A temperature-controlled nano drug release system and its application

Abstract

The present invention provides a temperature-controlled nano drug release system and its application; the nano drug release system comprises: component A and component B; component A is a temperature-controlled nano drug carrier obtained by connecting a temperature-sensitive DNA stem-loop structure with a DNA tetrahedron structure as the main body; component B is a free DNA single strand. The temperature-controlled nano drug release system provided by the present invention utilizes the temperature difference between tumor tissue and healthy tissue, and is designed to enable the nano drug release system to have the ability to respond to temperature changes. When around normal tissue, the temperature-sensitive DNA stem-loop structure part carrying the therapeutic drug is in a closed state, and when around tumor tissue, the temperature-sensitive DNA stem-loop structure part carrying the therapeutic drug is in an open state, so that the therapeutic drug is targeted and released to the tumor tissue, effectively reducing the toxic and side effects of the drug on normal tissue, and further improving the tumor healing effect.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a temperature-controlled nano drug release system and application thereof. Background Art

[0002] Tumor is one of the major diseases threatening human health in today's society. Every year, a large amount of manpower and material resources are consumed worldwide for related treatment and research. Common superficial tumors (tumors occurring in superficial parts of the body) include melanoma, breast cancer, lipoma, etc. The conventional treatment for superficial tumors is surgical resection, which can quickly achieve therapeutic effects by directly removing the tumor and surrounding tissues. However, large-scale tissue resection is prone to postoperative wound infection and difficulty in healing.

[0003] In order to avoid the problems caused by surgical treatment, thermotherapy is widely used to treat superficial tumors. Thermotherapy uses the different tolerance temperatures of normal cells and cancer cells to kill tumor cells by heating the lesion (setting the temperature within the range that is most lethal to cancer cells and least damaging to normal cells). Usually, infrared lamps, hot compresses, hot baths, etc. are used directly on the skin surface to raise the temperature of the target tissue to above 42°C using the heat transfer effect. Therefore, thermotherapy is a treatment method with easy-to-understand principles and simple operation. However, thermotherapy also has problems in treating superficial tumors. For example, when using higher temperature thermotherapy, the high temperature will not only kill cancer cells but also damage normal tissues; while the treatment temperature is low, it may not kill enough cancer cells and fail to achieve the treatment purpose.

[0004] Therefore, improving the effectiveness of thermotherapy in treating superficial tumors is a difficult problem that needs to be overcome urgently in current tumor research. Summary of the invention

[0005] In view of the above-mentioned problems existing in the prior art, the present invention provides a temperature-controlled nano-drug release system and its application. When using thermotherapy to treat superficial tumors, it is assisted by a temperature-controlled nano-drug carrier carrying tumor drugs to accurately and effectively improve the effect of thermotherapy in treating superficial tumors.

[0006] In a first aspect, the present invention provides a temperature-controlled nano drug release system, the nano drug release system comprising: component A and component B;

[0007] Among them, the component A is a temperature-controlled nano drug carrier with a DNA tetrahedron structure as the main body, which is obtained by connecting a thermosensitive DNA stem-loop structure; the component B is a free DNA single strand.

[0008] Preferably, the preparation method of component A comprises:

[0009] Four DNA long chains numbered Th-A, Th-B, Th-C, and Th-D were mixed in equal proportions, and then 1×PBS solution was added. After mixing evenly, the mixture was placed in a PCR instrument and self-assembled through annealing reaction.

[0010] Preferably, the concentrations of the four long DNA chains are all 1 uM.

[0011] Preferably, the pH of the 1×PBS solution is 7.4; the 1×PBS solution is composed of a mixture of 136.89 mM NaCl, 2.67 mM KCl, 8.1 mM Na2HPO4 and 1.76 mM KH2PO4.

[0012] Preferably, the nucleotide sequence of the long DNA chain numbered Th-A is shown in SEQ ID NO: 1;

[0013] The nucleotide sequence of the long DNA chain numbered Th-B is shown in SEQ ID NO: 2;

[0014] The nucleotide sequence of the long DNA chain numbered Th-C is shown in SEQ ID NO: 3;

[0015] The nucleotide sequence of the long DNA chain numbered Th-D is shown in SEQ ID NO:4.

[0016] Preferably, the temperature-sensitive DNA stem-loop structure changes from a closed state to an open state at 45° C. under the action of the free DNA single strand.

[0017] Preferably, the nucleotide sequence of the free single-stranded DNA is shown in SEQ ID NO:5.

