA nanosheet structure. The scaffold strand is the M13 genome, and the short staple strand is a nucleic acid sequence that is complementary paired with the scaffold strand. The upper and lower capture chains are used for hybridizing two different functional groups respectively.
The specific operation is as follows: mixing scaffold chain, staple short chain, upper layer capturing chain and lower layer capturing chain in TAE-Mg ratio of 1:5:5:52+In solution (Tris-Base, 40 mM; acetic acid, 20 mM; EDTA, 2 mM; magnesium acetate, 12.5 mM; pH 8.0), the temperature was raised to 95 ℃ and then slowly annealed to room temperature (25 ℃) for 12 hours. After the self-assembly is finished, a centrifugal purification column with the molecular weight cutoff of 100k is adopted to remove redundant stapled chains and capture chains by centrifugation for 5 minutes at 5000r/min, and a purified DNA origami structure template (namely a two-dimensional DNA nanosheet layer) is obtained.
(2) Synthesis of polypeptide-DNA conjugates
The polypeptide pHLIP (SEQ ID NO. 1: ACEQNPIYWARYADWLFTTPLLLLDLALLVDADET) with the terminal propargyl glycine modified and the subacid response characteristic is covalently coupled with the azide modified DNA single strand (SEQ ID NO. 2: 5'-TAAACTCTTTGCGCACTT-3') through a Click reaction.
The specific operation is as follows: the polypeptide pHLIP was first dissolved in PBS to prepare a stock solution at a concentration of 200. mu.M. The DNA was dissolved in water to prepare a stock solution having a concentration of 200. mu.M. Mixing 200 mu L of DNA stock solution with 400 mu L of polypeptide stock solution, then adding 200 mu L of dimethyl sulfoxide (DMSO), blowing, stirring and mixing uniformly, then adding 200 mu L of 10mM TBTA-Cu (II), mixing uniformly, finally adding 200 mu L of 10mM ascorbic acid, oscillating, and placing in a constant-temperature (25 ℃) shaking instrument in a dark place for 12 hours to obtain a crude product. Separating and purifying by 12% modified polyacrylamide gel electrophoresis to obtain the pHLIP-ssDNA conjugate.
(3) Preparation of bifunctional two-dimensional DNA nanosheet layer
Adding pHLIP-ssDNA obtained in the step (2) with 5-fold excess of the upper capture chain and Biotin-modified single-stranded DNA (Biotin-ssDNA, 5 '-TTTTTTTTTTTTTTTAGCG-Biotin-3', the base sequence number of which is SEQ ID NO.3) with 5-fold excess of the lower capture chain into the purified DNA origami structure template solution obtained in the step (1) for assembly, wherein the final concentration of the DNA origami is 20nM, and the final concentration of the pHLIP-ssDNA is 6 μ M (the reaction system is 100 μ L, 1 × TAE/Mg2+A solution). The mixed solution was warmed to 45 ℃ and then slowly annealed to 25 ℃ for 5 minutes per degree celsius, repeating 5 cycles. After the reaction, excess pHLIP-ssDNA and Biotin-ssDNA were removed by purification using polyethylene glycol (PEG) precipitation. The PEG precipitation step comprises: mu.L of the crude product solution (20nM concentration) was mixed with 600. mu.L of Base buffer (Tris-Base 5mM, EDTA-2Na 1mM, MgCl)220mM, NaCl 5mM) and 800. mu.L of sedimentation buffer (PEG 800015%, EDTA-2Na 1mM, NaCl 500mM), centrifuged at 16000g for 25 minutes at 25 ℃ and the supernatant removed and treated with TAE/Mg2+And (4) resuspending and precipitating the buffer solution to obtain the bifunctional two-dimensional DNA nanosheet layer.
