CN114392354B - 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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- CN114392354B CN114392354B CN202210042758.6A CN202210042758A CN114392354B CN 114392354 B CN114392354 B CN 114392354B CN 202210042758 A CN202210042758 A CN 202210042758A CN 114392354 B CN114392354 B CN 114392354B
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- Medicinal Preparation (AREA)
Abstract
The invention provides a DNA nano system for tumor targeting, a preparation method and application thereof, wherein the DNA nano system for tumor targeting comprises a DNA nano lamellar, single-stranded DNA coupled with low pH insertion peptide, biotin modified single-stranded DNA and streptavidin-functional molecule conjugate; wherein the DNA nano-sheet 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 mode to form a double-functional DNA nano-sheet layer; the streptavidin-functional molecule conjugate is combined with the difunctional 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 purpose 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 in particular 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, clinical treatment of cancer mainly depends on radiotherapy, chemotherapy and operation treatment, and the methods relieve the pain of patients to a certain extent, but have certain limitations and lower cure rate. With the continuous development of nano technology, the nano technology is continuously fused with modern medicine, so that the interdisciplinary science of nano medicine is formed, and the potential application of the interdisciplinary science relates to the aspects of diagnosis, monitoring, treatment and the like of diseases. The nano carrier is used for loading the chemotherapeutic drugs, so that the damage of the chemotherapeutic drugs to normal tissues and cells of a human body can be reduced, and toxic and side effects are reduced. Meanwhile, the long circulation time of the medicine molecules in the body is improved, and the bioavailability of the medicine 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, combined with the superior properties of nanosystems, a variety of nanosystems for tumor targeting have been developed.
For example, CN109806402a discloses a tumor targeted fluorescent nano-chain probe, a preparation method and application thereof. The tumor targeting fluorescent nano-chain probe comprises a self-assembled nano-chain serving as a carrier, and a fluorescent material and a tumor targeting ligand which are loaded on the self-assembled nano-chain, wherein the fluorescent material and the tumor targeting 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 respectively chemically crosslinking the fluorescent material and the tumor targeting ligand to the self-assembled nano chains by using a chemical crosslinking mode. The tumor targeted fluorescent nano-chain probe has the characteristic of near infrared fluorescent tumor targeting, can realize the efficient in-vivo and in-vitro targeting of tumor cells, can overcome the difficulty that the traditional operation is difficult to monitor and guide in real time in the diagnosis and operation navigation of tumors, and provides a visual and accurate navigation tool for the operation treatment of a series of tumors such as peritoneal metastasis.
CN105288620a discloses a method for preparing 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 by mercapto groups, selectively connecting different tumor targeted molecules on the hydrophilic polymers by the reaction of the mercapto groups and succinimide, and connecting different amounts of antitumor drugs to the surfaces of the gold nanoflowers by stimulating responsive acylhydrazone bonds, thereby obtaining the gold nanoflowers with tumor targeted photo-thermal therapy and chemotherapy combined. 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 nano system for tumor targeting provided in the prior art is very limited, and the problems of low targeting efficiency, poor therapeutic effect, obvious drug resistance and the like generally exist, and the nano system is not suitable for some tumor bundles lacking specific targets.
Therefore, how to provide a nano system for tumor targeting to promote tumor specific targeting becomes a problem to be solved in the art.
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, in particular to a DNA nano system for tumor targeting and a preparation method and application thereof.
In order to achieve the aim of the invention, 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, single-stranded DNA coupled to a low pH intercalating peptide, biotin-modified single-stranded DNA and a streptavidin-functional molecule conjugate;
wherein the DNA nano-sheet 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 mode, and the double-functional DNA nano-sheet layer is formed by assembly; the streptavidin-functional molecule conjugate is combined with the difunctional DNA nanosheet layer through the specific recognition effect of streptavidin and biotin to form the DNA nanosystem for tumor targeting.
The invention creatively constructs a DNA nano system for tumor targeting, and the construction principle is shown in figure 1. Wherein, the low pH insertion peptide (pHLIP) is a water-soluble polypeptide derived from bacteriorhodopsin C helix, is a slightly acidic environment (tumor environment is slightly acidic, pH is below 6.5; normal physiological environment pH is 7.4) responsive polypeptide, and can be spontaneously inserted into cell membranes through conformational transformation (formation of alpha-helix conformation) under slightly acidic conditions without being internalized by the cells. pHLIP can be coupled with DNA single-chain through covalent function, DNA single-chain can guide it to hybridize at the specific site of DNA nano-plane structure, thus make two-dimensional DNA plane can insert on the surface of tumor cell. The other side of the two-dimensional DNA nano-planar structure hybridizes with a biotin-modified DNA strand (the biotin-modified DNA strand is easy to obtain; the DNA single strand can guide hybridization at a specific site of the DNA nano-planar structure). Since biotin can be specifically bound with streptavidin, after pHLIP is inserted into cell membrane under slightly acidic condition, biotin site on the other side of two-dimensional DNA nano-plane structure is exposed as artificial target for recruiting streptavidin-functional molecule conjugate. The functional molecules can be adjusted as required, for example, a photosensitizer is modified to realize targeted photothermal therapy, or a fluorescent dye is modified to realize tumor imaging. According to the invention, the nucleic acid polypeptide nanostructure is used as a targeting vector, and a DNA nano system is constructed by means of high affinity of a biotin-streptavidin system, so that the purpose of targeted treatment or imaging of tumors lacking specific targets is achieved. Compared with other therapeutic carriers such as inorganic nano particles, liposome, polymer micelle and the like, the DNA nano system provided by the invention has better biological safety and low toxic and side effect, and can be used as a drug delivery system. The system can construct artificial targets on the surfaces of tumor cells, and the targets can further guide the enrichment of drug molecules on the surfaces of the tumor cells, so that the system has potential application value in tumor treatment and early diagnosis.