[0018] Preferably, the concentration of the free single-stranded DNA is 6 uM.

[0019] In a second aspect, the present invention provides an application of a temperature-controlled nano-drug release system, wherein the nano-drug release system responds to temperature changes in superficial tumor tissue and releases therapeutic drugs in a targeted manner in the superficial tumor tissue.

[0020] Compared with the prior art, this application has the following advantages:

[0021] (1) The nano drug release system provided by the present invention utilizes the temperature difference between tumor tissue and healthy tissue and is designed to enable the nano drug release system to respond to temperature changes. When around normal tissue, the thermosensitive DNA stem-loop structure carrying the therapeutic drug is in a closed state. When in the tumor tissue environment, the thermosensitive DNA stem-loop structure carrying the therapeutic drug is in an open state, so that the therapeutic drug is released in a targeted manner to the tumor tissue, effectively reducing the toxic and side effects of the drug on normal tissue and further improving the tumor cure effect.

[0022] (2) The present invention selects DNA nanomaterials to design a drug release system for treating superficial tumors, wherein the DNA tetrahedron structure is used as the main structure. Because of its natural biocompatibility and cell penetration ability, it can carry the thermosensitive DNA stem-loop structure after drug loading, penetrate the cell membrane through endocytosis and enter living cells, so that the DNA stem-loop structure can smoothly respond to the temperature difference between tumor tissue and healthy tissue, thereby completing the task of targeted release of therapeutic drugs to tumor tissue. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The schematic diagram of the structure of the temperature-controlled nano-drug carrier in the temperature-controlled nano-drug release system provided by the embodiment of the present invention is shown;

[0024] Figure 2 A schematic diagram showing the process of targeting tumor cells with a temperature-sensitive DNA stem-loop structure in the temperature-controlled nano-drug release system provided by an embodiment of the present invention is shown;

[0025] Figure 3 The figure shows the melting degree curve of the DNA stem-loop structure as the temperature changes in the temperature-controlled nano drug release system provided by the embodiment of the present invention. DETAILED DESCRIPTION

[0026] The following examples are provided for a better understanding of the present invention, but are not intended to limit the best mode of implementation, nor to limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior arts shall fall within the protection scope of the present invention.

[0027] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the prior art can be used. The reagents and other instruments used without specifying the manufacturer are all conventional reagent products that can be purchased commercially.

[0028] In order to further improve the efficacy of non-surgical treatment of superficial tumors and reduce the side effects of systemic administration, the technical concept proposed by the present invention is: using DNA as raw material to construct a temperature-controlled nano drug release system. Among them, the temperature-sensitive DNA stem-loop structure is connected with DNA tetrahedron, which constitutes the core part of the drug release system. The system can respond automatically according to temperature changes (the temperature-sensitive DNA stem-loop structure loaded with drugs automatically binds to the free DNA single strand at a preset temperature, so that the stem-loop structure changes from a closed state to an open state, and the therapeutic drugs adapted to the closed stem-loop structure are targeted and released to the tumor tissue with a higher temperature, thereby reducing the damage of the drugs to normal tissue cells. When used in conjunction with thermotherapy, the therapeutic effect of tumors can be further improved. Based on the above technical concept, the inventor provides a temperature-controlled nano drug release system and its application, and the specific implementation content is as follows:

[0029] In a first aspect, an embodiment of the present invention provides a temperature-controlled nano drug release system, the nano drug release system comprising: component A and component B;

[0030] Among them, component A is a temperature-controlled nano drug carrier with a DNA tetrahedron structure as the main body and connected to a temperature-sensitive DNA stem-loop structure;

[0031] Component B is a free single-stranded DNA.

[0032] During the specific implementation, the inventors found that after understanding the metabolic process of tumor cells, the metabolic process of tumor cells is different from that of normal cells. It can adapt to changes in the metabolic environment through the conversion between glycolysis and oxidative phosphorylation, and can also change the surrounding environment through cell secretion and other effects to make it conducive to the growth and development of tumors. On the one hand, these metabolic pathways increase the intensity of the immune response near the tumor tissue, causing it to generate a large amount of heat. On the other hand, tumor tissue requires more nutrient supply, so there are more blood vessels and they are distributed in a disorderly manner, which leads to poor heat dissipation of tumor tissue, causing an "endogenous" high temperature phenomenon near the tumor tissue, and the temperature of the tumor tissue is higher than that of healthy tissue. In addition, when using thermotherapy to treat superficial tumors (the temperature is determined to be in the range that is most lethal to cancer cells and least damaging to normal cells), the temperature of the target tissue will also rise to 42°C-45°C, resulting in an "exogenous" high temperature phenomenon, thereby killing tumor cells.

