A method for preparing biomolecular nanoparticles by freezing

Abstract

The present invention discloses a method for preparing biomolecule nanoparticles by freezing, and proposes for the first time to drive biomolecule self-assembly to form nanoparticles by freezing method. With biomolecules as raw materials, whether or not additives are added, after the solution is frozen by cooling, water molecules crystallize, thereby repelling biomolecules and additives from growing ice crystals, resulting in an increase in their local concentration in non-ice areas, thereby enhancing the attachment dynamics of biomolecules, and this concentration effect strengthens the intramolecular and intermolecular electrostatic attraction, π-π stacking and hydrophilic-hydrophobic interaction between biomolecules or between biomolecules and additives, resulting in biomolecule self-assembly to form nanoparticles. The preparation method of the present invention is simple, and the required refrigerator is available in ordinary households, and is suitable for the assembly of antigen peptides that are unstable at high temperatures and high-temperature unstable drugs that need to be assembled and used in conjunction with biomolecules, and is particularly suitable for the preparation of nano vaccines.

Description

Technical Field

[0001] The invention belongs to the field of nano materials, and in particular relates to a method for preparing biomolecular nanoparticles by freezing. Background Art

[0002] Cryo-processing technology is a wet processing technology that uses the directional solidification of water-based (or other solvent) suspensions (metals or polymers) to cause the particles to directly self-assemble and form highly porous bulk materials. In frozen aqueous solutions, as the ice crystals grow, they expel nanoparticles, macromolecules and nanosheets that are gathered between the ice crystals, thereby obtaining a layered ice crystal structure. In the gaps of the ice crystal structure, the local concentration of solute molecules increases, resulting in an increase in the interaction between its molecules, mainly molecular electrostatic attraction, electrostatic attraction, π-π stacking and hydrophilic and hydrophobic interactions. The solutes form nanomaterials between the ice crystals, including nanoparticles, nanofibers and nanosheets. In addition, the addition of additives will affect the generation and growth of ice crystals. For example, glycerol can inhibit ice recrystallization (IRI), lower the freezing point and control the shape and growth of ice, thereby affecting the morphology of nanoparticles. Therefore, various types of microstructures can be obtained depending on the additives, freezing conditions, and solvents used.

[0003] Biomolecules are a general term for molecules that exist naturally in living organisms. Most biomolecules are organic compounds containing carbon, hydrogen, oxygen, nitrogen, phosphorus and sulfur, and sometimes other elements. Common biomacromolecules mainly include: (1) Nucleic acids, mainly including one or more of DNA, siRNA, mRNA, oligonucleotides, circular RNA / DNA, double-stranded RNA / DNA, and plasmids. (2) Amino acid peptides: one or more of amino acids, dipeptides, cyclic peptides, oligopeptides, polypeptides, and proteins. These biomolecules have certain functions in organisms, so biomolecules are widely used in the field of biomedicine. However, due to the easy degradation of biomolecules themselves, they are often degraded by biological enzymes during metabolism in the body and cannot achieve the expected effect. For example, nucleic acids have been widely used in the prevention, diagnosis and treatment of cancer. Various oligoribonucleotides (RNA) or oligodeoxyribonucleotides (DNA) with different functions mainly play a role at the gene level. Nucleic acid drugs are developed to treat diseases that are specific to pathogenic genes, that is, they have specific targets and mechanisms of action. Therefore, nucleic acid drugs, especially small nucleic acid drugs, show excellent application prospects in the field of biological diagnosis and treatment. However, due to the instability of chain nucleic acids, they are easily degraded by nucleases, and the transfection efficiency of circular nucleic acids is low due to the negative charge of nucleic acids themselves, which seriously restricts the application of functional nucleic acids. In addition, for amino acid peptides, the in vivo polypeptide is biologically active after synthesis or protein hydrolysis, and it can play various physiological functions, such as enzyme activity, antibacterial, immunomodulatory, antihypertensive and antioxidant, etc. Based on the biological multifunctionality of polypeptides, polypeptides have been developed and widely used in various fields such as catalysis, antibacterial, vaccines, cancer immunotherapy, etc. However, polypeptides are easily affected by enzymes and biological environments during application and lose their activity.