(4) Coupling of streptavidin functional Components
Photosensitizer chlorin (Ce6) is used as a functional component, and Ce6 and Streptavidin (SA) are covalently linked through condensation reaction. SA was dissolved in PBS to make a stock solution at a concentration of 30. mu.M, and Ce6 was dissolved in DMSO to make a stock solution at a concentration of 12.5 mM. 30 μ L of Ce6 stock solution was mixed with equimolar amounts of NHS and EDC and shaken for 1h to activate the carboxyl groups on the Ce6 molecule. Then 200. mu.L of SA solution (60: 1 molar ratio of Ce6 to SA) was added and 20. mu.L of DMSO was added to increase the solubility of Ce6, and shaken at 25 ℃ for 2 h. After the reaction is finished, a desalting column with the molecular weight cutoff of 40KDa is adopted to remove redundant Ce6, and the SA-Ce6 coupling product is obtained.
(5) Construction of DNA nanosystems
And (3) simply and physically mixing the difunctional two-dimensional DNA nano-sheet layer obtained in the step (3) and the SA-Ce6 coupled product obtained in the step (4), and identifying and combining the difunctional two-dimensional DNA nano-sheet layer and the SA-Ce6 coupled product through the interaction of biotin and streptavidin to obtain the DNA nano-system for tumor targeting. The identification binding can be completed at the test tube level, the cell level and in the animal body. The specific operation of the test tube level is as follows:
mixing the bifunctional two-dimensional DNA nanosheet layer obtained in the step (3) and the SA-Ce6 coupled product obtained in the step (4) in 100 mu L of 1 XTAE/Mg2+And (3) keeping the solution (the final concentration of the bifunctional two-dimensional DNA nano-sheet layer is 10nM, and the final concentration of the SA-Ce6 coupled product is 2 mu M) at 37 ℃ for 30 minutes to identify and combine the bifunctional two-dimensional DNA nano-sheet layer and the SA-Ce6 coupled product, thus obtaining the DNA nano-system for tumor targeting.
Example 2
This example provides a DNA nano-system for tumor targeting, and the preparation method thereof is different from that of example 1 only in that the photosensitizer Ce6 in step (4) is replaced by fluorescent dye indocyanine green (ICG), so as to obtain an SA-ICG coupled product, which is specifically performed as follows:
SA was dissolved in PBS to make 200. mu.M stock solution, and NHS-activated indocyanine green was dissolved in DMSO to make 6mM ICG-NHS stock solution. mu.L of SA stock was mixed with 20. mu.L of ICG-NHS stock and shaken for 2h at 25 ℃. After completion of the reaction, excess ICG was removed using a desalting column with a molecular weight cut-off of 40kDa to give the SA-ICG coupled product.
Other steps refer to example 1.
Comparative example 1
This comparative example provides a DNA nano-system which is different from example 2 only in that pHLIP is not contained, and is prepared by:
(1) mixing a long scaffold chain with a pre-designed staple short chain, an upper layer capturing chain and a lower layer capturing chain, cooling by a PCR program, and assembling according to the base complementary pairing principle to obtain the two-dimensional DNA nanosheet structure. The scaffold strand is the M13 genome, and the short staple strand is a nucleic acid sequence that is complementary paired with the scaffold strand. The upper and lower capture chains are used for hybridizing two different functional groups respectively.
The specific operation is as follows: mixing scaffold chain, staple short chain, upper layer capturing chain and lower layer capturing chain in TAE-Mg ratio of 1:5:5:52+In solution (Tris-Base, 40 mM; acetic acid, 20 mM; EDTA, 2 mM; magnesium acetate, 12.5 mM; pH 8.0), the temperature was raised to 95 ℃ and then slowly annealed to room temperature (25 ℃) for 12 hours. After the self-assembly is finished, a centrifugal purification column with the molecular weight cutoff of 100k is adopted to remove the redundant staple chains and capture chains by centrifugation for 5 minutes at 5000r/min, and the purified DNA origami structure template is obtained.