Preferably, the DNA nanoplatelets are constructed from scaffold strands, staple strands, upper capture strands and lower capture strands by base complementary pairing.
Preferably, the functional molecule comprises a drug molecule or an imaging molecule.
Preferably, the drug molecule chlorin and/or pheophorbide a.
Preferably, the imaging molecule comprises any one or a combination of at least two of Cy series fluorescent dyes, AF series fluorescent dyes or indocyanine green. Such as Cy3, cy5.5, cy7, etc., and such as AF488, AF595, AF750, etc.
In a second aspect, the present invention provides a method for preparing a DNA nanosystem for tumor targeting according to the first aspect, the method comprising the steps of:
(1) Mixing the DNA nano-sheet layer, the single-stranded DNA coupled with the low pH insertion peptide and the biotin-modified single-stranded DNA, heating and annealing to obtain a double-function two-dimensional DNA nano-sheet layer;
(2) And mixing the difunctional two-dimensional DNA nanosheets with the streptavidin-functional molecule conjugate to obtain the DNA nanosystem for tumor targeting.
Preferably, the preparation method of the DNA nanosheet layer in the step (1) comprises mixing a scaffold chain, a staple chain, an upper capturing chain and a lower capturing chain, heating, and annealing.
Preferably, the heating temperature is 92-98deg.C, such as 92 deg.C, 93 deg.C, 94 deg.C, 95 deg.C, 97 deg.C, 98 deg.C, etc., and the heating time is 2-8min, such as 2min, 3min, 4min, 5min, 6min, 7min, 8min, etc.
Preferably, the end temperature of the annealing is 15-40 ℃, e.g., 15 ℃, 17 ℃,20 ℃, 22 ℃,25 ℃, 27 ℃, 30 ℃, 32 ℃, 35 ℃, 37 ℃,40 ℃, etc.
Preferably, the annealing is performed for a period of time ranging from 10 to 16 hours, such as 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, etc.
Preferably, the annealing is followed by purification.
Preferably, the purification refers to passing through a centrifugal purification column (molecular weight cut-off 100 k).
Preferably, the rotational speed of the centrifugation is 3000-8000r/min, e.g. 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 preparation method of the single-stranded DNA coupled with the low pH intercalating peptide in the step (1) comprises the following steps: mixing the low pH insertion peptide with the azide-modified DNA single strand.
Preferably, the mixing is carried out in the absence of light.
Preferably, the temperature of the mixing is 20-40 ℃, e.g., 20 ℃, 22 ℃,25 ℃, 27 ℃, 30 ℃, 32 ℃, 35 ℃, 37 ℃,40 ℃, etc., and the time of the mixing is 12-24 hours, e.g., 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, etc.
Preferably, the mixing further comprises separation and purification.
Preferably, the separation and purification means separation and purification by denaturing polyacrylamide gel electrophoresis.
Preferably, the heating in step (1) is performed at a temperature of 40-50deg.C, such as 40deg.C, 41 deg.C, 42 deg.C, 43 deg.C, 44 deg.C, 46 deg.C, 47 deg.C, 48 deg.C, 49 deg.C, 50 deg.C, etc., and the heating is performed for a time of 2-8min, such as 2min, 3min, 4min, 5min, 6min, 7min, 8min, etc.
Preferably, the end temperature of the annealing is 20-30 ℃, e.g., 20 ℃, 21 ℃, 22 ℃, 23 ℃,24 ℃,25 ℃, 26 ℃, 27 ℃, 28 ℃, 29 ℃, 30 ℃, etc.
Preferably, the annealing speed is 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, etc.
Preferably, the annealing is followed by purification.
Preferably, the purification refers to purification by polyethylene glycol precipitation.
Preferably, the preparation method of the streptavidin-functional molecule conjugate in the step (2) comprises the following steps: mixing streptavidin with functional molecule.