[0033] In addition, in existing drug delivery methods, the nano-drug carriers used are generally modified with specific recognition groups on the surface. The recognition groups will randomly and non-specifically bind to normal cells, ultimately resulting in low aggregation efficiency of the drug carriers near the target cells; conventional nano-structured carriers have the characteristics of structural stability within the temperature range that the human body can adapt to, and cannot respond to slight changes in body temperature, cannot change the spatial conformation of the nano-carriers to control the release of drugs, and cannot respond to this widely existing temperature difference phenomenon, thereby limiting the effectiveness and reliability of nano-drug carriers.

[0034] Therefore, in the specific implementation, the inventor hopes to combine the thermotherapy with a carrier administration method that can respond to temperature changes, so as to release drugs in a targeted manner to tumor cells at higher temperatures, and further improve the therapeutic effect of thermotherapy on superficial tumors.

[0035] In specific implementation, in the temperature-controlled nano drug release system provided by the present invention, the DNA stem-loop structure has a special temperature-sensitive property. The stem-loop structure is a long chain with complementary sequences at both ends. At room temperature, the two ends of the stem-loop structure are paired to form a special structure with a closed cavity, but at a higher temperature, the special structure will unwind and return to a single-stranded state.

[0036] During specific implementation, due to the small particle size of the single DNA stem-loop structure itself, when used as a drug carrier, it is easily metabolized and excreted from the body, and it is difficult to exert a specific therapeutic effect. Therefore, the inventor connects the DNA stem-loop structure with a smaller particle size to a DNA tetrahedron structure with a larger particle size and stronger stability, so as to increase the particle size and structural stability of the DNA stem-loop structure, and prevent the drug-carrying DNA stem-loop structure from being metabolized and excreted from the body and unable to reach the patient. In the temperature-controlled nano drug release system provided by the present invention, the DNA tetrahedron structure has excellent cell membrane permeability because it can penetrate the cell membrane into living cells through cellular endocytosis without using any ligand or transfection agent. At the same time, the DNA tetrahedron structure is based on double-stranded DNA, which can effectively resist the attack of nucleases and proteases without being hydrolyzed by them, and has the ability to maintain structural integrity and stability for a long time. Therefore, the temperature-controlled nano drug carrier obtained by connecting the DNA stem-loop structure and taking the DNA tetrahedron structure as the main body can ensure the integrity and stability of the DNA stem-loop structure, so that the DNA stem-loop structure can smoothly respond to the temperature difference between tumor tissue and healthy tissue, and complete the task of targeted release of therapeutic drugs to the tumor tissue.

[0037] Figure 1 FIG. 4 shows a schematic diagram of the structure of a temperature-controlled nano-drug carrier in a temperature-controlled nano-drug release system provided by an embodiment of the present invention. Figure 1As shown, the present invention has the unique advantages of good biocompatibility, easy modification, and easy functionalization by means of the DNA tetrahedron structure. In the sequence design stage, the temperature-sensitive DNA stem-loop structure is simply and efficiently connected to the DNA tetrahedron structure by the base pairing method. Among them, the temperature-sensitive DNA stem-loop structure reserves a region in its base sequence, which is set as the relevant sequence of the nucleic acid aptamer to obtain a temperature-controlled nano drug carrier, and the relevant functions of the aptamer can be realized when its stem-loop structure is in an open state. The temperature-controlled nano drug carrier is made to have the ability to maintain structural integrity and stability for a long time and the function of temperature self-adaptation.

[0038] In specific implementation, the temperature-controlled nano-drug carrier is mixed with a certain concentration of free DNA single strands to form a temperature-controlled intelligent drug release system. This system can monitor the body's environmental temperature. After determining that the environment is tumor tissue, it can target and bind to tumor cells and induce cell endocytosis through the conformational change of the temperature-sensitive DNA stem-loop structure (from closed state to open state), thereby completing the intelligent drug release controlled by body temperature changes.