(2) Adding Biotin-modified single-stranded DNA (Biotin-ssDNA, 5 '-TTTTTTTTTTTTTTTAGCG-Biotin-3', the base sequence number of which is SEQ ID NO.3) in 5-fold excess to the lower capture chain into the purified DNA origami structure template solution obtained in the step (1) for assembly, wherein the final concentration of the DNA origami is 20nM, (reaction system 100 uL, 1 XTAE/Mg2+A solution). The mixed solution was warmed to 45 ℃ and then slowly annealed to 25 ℃ for 5 minutes per degree celsius, repeating 5 cycles. After the reaction, the excess Biotin-ssDNA was purified by means of polyethylene glycol (PEG) precipitation. The PEG precipitation step comprises: mu.L of the crude product solution (20 nM) was centrifuged at 25 ℃ and 16000g for 25 min with 600. mu.L of Base buffer (Tris-Base 5mM, EDTA-2Na 1mM, MgCl 220 mM, NaCl 5mM) and 800. mu.L of sedimentation buffer (PEG 800015%, EDTA-2Na 1mM, NaCl 500mM), followed by removal of the supernatant and application of TAE/Mg2+And (4) resuspending the precipitate in a buffer solution to obtain the functional DNA nanosheet layer.
Streptavidin-functional component coupling and construction of DNA nanosystems reference example 2.
Test example 1
Morphological characterization of DNA nanosystems
The DNA origami structure template obtained in the step (1) of example 1 was subjected to morphology characterization by an atomic force microscope, and the result is shown in FIG. 2.
The bifunctional two-dimensional DNA nanosheets obtained in step (3) of example 1 were topographically characterized by using an atomic force microscope, and the results are shown in FIG. 3.
The DNA nano system obtained in the step (5) of example 1 was subjected to morphological characterization by an atomic force microscope, and the results are shown in FIG. 4.
As shown in fig. 2: the two-dimensional DNA nanosheet layer is in a two-dimensional triangular structure; as shown in FIG. 3, the white and highlighted structures regularly arranged on the DNA nanosheet layer prove the successful synthesis of the bifunctional DNA nanosheet layer; as shown in fig. 4, the highlighted structures regularly arranged on the DNA nanosheet layer after incubation with streptavidin proved successful recognition and binding of the coupled product of the bifunctional DNA nanosheet layer and the streptavidin-functional component, i.e., successful construction of the DNA nanosystem for tumor targeting of the present invention.
Test example 2
SDS-PAGE characterization of streptavidin-functional component conjugate products
The SA-to-Ce 6 coupled product was characterized using SDS-PAGE electrophoresis.
The results are shown in FIG. 5.
As shown, the left side shows SDS-PAGE patterns of the products of SA-Ce6 coupling in bright field, and the left-to-right bands are taken as a standard band, an SA-Ce6 band and an SA band. It can be seen that the SA-Ce6 band lags to some extent relative to the SA band, demonstrating successful coupling of the Ce6 molecule. The right side is an SDS-PAGE picture of the SA-SA 6 coupled product under a fluorescence channel, and the fluorescence phenomenon of an SA-Ce6 band also proves the successful coupling of SA-Ce 6.
Test example 3
Yield test
Taking example 1 as an example, the yields of the DNA nano system and the products obtained in each step during the assembly process thereof were tested, and the yields of the products 1 to 3 in the table are the concentration of the DNA origami template after reaction and purification/the assembly concentration of the DNA origami template before purification; the yield of product 4 in the table was calculated from the amount of SA before and after the reaction, and the results are shown in Table 1.
TABLE 1
The result shows that the DNA nano system constructed by adopting the design mode of S1+ S2 has high yield.