Preferably, the mixing is performed at 20-40 ℃, e.g., 20 ℃, 22 ℃,25 ℃, 27 ℃, 30 ℃, 32 ℃, 35 ℃, 37 ℃,40 ℃, etc.
Preferably, the mixing time is 1-3 hours, such as 1h, 1.2h, 1.5h, 1.7h, 2h, 2.2h, 2.5h, 2.7h, 3h, etc.
Preferably, the mixing is followed by purification.
Preferably, the purification refers to purification by a desalting column (molecular weight cut-off 40 k).
Preferably, the temperature of the mixing in step (2) is 25-40 ℃, e.g. 25 ℃, 26 ℃, 27 ℃, 28 ℃, 29 ℃, 30 ℃, 31 ℃, 32 ℃, 33 ℃, 34 ℃, 35 ℃, 36 ℃, 37 ℃, 38 ℃, 39 ℃,40 ℃, etc., and the time of the mixing is 20-120min, e.g. 20min, 30min, 40min, 50min, 60min, 70min, 80min, 90min, 100min, 110min, 120min, etc.
As a preferred scheme of the invention, the preparation method of the DNA nanosystem for tumor targeting comprises the following steps:
(1) Mixing scaffold chains, stapling short chains, upper layer capturing chains and lower layer capturing chains, heating to 92-98 ℃, maintaining for 2-8min, annealing to 15-40 ℃, and purifying by a centrifugal purification column to obtain a DNA nano sheet layer;
mixing the low pH insertion peptide and the azide-modified DNA single strand for 12-24 hours at 20-40 ℃ in the dark, and separating and purifying by using modified polyacrylamide gel electrophoresis to obtain single strand DNA coupled with the 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 nano-sheet layer, the single-stranded DNA coupled with the low pH insertion peptide and the biotin-modified single-stranded DNA, heating to 40-50 ℃, keeping for 2-8min, annealing to 20-30 ℃ at a speed of 3-8 min/DEG C, and purifying by a polyethylene glycol precipitation method to obtain a difunctional two-dimensional DNA nano-sheet layer;
(3) Mixing the difunctional two-dimensional DNA nanosheets and the streptavidin-functional molecule conjugate at 25-40 ℃ for 20-120min to obtain the DNA nanosystem for tumor targeting.
In a third aspect, the present invention provides the use of a DNA nanosystem for tumor targeting as described in the first aspect or a method of preparing a DNA nanosystem for tumor targeting as described in the second aspect for the preparation of a medicament for treating a tumor.
The numerical ranges recited herein include not only the recited point values, but also any point values between the recited numerical ranges that are not recited, and are limited to, and for the sake of brevity, the invention is not intended to be exhaustive of the specific point values that the recited range includes.
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 using a DNA paper folding technology, the safety of living body application is ensured by a DNA material, a certain degree of tumor passive targeting is provided 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 locatable modification of the DNA material. Then, respectively and accurately positioning and quantifying low pH insertion peptide (pHLIP) and biotin on two sides of the two-dimensional DNA nano plane to obtain the dual-function two-dimensional DNA nano plane, and realizing tumor microenvironment positioning (pHLIP) and artificial target construction (biotin). Second stage (S2): the coupling design of streptavidin-functional molecules ensures that the streptavidin-functional molecules have smaller nano-size, so that the streptavidin-functional molecules can permeate into tumor tissues more easily, the functional molecules can be adjusted according to requirements, for example, the coupling of drug molecules or imaging elements can be realized by a chemical modification method (carboxyl-amino coupling reaction), so that a plurality of different functions are realized, and the same S1 can be adapted to different S2, so that the application range of the DNA nanosystem is greatly expanded. Finally, S1 and S2 are assembled based on the interaction of biotin and streptavidin (the two have extremely high affinity, the combination is rapid and specific, and the wide range of pH and temperature changes can be tolerated), S2 'finding' for S1 in vivo is based on receptor-ligand specific recognition of streptavidin and biotin, similar to the recruitment amplification process of biological signals in vivo, and the effect of artificial target amplification can be regulated and controlled according to the number of biotin sites on the surface of S1, the density, the use concentration of S1 and S2, the frequency, the interval time and other parameters, so that the targeting performance of living bodies is optimized. Compared with a single system formed by all functional elements through multi-stage positioning and co-assembly, the preparation method of the DNA nano system is higher in yield, simpler and more convenient, avoids complicated material design and preparation steps, improves the pharmacokinetics of streptavidin-functional components, greatly improves the effect of the functional components on reaching/retaining tumor tissues, and enhances the corresponding functions of drug molecules/imaging molecules. The invention realizes the positioning of tumor slightly acidic environment based on the DNA two-dimensional nano-plane by utilizing pHLIP, artificially constructs a biotin target spot, realizes targeted delivery at the cellular and living levels by utilizing streptavidin-functional molecules, brings specific molecular targets for tumors, greatly improves the targeting function of the functional molecules, has good targeting effect on tumors, and has important application value in the fields of tumor imaging, treatment and the like.