[0039] Figure 2 The schematic diagram of the process of targeting tumor cells with a temperature-sensitive DNA stem-loop structure in the temperature-controlled nano-drug release system provided by an embodiment of the present invention is shown. Figure 2 As shown in the figure, the stem-loop structure is composed of a long single-stranded DNA, which is divided into two parts. The dotted part is the functional nucleic acid molecule sequence, and the double solid line part is the sequence that is complementary to the dotted part. Under low temperature conditions, the two ends of the long single strand self-pair to form a stem-loop structure. When the ambient temperature rises, the stem-loop structure can react with a DNA of a specific sequence to form a double-stranded binding region. The stem-loop structure changes from a closed state to an open state, and the functional nucleic acid molecule sequence is released to bind to the target molecule.

[0040] In specific implementation, preferably, the preparation method of component A comprises:

[0041] Four DNA long chains numbered Th-A, Th-B, Th-C, and Th-D were mixed in equal proportions, and then 1×PBS solution was added. After mixing evenly, the mixture was placed in a PCR instrument and self-assembled through annealing reaction.

[0042] In specific implementation, preferably, the concentration of the four long DNA chains is 1 uM.

[0043] In specific implementation, preferably, the pH of 1×PBS solution is 7.4; 1×PBS solution is composed of a mixture of 136.89 mM NaCl, 2.67 mM KCl, 8.1 mM Na2HPO4 and 1.76 mM KH2PO4.

[0044] In a specific implementation, preferably, the nucleotide sequence of the DNA long chain numbered as Th-A is shown in SEQ ID NO: 1;

[0045] The nucleotide sequence of the long DNA chain numbered Th-B is shown in SEQ ID NO: 2;

[0046] The nucleotide sequence of the long DNA chain numbered Th-C is shown in SEQ ID NO: 3;

[0047] The nucleotide sequence of the long DNA chain numbered Th-D is shown in SEQ ID NO:4.

[0048] In specific implementation, preferably, the temperature-sensitive DNA stem-loop structure changes from a closed state to an open state under the action of a free DNA single strand at 45°C.

[0049] In specific implementation, the temperature-controlled nano-drug release system provided by the present invention has an inactive drug carrier under normal body temperature, which effectively reduces the endocytosis of drug carriers by normal cells; near tumor tissue with higher temperature, the nano-drug carrier is activated as the temperature rises, and the specific binding with the tumor tissue is completed through the change of the drug carrier conformation.

[0050] In specific implementation, the temperature at which the thermosensitive DNA stem-loop structure melts is determined by the base sequence, and stem-loops composed of different base sequences have completely different temperature curves.

[0051] Figure 3 The figure shows the melting degree curve of the DNA stem-loop structure as the temperature changes in the temperature-controlled nano drug release system provided by the embodiment of the present invention. Figure 3As shown, in the temperature-controlled nano drug release system provided by the present invention, the DNA stem-loop structure is designed through sequence optimization, and the FAM fluorescent group and the BHQ1 quenching group are modified at both ends of the stem-loop region respectively. The spatial conformational changes of the drug carrier assembled by DNA can cause changes in the fluorescence signal: when the temperature is lower than 37°C, the stem-loop is in a closed state, and the spatial distance between the fluorescent group FAM and the quenching group BHQ1 at both ends of the stem-loop is relatively close, and the fluorescence signal is quenched; when the temperature rises to about 45°C, the free DNA single strand binds to the stem-loop, the stem-loop is in an open state, and the spatial distance between the fluorescent group and the quenching group at both ends of the stem-loop increases, the quenching effect gradually disappears, and the system produces a strong fluorescence signal. By monitoring the changes in the fluorescence signal, the drug release of the nanocarrier can be reflected in real time. In specific implementation, the DNA stem-loop structure can control the melting percentage to about 24% at 37°C. When the temperature reaches 45°C, the DNA stem-loop structure is in a completely open state. Such a reaction temperature node has a certain similarity with the temperature change related to tumors, that is, under the normal body temperature of 37°C for healthy tissues, the stem-loop is in a closed state, and under the condition of endogenous or exogenous temperature rise to 45°C for tumor tissues, the stem-loop is in an open state. The closed and open states of the stem-loop structure correspond to different nanostructure conformations, and on this basis, an intelligent controlled-release nanodevice based on temperature changes is constructed.

[0052] In specific implementation, preferably, the nucleotide sequence of the free single-stranded DNA is shown in SEQ ID NO: 5.

[0053] In specific implementation, preferably, the concentration of free single-stranded DNA is 6 uM.

[0054] In a second aspect, the present invention provides an application of a temperature-controlled nano-drug release system, which responds to temperature changes in superficial tumor tissue and releases therapeutic drugs in a targeted manner in the superficial tumor tissue.