Test example 4
Targeting effect test of DNA nano system on cellular level
(1) Construction of DNA nano system on surface of cancer cell membrane
Mouse breast cancer cells 4T1 were inoculated into 24-well plates, 500. mu.L of medium per well, containing 1X 105And (4) cells. The cells were cultured overnight, the medium was aspirated, PBS (10. mu.L), the functional DNA nanosheet layer of comparative example 2 (20nM, 10. mu.L, pH 7.4), the functional DNA nanosheet layer of comparative example 2 (20nM, 10. mu.L, pH 6.5), the bifunctional two-dimensional DNA nanosheet layer of example 2 (20nM, 10. mu.L, pH 7.4), the bifunctional two-dimensional DNA nanosheet layer of example 2 (20nM, 10. mu.L, pH 6.5), incubated for 1h, the medium was aspirated, each well was washed 3 times with PBS corresponding to pH, then medium containing 0.4. mu.M of the coupled product of SA-ICG at corresponding pH was added to each well, incubation was continued for 0.5h, then the medium was removed, changed to PBS at different pH, the cells were blown down with a gun, centrifuged (1000 rpm, 4min) and resuspended with PBS at corresponding pH. Wherein pH 7.4 is a condition simulating normal physiological conditions, and pH 6.5 is a condition simulating tumor microenvironment.
The fluorescence intensity of each group was measured by flow cytometry, and the results are shown in FIG. 6.
As shown in the figure, the fluorescence intensity of the pH 6.5-example 2 treatment group (6.5-TOBP) is significantly stronger than that of the PBS group, the pH 7.4-comparative example 1 treatment group (7.4-TOB), the pH 6.5-comparative example 1 treatment group (6.5-TOB) and the pH 7.4-example 2 treatment group (7.4-TOBP), and the results show that the pHLIP polypeptide-assembled bifunctional DNA nanosheets of the present invention spontaneously insert into the cancer cell membrane through conformational transformation under the tumor microenvironment of pH 6.5 and are successfully identified and combined with the coupling product of SA-ICG, thereby proving the successful construction of the DNA nanosystem of the present invention on the surface of the cancer cell membrane. The nano system does not respond under the normal physiological condition of pH 7.4, and is only embedded into a cell membrane under a slightly acidic tumor microenvironment, so that the nano system has an excellent targeting effect on cancer cells.
(2) Photothermal therapeutic effect
Mouse breast cancer cells 4T1 were inoculated into black 96-well plates, 100. mu.L of medium per well, containing 1X 104The cells were incubated overnight, the medium aspirated and treated as follows:
PBS treatment group: PBS (10. mu.L) was added and incubated for 1.5 h;
② separate SA-Ce6 treatment group: PBS (25. mu.M, 10. mu.L) containing the SA-Ce6 coupled product obtained in step (4) of example 1 was added and incubated for 1.5 h;
③ pH 7.4-DNA nanosystems treatment group (7.4TO + SA-Ce 6): adding PBS (100nM, 10. mu.L, pH 7.4) containing the DNA bifunctional nanosheets obtained in step (3) of example 1, incubating for 1h, washing, adding PBS (25. mu.M, 10. mu.L, pH 7.4) containing the SA-Ce6 coupled product obtained in step (4) of example 1, and further incubating for 0.5 h;
pH 6.5-DNA nanosystem treatment group (6.5TO + SA-Ce 6): adding PBS (100nM, 10. mu.L, pH 6.5) containing the DNA bifunctional nanosheets obtained in step (3) of example 1, incubating for 1h, washing, adding PBS (25. mu.M, 10. mu.L, pH 6.5) containing the SA-Ce6 coupled product obtained in step (4) of example 1, and further incubating for 0.5 h;
after the above treatment, each group was irradiated with a laser having a wavelength of 650nm at a laser intensity of 0.7W/cm for 4 minutes2(in the absence of light for each group). After 20h the medium was aspirated off and incubation continued for 1h with the addition of 100. mu.L of CCK solution. The survival rate of cancer cells of each group was measured by a microplate reader.
The results are shown in FIG. 7.
As shown in the figure, the survival rate of the cancer cells after the photo-thermal treatment of the pH 6.5-DNA nano system treatment group (6.5TO + SA-Ce6) is less than 40%, which is far lower than that of other treatment groups, and the excellent targeting effect of the DNA nano system on the cancer cells is proved, so that the DNA nano system has huge application potential in photo-thermal treatment.