Drawings
FIG. 1 is a schematic diagram of the construction of a DNA nanosystem of the present invention.
FIG. 2 is a diagram showing the morphology of the template of the DNA paper folding structure obtained in the step (1) of example 1.
FIG. 3 is a graph showing the morphology of the bifunctional two-dimensional DNA nanosheets obtained in step (3) of example 1.
FIG. 4 is a graph showing the morphology of the DNA nanosystem obtained in the step (5) of example 1.
FIG. 5 is a SDS-PAGE map of SA-Ce6 coupled products.
FIG. 6 is a graph showing the comparison of fluorescence intensity for each treatment group.
Fig. 7 is a graph comparing the survival rates of cancer cells in each treatment group.
Fig. 8 is a tumor imaging of mice from the experimental and control groups.
Fig. 9 is a graph showing the fluorescence intensity of tumor regions after treatment of experimental group and control group with time.
Fig. 10 is a graph of tumor volume versus time for mice of each treatment group.
Detailed Description
The technical scheme of the invention is further described by the following specific embodiments. It will be apparent to those skilled in the art that the examples are merely to aid in understanding the invention and are not to be construed as a specific limitation thereof.
Example 1
The embodiment provides a DNA nano system for tumor targeting, and the preparation method thereof is as follows:
(1) Preparation of two-dimensional DNA nanosheets
Mixing a long scaffold chain with a pre-designed stapling short chain, an upper layer capturing chain and a lower layer capturing chain, cooling by a PCR program, and assembling according to a base complementary pairing principle to obtain the two-dimensional DNA nano lamellar structure. The scaffold strand is the M13 genome, and the stitched strand is a nucleic acid sequence complementary to the scaffold strand. The upper and lower capture chains are used to hybridize to two different functional groups, respectively.
The specific operation is as follows: the scaffold chain, the staple chain and the upper partThe layer capturing chain and the lower layer capturing chain are mixed in the TAE-Mg in the ratio of 1:5:5:5 2+ In solution (Tris-Base, 40mM; acetic acid, 20mM; EDTA,2mM; magnesium acetate, 12.5mM; pH 8.0), the temperature was raised to 95℃and then slowly annealed to room temperature (25 ℃) for 12h. After self-assembly, a centrifugal purification column with the molecular weight cut-off of 100k is adopted to remove redundant staple chains and capture chains after centrifugation for 5 minutes at 5000 revolutions per minute, so as to obtain the purified DNA paper folding structure template (namely the two-dimensional DNA nano sheet layer).
(2) Synthesis of polypeptide-DNA conjugates
A propargylglycine-modified polypeptide pHLIP (SEQ ID NO.1: ACEQNPIYWARYADWLFTTPLLLLDLALLVDADET) having slightly acidic response properties at the end was covalently coupled to an azide-modified single strand DNA (SEQ ID NO.2: 5'-TAAACTCTTTGCGCACTT-3') by a Click reaction.
The specific operation is as follows: 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. 200 mu L of DNA stock solution and 400 mu L of polypeptide stock solution are mixed, then 200 mu L of dimethyl sulfoxide (DMSO) is added for blowing and mixing uniformly, then 200 mu L of 10mM TBTA-Cu (II) is added for mixing uniformly, finally 200 mu L of 10mM ascorbic acid is added, oscillation and light shielding are carried out, and the mixture is placed in a constant temperature (25 ℃) oscillation instrument for 12 hours, so that a crude product is obtained. Separating and purifying by 12% modified polyacrylamide gel electrophoresis to obtain pHLIP-ssDNA conjugate.
(3) Preparation of difunctional two-dimensional DNA nanosheets
Adding 5 times of pHLIP-ssDNA obtained in step (2) exceeding the upper capturing chain and 5 times of Biotin-modified single-stranded DNA (Biotin-ssDNA, 5'-TTTTTTTTTTTTTTTAGCG-Biotin-3', the base sequence of which is SEQ ID NO. 3) exceeding the lower capturing chain to the purified DNA paper folding structure template solution obtained in step (1) for assembly, wherein the final concentration of DNA paper folding is 20nM, and the final concentration of pHLIP-ssDNA is 6. Mu.M (reaction system 100. Mu.L, 1 xTAE/Mg) 2+ 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 purified by means of polyethylene glycol (PEG) precipitation. PEG precipitationThe method comprises the following steps: mu.L of the crude product solution (concentration 20 nM) was mixed with 600. Mu.L of Base buffer (Tris-Base 5mM, EDTA-2Na 1mM, mgCl) 2 20mM, 5mM NaCl) and 800. Mu.L of sedimentation buffer (PEG 8000 15%, EDTA-2Na 1mM,NaCl 500mM), were centrifuged at 16000g for 25 min at 25℃and the supernatant was subsequently removed using TAE/Mg 2+ And re-suspending and precipitating with buffer solution to obtain the double-function two-dimensional DNA nano-sheet layer.