[0055] In specific implementation, the DNA tetrahedron structure is used as the main structure of the nanodrug carrier, and the temperature-sensitive stem-loop structure is used as the functional element. The combination of the two can construct a nanodrug release system with temperature change control. The nanodrug release system responds to the temperature changes of superficial tumor tissues and targets the release of therapeutic drugs in superficial tumor tissues, effectively reducing the side effects of general systemic administration of nanodrugs and improving the therapeutic effect of hyperthermia therapy for superficial tumors.

[0056] In order to enable those skilled in the art to more clearly understand the present invention, the method of the present invention is now described in detail through the following examples.

[0057] Embodiment 1:

[0058] (1) The dry powdered single-stranded DNA ordered with the numbers Th-A, Th-B, Th-C and Th-D were dissolved and the concentration was measured.

[0059] First, the dry fractionated DNA molecule numbered Th-A was centrifuged at 8000 rpm for 3 minutes. After centrifugation, the sample tube was carefully removed, and an appropriate amount of deionized water (the amount added is indicated on the wall of the packaging tube) was added, vortexed for 3 minutes, and the solution was collected to the bottom of the tube using a hand-held centrifuge for measuring the concentration of dissolved DNA.

[0060] Then, use Thermo NanoDrop micro-spectrophotometer measures the OD value of the solution, cleans the sample stage with anhydrous ethanol, and drops 2 μL of deionized water as a blank control. Test the OD value of the DNA molecule at a wavelength of 260 nm, and calculate the concentration of the DNA molecule based on its molecular weight.

[0061] Finally, the four long DNA chains numbered Th-B, Th-C and Th-D were dissolved and their concentrations were measured using the same method as that for the single DNA chain numbered T1.

[0062] (2) Preparation of temperature-controlled nano-drug release system

[0063] The four DNA long chains Th-A, Th-B, Th-C and Th-D were mixed in 1×PBS solution (136.89mM NaCl; 2.67mM KCl; 8.1mM Na2HPO4; 1.76mM KH2PO4; pH=7.4) in equal proportions, and the concentration of each chain was 1uM. The mixture was dispensed into PCR tubes (EP tubes with a volume of 200μL), and annealing was completed according to the set temperature program. After annealing, the DNA short chain CS was added to make its concentration ratio with the long chain 6:1. The specific sequence is shown in Table 1, where the 5' end and the appropriate position in the middle of the four long chains Th-A, Th-B, Th-C and Th-D were modified with FAM fluorescent groups and BHQ1 quenching groups. The mixed solution obtained a temperature-controlled nano drug release system and was stored at 4°C.

[0064] Table 1: DNA tetrahedron sequence design

[0065]

[0066]

Claims

1. A temperature-controlled nano drug release system, characterized in that: The nano drug release system comprises: component A and component B; Wherein, the component A is a temperature-controlled nano drug carrier based on a DNA tetrahedron structure and obtained by connecting a temperature-sensitive DNA stem-loop structure; The component B is a free single-stranded DNA; The component A is formed by self-assembly of four DNA long chains numbered Th-A, Th-B, Th-C, and Th-D; the nucleotide sequence of the DNA long chain numbered Th-A is shown in SEQ ID NO: 1; the nucleotide sequence of the DNA long chain numbered Th-B is shown in SEQ ID NO: 2; the nucleotide sequence of the DNA long chain numbered Th-C is shown in SEQ ID NO: 3; the nucleotide sequence of the DNA long chain numbered Th-D is shown in SEQ ID NO: 4; The nucleotide sequence of the free single-stranded DNA is shown in SEQ ID NO: 5; The temperature-sensitive DNA stem-loop structure changes from a closed state to an open state under the action of the free DNA single strand at 45°C.

2. The nano drug release system according to claim 1, characterized in that: The preparation method of component A comprises: Four DNA long chains numbered Th-A, Th-B, Th-C, and Th-D were mixed in equal proportions, and then 1×PBS solution was added. After mixing evenly, the mixture was placed in a PCR instrument and self-assembled through annealing reaction.

3. The nano drug release system according to claim 2, characterized in that: The concentrations of the four DNA long chains were all 1 μM.

4. The nano drug release system according to claim 2, characterized in that: The pH value of the 1×PBS solution is 7.4; the 1×PBS solution is composed of a mixture of 136.89 mM NaCl, 2.67 mM KCl, 8.1 mM Na2HPO4 and 1.76 mM KH2PO4.

5. The nano drug release system according to claim 4, characterized in that: The concentration of the free single-stranded DNA was 6 μM.

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