Test example 5
Tumor targeting effect test of DNA nano system on animal level
(1) Construction of DNA nanosystems in animals
Dissolving mouse breast cancer cell 4T1 in PBS and matrigel mixed solution (PBS: matrigel is 1:1), subcutaneously injecting into upper part of right leg of BALB/c female nude mouse (mouse age 6 weeks), injecting 100 μ L each mixed solution containing 5 × 105And (4) cells. When the tumor grows to 150mm3The mice were divided into 2 groups, and group 1 was an experimental group, and PBS (30nM, 100. mu.L) containing the DNA bifunctional nanosheets of example 2 was injected first; group 2 was a control group, which was injected with PBS (100. mu.L) first, and 12 hours later, two groups were injected with SA-ICG-containing PBS (5. mu.M, 100. mu.L) simultaneously. The results of the observation imaging with the small animal optical in vivo imaging system were obtained at 1, 6, 12, 24h, and 48h after the SA-ICG injection, respectively, and are shown in FIG. 8. The fluorescence intensity of the tumor area of the mice at different time points was recorded. The results are shown in FIG. 9.
As shown in the figure, the fluorescence of the tumor region of the DNA nano system treatment group (TOBP + SA) is obviously stronger than that of the control group (PBS + SA), and the successful construction of the DNA nano system in the animal body and the excellent tumor targeting effect are proved.
(2) Photothermal therapy
The mouse mammary cancer cell 4T1 was dissolved in a mixed solution of PBS and matrigel (PBS: matrigel volume ratio: 1), and was subcutaneously injected into the upper part of the right leg of BALB/c female nude mice (aged 6 weeks) with 100. mu.L of each mixed solution containing 5X 105And (4) cells. When the tumor grows to 60mm3Mice were divided into 5 groups. The following treatments were respectively carried out:
control group (PBS): PBS (100. mu.L) was injected without light;
② PBS treatment group (PBS + L): injecting PBS (100 μ L), and irradiating 12h later;
③ single DNA bifunctional nanosheet layer processing group (TOBP): injecting PBS (150nM,100 uL) containing the DNA bifunctional nanosheet layer obtained in step (3) of example 1, and irradiating after 12 h;
treatment group of SA-Ce6 alone (SA-Ce 6): PBS (30. mu.M, 100. mu.L) containing the SA-Ce6 coupled product obtained in the step (4) of example 1 was injected and irradiated after 12 hours;
DNA nano-system treatment group (TOBP + SA-Ce 6): PBS (150nM, 100. mu.L) containing the DNA bifunctional nanosheet layer obtained in step (3) of example 1 was injected first, PBS (30. mu.M, 100. mu.L) containing the SA-Ce6 coupled product obtained in step (4) of example 1 was injected 24 hours later, and light irradiation was performed 12 hours later;
the illumination conditions are unified as follows: 650nm laser, irradiation time 30min, intensity 0.7W/cm2。
FIG. 10 is a statistical plot of tumor volume versus time for groups of mice 16 days after light exposure. The tumor volume calculation method comprises the following steps: tumor volume is L × W2And/2, L is the length of the long side of the tumor, and W is the length of the short side of the tumor.
As shown, the tumor volume of the DNA nanosystem treated group (TOBP + SA-Ce6) was significantly lower than that of the other treated groups, indicating that the tumor growth of the DNA nanosystem treated group was significantly inhibited. The DNA nano system has excellent targeting effect on tumors, has obvious photothermal treatment effect on the tumors, and has huge clinical application potential.
The applicant states that the present invention is illustrated by the above embodiments, but the present invention is not limited to the above embodiments, i.e. the present invention does not mean that the present invention must be implemented by the above embodiments. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.
The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.