(4) Coupling of streptavidin-functional components
The photosensitizer chlorin (Ce 6) is used as a functional component, and the Ce6 and Streptavidin (SA) are covalently connected through a condensation reaction. SA was dissolved in PBS to give a stock solution at a concentration of 30. Mu.M, and Ce6 was dissolved in DMSO to give a stock solution at a concentration of 12.5 mM. 30. Mu.L of Ce6 stock was mixed with equimolar amounts of NHS and EDC and shaken for 1h to activate the carboxyl groups on the Ce6 molecule. 200. Mu.L of SA solution (molar ratio of Ce6 to SA 60:1) was then added, and 20. Mu.L of DMSO was added to increase the solubility of Ce6, and shaking was performed at 25℃for 2h. After the reaction is completed, a desalting column with a cut-off molecular weight of 40kDa is used for removing excessive Ce6, and the SA-Ce6 coupled product is obtained.
(5) Construction of DNA nanosystems
And (3) simply and physically mixing the difunctional two-dimensional DNA nanosheet layer obtained in the step (3) with the SA-Ce6 coupling product obtained in the step (4), and carrying out recognition and combination on the difunctional two-dimensional DNA nanosheet layer and the SA-Ce6 coupling product through interaction of biotin and streptavidin, so that the DNA nanosystem for tumor targeting is obtained. The recognition can be accomplished at the tube level, at the cellular level, and in vivo in animals. The specific operation of the test tube level is as follows:
mixing the difunctional two-dimensional DNA nano-sheet layer obtained in the step (3) and the SA-Ce6 coupling product obtained in the step (4) into 100 mu L of 1 xTAE/Mg 2+ The solution (the final concentration of the difunctional two-dimensional DNA nano-sheet layer is 10nM, the final concentration of the SA-Ce6 coupled product is 2 mu M) is kept at 37 ℃ for 30 minutes to enable the solution to be identified and combined, and the DNA nano-system for tumor targeting is obtained.
Example 2
The present example provides a DNA nanosystem for tumor targeting, and the preparation method thereof differs from that of example 1 only in that the photosensitizer Ce6 in step (4) is replaced with the fluorescent dye indocyanine green (ICG), thereby obtaining a SA-ICG coupled product, which specifically comprises the following steps:
SA was dissolved in PBS to make 200. Mu.M stock solution, NHS activated indocyanine green was dissolved in DMSO to make 6mM ICG-NHS stock solution. 100. Mu.L of SA stock was mixed with 20. Mu.L of ICG-NHS stock and shaken at 25℃for 2h. After the reaction was completed, excess ICG was removed using a desalting column having a cut-off molecular weight of 40kDa to give an SA-ICG coupled product.
For further steps reference is made to example 1.
Comparative example 1
This comparative example provides a DNA nanosystem which differs from example 2 only in that it does not contain pHLIP, and is prepared by:
(1) Mixing a long scaffold chain with a pre-designed stapling short chain, an upper layer capturing chain and a lower layer capturing chain, cooling by a PCR program, and assembling according to a base complementary pairing principle to obtain the two-dimensional DNA nano lamellar structure. The scaffold strand is the M13 genome, and the stitched strand is a nucleic acid sequence complementary to the scaffold strand. The upper and lower capture chains are used to hybridize to two different functional groups, respectively.
The specific operation is as follows: the scaffold chain, the stapling chain, the upper capturing chain and the lower capturing chain are mixed in a ratio of 1:5:5:5 to TAE-Mg 2+ In solution (Tris-Base, 40mM; acetic acid, 20mM; EDTA,2mM; magnesium acetate, 12.5mM; pH 8.0), the temperature was raised to 95℃and then slowly annealed to room temperature (25 ℃) for 12h. After self-assembly, a centrifugal purification column with the molecular weight cut-off of 100k is adopted to remove redundant staple chains and capturing chains after centrifugation for 5 minutes at 5000 revolutions per minute, and the purified DNA paper folding structure template is obtained.
(2) Adding Biotin-modified single-stranded DNA (Biotin-ssDNA, 5'-TTTTTTTTTTTTTTTAGCG-Biotin-3', the base sequence of which is SEQ ID NO. 3) which is 5 times more than the lower capture chain into the purified DNA paper folding structure template solution obtained in the step (1) for assembly, wherein the final concentration of the DNA paper folding is 20nM (reaction system 100 mu L,1 xTAE/Mg) 2+ 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, makeExcess Biotin-ssDNA was purified by means of polyethylene glycol (PEG) precipitation. The PEG precipitation step is: 200. Mu.L of the crude product solution (concentration 20 nM) was centrifuged with 600. Mu.L of Base buffer (Tris-Base 5mM, EDTA-2Na 1mM,MgCl2 20mM,NaCl 5mM) and 800. Mu.L of sedimentation buffer (PEG 8000 15%, EDTA-2Na 1mM,NaCl 500mM) at 25℃for 25 minutes at 16000g, and the supernatant was removed, using TAE/Mg 2+ And re-suspending and precipitating with buffer solution to obtain the functional DNA nano-sheet layer.
Coupling of streptavidin-functional components and construction of DNA nanosystems reference example 2.
Test example 1
Topography characterization of DNA nanosystems
The morphology of the DNA origami structure template obtained in the step (1) of the example 1 is characterized by an atomic force microscope, and the result is shown in fig. 2.
The morphology of the bifunctional two-dimensional DNA nanosheets obtained in the step (3) of the example 1 is characterized by an atomic force microscope, and the result is shown in FIG. 3.
The appearance of the DNA nanosystem obtained in step (5) of example 1 was characterized by using an atomic force microscope, and the result is shown in fig. 4.
As shown in fig. 2: the two-dimensional DNA nano sheet layer is in a two-dimensional triangle structure; as shown in fig. 3, white highlighted structures regularly arranged on the DNA nanoplatelets demonstrate successful synthesis of the bifunctional DNA nanoplatelets; as shown in FIG. 4, the regularly arranged highlight structures on the DNA nanosheets after incubation with streptavidin prove that the successful identification and combination of the double-function DNA nanosheets and the coupling products of streptavidin-functional components, namely the successful construction of the DNA nanosystem for tumor targeting.
Test example 2
SDS-PAGE characterization of streptavidin-functional component conjugate products
The SA and SA-Ce6 coupled products were characterized using SDS-PAGE electrophoresis.
The results are shown in FIG. 5.
As shown in the figure, the left side is an SDS-PAGE image of SA-Ce6 coupled products under bright field, and the left to right bands are the standard bands, the SA-Ce6 band and the SA band. It can be seen that the SA-Ce6 band has a degree of hysteresis relative to the SA band, demonstrating successful coupling of the Ce6 molecule. On the right is SDS-PAGE of SA-Ce6 coupled products under fluorescent channel, and the fluorescence phenomenon of SA-Ce6 band also demonstrates the successful coupling of SA-Ce 6.
Test example 3
Yield test
Taking example 1 as an example, the yields of the DNA nanosystems and the products of each step during the assembly process were tested, the yields of the products 1-3 in the table = concentration of DNA folding templates after reaction purification/assembly concentration of DNA folding templates before purification; the yields of product 4 in the table were calculated as SA before and after the reaction, and the results are shown in Table 1.
TABLE 1
The result shows that the DNA nano system has high yield by adopting the design mode of S1+S2.
Test example 4
Targeting effect test of DNA nanosystems at cellular level
(1) Construction of DNA nanosystems on the surface of cancer cell membranes
Mouse breast cancer cells 4T1 were inoculated into 24-well plates with 500. Mu.L of medium containing 1X 10 per well 5 Individual cells. Cells were incubated overnight, medium was aspirated, PBS (10. Mu.L), functional DNA nanoplatelets of comparative example 2 (20 nM, 10. Mu.L, pH 7.4), functional DNA nanoplatelets of comparative example 2 (20 nM, 10. Mu.L, pH 6.5), bifunctional two-dimensional DNA nanoplatelets of example 2 (20 nM, 10. Mu.L, pH 7.4), bifunctional two-dimensional DNA nanoplatelets of example 2 (20 nM, 10. Mu.L, pH 6.5), incubated for 1h, medium was aspirated, each well was rinsed 3 times with PBS of the corresponding pH, medium of the corresponding pH containing the coupling product of SA-ICG of 0.4. Mu.M was then added to each well, incubation was continued for 0.5h, medium was then removed, the cells were switched to PBS of different pH, blown down with a gun head, centrifuged (1000 rpm, 4 min), and resuspended with PBS of the corresponding pH. Wherein pH 7.4 is simulated normal physiological condition, and pH 6.5 is simulated tumorConditions of the 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 result shows that the bifunctional two-dimensional DNA nanosheets assembled with pHLIP polypeptide of the invention spontaneously intercalate into cancer cell membranes through conformational transformation in the tumor microenvironment of pH 6.5 and successfully identify and combine with the coupling products of SA-ICG, thereby proving the successful construction of the DNA nanosystems of the invention on the surfaces of the cancer cell membranes. Moreover, the nano system does not respond under the normal physiological condition of pH 7.4, and is embedded into cell membranes only under the 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 with 100. Mu.L of medium per well containing 1X 10 4 Cells were incubated overnight, medium was aspirated and the following treatments were performed, respectively:
(1) PBS treatment group: PBS (10. Mu.L) was added and incubated for 1.5h;
(2) SA-Ce6 treatment group alone: PBS (25. Mu.M, 10. Mu.L) containing the SA-Ce6 conjugate product from step (4) of example 1 was added and incubated for 1.5h;
(3) pH 7.4-DNA nanosystems treatment group (7.4TO+SA-Ce 6): adding PBS (100 nM,10 μL, pH 7.4) containing the DNA bifunctional nanoplatelets obtained in step (3) of example 1, incubating for 1h, washing, adding PBS (25 μM,10 μL, pH 7.4) containing the SA-Ce6 coupled product obtained in step (4) of example 1, and incubating for 0.5h;
(4) pH 6.5-DNA nanosystems treatment group (6.5TO+SA-Ce 6): adding PBS (100 nM,10 μL, pH 6.5) containing the DNA bifunctional nanoplatelets obtained in step (3) of example 1, incubating for 1h, washing, adding PBS (25 μM,10 μL, pH 6.5) containing the SA-Ce6 coupled product obtained in step (4) of example 1, and incubating for 0.5h;
each group was subjected to the above treatment and irradiated with a laser having a wavelength of 650nm for 4 minutes at a laser intensity of 0.7W/cm 2 (while each group was not illuminated as a control). After 20h the medium was aspirated and incubation was continued for 1h with the addition of 100. Mu.L of CCK solution. The viability of each group of cancer cells was measured using an enzyme-labeled instrument.