SEQUENCE LISTING
<110> national center for Nano science
<120> DNA nano system for tumor targeting and preparation method and application thereof
<130> 2022-1-14
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Claims (10)
1. A DNA nanosystem for tumor targeting comprising a DNA nanosheet layer, single stranded DNA coupled to a low pH insertion peptide, biotin-modified single stranded DNA, and a streptavidin-functional molecule conjugate;
wherein the DNA nanosheet layer, the single-stranded DNA coupled with the low-pH insertion peptide and the biotin-modified single-stranded DNA are combined in a base complementary pairing manner and assembled to form the bifunctional DNA nanosheet layer; the streptavidin-functional molecule conjugate is combined with the bifunctional DNA nano-sheet layer through the specific recognition effect of streptavidin and biotin to form the DNA nano-system for tumor targeting.
2. The DNA nanosystem for tumor targeting of claim 1, wherein the DNA nanosheet is constructed from scaffold strands, stapled short strands, upper capture strands and lower capture strands by base complementary pairing.
3. The DNA nanosystem for tumor targeting of claim 1 or 2, wherein the functional molecule comprises a drug molecule or an imaging molecule;
preferably, the drug molecule comprises chlorin and/or pheophorbide a;
preferably, the imaging molecule includes any one of Cy series fluorescent dye, AF series fluorescent dye, or indocyanine green, or a combination of at least two thereof.
4. The method for preparing a DNA nanosystem for tumor targeting according to any of claims 1 to 3, characterized in that it comprises the following steps:
(1) mixing the DNA nanosheet layer, the single-stranded DNA coupled with the low-pH insertion peptide and the biotin-modified single-stranded DNA, heating and annealing to obtain the difunctional two-dimensional DNA nanosheet layer;
(2) and mixing the difunctional two-dimensional DNA nano-sheet layer with the streptavidin-functional molecule conjugate to obtain the DNA nano-system for tumor targeting.
5. The method according to claim 4, wherein the DNA nanosheet layer of step (1) is prepared by mixing scaffold strands, staple short strands, upper capture strands and lower capture strands, heating, and annealing;
preferably, the heating temperature is 92-98 ℃, and the heating time is 2-8 min;
preferably, the end point temperature of the annealing is 15-40 ℃;
preferably, the annealing time is 10-16 h;
preferably, the annealing further comprises purification;
preferably, the purification is performed by a centrifugal purification column;
preferably, the rotation speed of the centrifugation is 3000-.
6. The method according to claim 4 or 5, wherein the single-stranded DNA to which the low pH insertion peptide is coupled in step (1) is prepared by: mixing the low-pH insertion peptide with the azide-modified DNA single chain to obtain the product;
preferably, the mixing is carried out under protection from light;
preferably, the mixing temperature is 20-40 ℃, and the mixing time is 12-24 h;
preferably, the mixing further comprises separation and purification;
preferably, the separation and purification refers to separation and purification by denaturing polyacrylamide gel electrophoresis.
7. The method according to any one of claims 4to 6, wherein the heating temperature in step (1) is 40 to 50 ℃ and the heating time is 2 to 8 min;
preferably, the end point temperature of the annealing is 20-30 ℃;
preferably, the annealing speed is 3-8 min/DEG C;
preferably, the annealing further comprises purification;
preferably, the purification is carried out by polyethylene glycol precipitation.
8. The method of any one of claims 4-7, wherein the streptavidin-functional molecule conjugate of step (2) is prepared by a method comprising: mixing streptavidin with functional molecules to obtain the product;
preferably, the mixing is carried out at 20-40 ℃ for 1-3 h;
preferably, the mixing further comprises purification;
preferably, the purification is performed by passing through a desalting column.
9. The method according to any one of claims 4to 8, wherein the temperature of the mixing in the step (2) is 25 to 40 ℃ and the time of the mixing is 20 to 120 min.
10. Use of the DNA nanosystems for tumor targeting of any of claims 1 to 3 or of the method for preparation of the DNA nanosystems for tumor targeting of any of claims 4to 9 for the preparation of a medicament for the treatment of tumors.
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