The results are shown in FIG. 7.
As shown in the figure, the survival rate of the cancer cells is lower than 40% after the photo-thermal treatment by the pH 6.5-DNA nanosystem treatment group (6.5TO+SA-Ce 6), which is far lower than that of other treatment groups, and the excellent targeting effect of the DNA nanosystem on the cancer cells is proved, so that the DNA nanosystem has great application potential in photo-thermal treatment.
Test example 5
Tumor targeting effect test of DNA nanosystems at animal level
(1) Construction of DNA nanosystems in animals
Mouse breast cancer cells 4T1 were dissolved in a mixed solution of PBS and matrigel (PBS: matrigel 1:1), inoculated subcutaneously into the upper right leg of BALB/c female nude mice (6 weeks old) and each injected with 100. Mu.L of the mixed solution containing 5X 10 5 Individual cells. When the tumor grows to 150mm 3 When the mice were divided into 2 groups, group 1 was the experimental group, PBS (30 nM, 100. Mu.L) containing the DNA bifunctional nanoplatelets of example 2 was injected first; group 2 was a control group, in which PBS (100. Mu.L) was injected first, and after 12 hours both groups were injected simultaneously with SA-ICG-containing PBS (5. Mu.M, 100. Mu.L). Observations were made with the small animal optical living imaging system at 1, 6, 12, 24h, 48h after SA-ICG injection, respectively, and the results are shown in FIG. 8. Fluorescence intensities of mouse tumor areas at different time points were recorded. The results are shown in FIG. 9.
As shown in the figure, the fluorescence of the tumor region of the DNA nanosystem treatment group (TOBP+SA) is obviously stronger than that of the control group (PBS+SA), and the DNA nanosystem of the invention is proved to be successfully constructed in animals and has excellent tumor targeting effect.
(2) Photothermal therapy
Mouse breast cancer cells 4T1 were dissolved in a mixed solution of PBS and matrigel (PBS: matrigel volume ratio 1:1), inoculated subcutaneously into the upper right leg of BALB/c female nude mice (6 weeks old) and each injected with 100. Mu.L of the mixed solution containing 5X 10 5 Individual cells. To the extent that the tumor grows60mm 3 Mice were divided into 5 groups. The following treatments were respectively carried out:
(1) control group (PBS): PBS (100. Mu.L) was injected, without illumination;
(2) PBS treatment group (pbs+l): PBS (100. Mu.L) was injected, and light was applied after 12 hours;
(3) DNA bifunctional nanoplatelet treatment group alone (TOBP): injecting PBS (150 nM,100 μL) containing the DNA bifunctional nanoplatelets obtained in step (3) of example 1, and irradiating after 12 hours;
(4) SA-Ce6 treatment group alone (SA-Ce 6): injecting PBS (30 mu M,100 mu L) containing the SA-Ce6 coupled product obtained in the step (4) of the example 1, and irradiating after 12 hours;
(5) DNA nanosystem treatment group (tobp+sa-Ce 6): firstly injecting PBS (150 nM,100 mu L) containing the DNA difunctional nanosheets obtained in the step (3) of the example 1, injecting PBS (30 mu M,100 mu L) containing the SA-Ce6 coupling product obtained in the step (4) of the example 1 after 24 hours, and carrying out illumination after 12 hours;
the illumination conditions are unified as follows: 650nm laser, irradiation time of 30min, intensity of 0.7W/cm 2 。
Figure 10 is a statistical plot of tumor volume versus time for each group of mice over 16 days post-illumination. The tumor volume calculation method comprises the following steps: tumor volume = L x W 2 And (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-Ce 6) was significantly lower than that of the other treated groups, indicating that DNA nanosystem treated tumor growth was significantly inhibited. The DNA nano system has excellent targeting effect on tumors, obvious photothermal treatment effect on the tumors and great clinical application potential.
The applicant states that the present invention is described by the above examples to illustrate a DNA nanosystem for tumor targeting and a method for preparing the same and application thereof, but the present invention is not limited to the above examples, i.e. it does not mean that the present invention must be implemented depending on the above examples. It should be apparent to those skilled in the art that any modification of the present invention, equivalent substitution of raw materials for the product of the present invention, addition of auxiliary components, selection of specific modes, etc., falls within the scope of the present invention and the scope of disclosure.
The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the specific details of the above embodiments, and various simple modifications can be made to the technical solution of the present invention within the scope of the technical concept of the present invention, and all the simple modifications belong to the protection scope of the present invention.
SEQUENCE LISTING
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<120> a DNA nanosystem for tumor targeting, and preparation method and application thereof
<130> 2022-1-14
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Claims (12)
1. A DNA nanosystem for tumor targeting, characterized in that the DNA nanosystem for tumor targeting comprises a two-dimensional DNA nanosheet, single-stranded DNA coupled to a low pH intercalating peptide, biotin-modified single-stranded DNA and a streptavidin-functional molecule conjugate;
wherein the two-dimensional DNA nano-sheet 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 mode, and the double-functional two-dimensional DNA nano-sheet layer is formed by assembly; the streptavidin-functional molecule conjugate is combined with the difunctional two-dimensional DNA nano-sheet layer through the specific recognition effect of the streptavidin and the biotin to form the DNA nano-system for tumor targeting;
the two-dimensional DNA nanosheet layer is constructed by a scaffold chain, a stapling short chain, an upper layer capturing chain and a lower layer capturing chain through base complementary pairing;
the functional molecule is a drug molecule or an imaging molecule;
the amino acid sequence of the low pH insertion peptide in the single-stranded DNA coupled with the low pH insertion peptide is shown as SEQ ID NO. 1;
the DNA sequence of the single-stranded DNA coupled with the low pH insertion peptide is shown as SEQ ID NO. 2;
the DNA sequence of the biotin modified single-stranded DNA is shown as SEQ ID NO. 3.
2. The DNA nanosystem for tumor targeting according to claim 1, characterized in that the drug molecule comprises chlorin and/or pheophorbide a.
3. The DNA nanosystem for tumor targeting of claim 1, wherein the imaging molecule comprises any one or a combination of at least two of Cy-series fluorescent dyes, AF-series fluorescent dyes, or indocyanine green.
4. A method of preparing a DNA nanosystem for tumor targeting as claimed in any one of claims 1-3, wherein the method of preparing comprises the steps of:
(1) Mixing the two-dimensional DNA nano-sheet, the single-stranded DNA coupled with the low pH insertion peptide and the biotin-modified single-stranded DNA, heating and annealing to obtain a double-functional two-dimensional DNA nano-sheet;
(2) And mixing the difunctional two-dimensional DNA nanosheets with the streptavidin-functional molecule conjugate to obtain the DNA nanosystem for tumor targeting.
5. The method of claim 4, wherein the two-dimensional DNA nanoplatelets in step (1) are prepared by mixing scaffold chains, staple chains, upper capture chains and lower capture chains, heating, and annealing.
6. The method of claim 5, wherein the heating is at 92-98 ℃ for 2-8min; the final temperature of the annealing is 15-40 ℃; the annealing time is 10-16h; the annealing is followed by purification; the purification refers to passing through a centrifugal purification column; the rotational speed of the centrifugation is 3000-8000r/min.
7. The method according to claim 4, wherein the method for preparing the single-stranded DNA coupled with the low pH intercalating peptide in the step (1) comprises the steps of: mixing the low pH insertion peptide with the azide-modified DNA single strand.
8. The method of claim 7, wherein the mixing is performed in the absence of light; the temperature of the mixing is 20-40 ℃, and the mixing time is 12-24 hours; the mixing step further comprises separation and purification; the separation and purification refers to separation and purification by using denaturing polyacrylamide gel electrophoresis.
9. The method of claim 4, wherein the method of preparing the streptavidin-functional molecule conjugate of step (2) comprises: mixing streptavidin with functional molecule.
10. The method of claim 9, wherein the mixing is performed at 20-40 ℃ for a period of 1-3 hours; the mixing step further comprises purification; the purification refers to purification by a desalting column.
11. The method of claim 10, wherein the temperature of the mixing in step (2) is 25-40 ℃ and the time of the mixing is 20-120min.
12. Use of a DNA nanosystem for tumor targeting according to any of claims 1-3 or a method of preparation of a DNA nanosystem for tumor targeting according to any of claims 4-11 for the preparation of a medicament for the treatment of tumors.
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