[0004] Therefore, biomolecules are often compounded with nanomaterials during delivery to resist the harsh biological environment. At the same time, the enhanced permeation retention effect (EPR effect) of nanomaterials is used to promote the selective accumulation of biomolecular drugs in tumors and increase the targeted delivery ability of biomolecules. In addition, since the bioaffinity of biomolecules improves the biocompatibility and dispersibility of nanomaterials, this biomolecule nanocomposite material also improves the optical, electrical, catalytic and other properties that biomolecules do not have. Although nanocarrier-assisted biomolecule delivery has many advantages compared with traditional biomolecule delivery, the use of inactive carriers also brings some negative effects. First, the introduction of a large number of inactive carriers not only reduces the drug loading (usually no more than 20%), but also increases the cost. Secondly, the side effects caused by the carrier may hinder its application. Micelle may cause immunogenicity, mesoporous silica shows hepatotoxicity, and the positively charged surface of liposomes, dendrimers and gold carriers also causes toxicity. Finally, the synthesis and surface functionalization modification of many carriers often involve complex processes and the use of toxic reagents. Therefore, it is of great significance to develop simple and safe biomolecule nanoformulations that do not contain toxic solvents and nanocarriers.

[0005] The present invention reports a method for preparing biomolecule nanoparticles by freezing. We use biomolecules as raw materials, and in the presence or absence of additives, the crystallization of water molecules through freezing will exclude biomolecules and additives from growing ice crystals, resulting in an increase in their local concentration in non-ice areas, thereby enhancing the attachment dynamics of biomolecules. This concentration effect strengthens the intramolecular and intermolecular electrostatic attraction, π-π stacking, and hydrophilic and hydrophobic interactions between biomolecules or between biomolecules and additives, resulting in the self-assembly of biomolecules to form nanoparticles. The method for preparing nanoparticles of the present invention is simple, green, mild and low-cost, and the resulting nanostructure has high stability. As a general method, the present invention provides a good reference for the development of the cold chain nano industry, especially the preparation of nano vaccines. Summary of the invention

[0006] The purpose of the present invention is to provide a method for preparing biomolecular nanoparticles by freezing, and for the first time proposes a method for driving biomolecular self-assembly to form nanoparticles by freezing. The preparation method of biomolecular nano self-assembly is simple, green, mild and low-cost.

[0007] To achieve the above object, the present invention adopts the following technical solution:

[0008] A method for preparing biomolecule nanoparticles by freezing, which uses a freezing method to drive the self-assembly of biomolecules to form nanoparticles; specifically, biomolecules are used as raw materials, and no matter whether additives are added, after the solution is cooled and frozen, water molecules crystallize, thereby repelling the biomolecules and additives from the growing ice crystals, resulting in an increase in local concentration in non-ice areas, thereby enhancing the attachment dynamics of the biomolecules. This concentration effect strengthens the intramolecular and intermolecular electrostatic attraction, π-π stacking and hydrophilic-hydrophobic interaction between biomolecules or between biomolecules and additives, resulting in the self-assembly of biomolecules to form nanoparticles.

[0009] In the present invention, preferably, the method for preparing biomolecular nanoparticles by freezing specifically comprises the following steps:

[0010] (1) dissolving the biomolecule raw material in a solvent to obtain a biomolecule stock solution with a concentration of 50 μM to 20 mM;

[0011] (2) dissolving the additive raw material in a solvent to obtain an additive stock solution with a concentration of 10 wt % to 100 wt %;

[0012] (3) taking 0.1 to 500 μL of the biomolecule stock solution, mixing it evenly with 0 to 500 μL of the additive stock solution, and then adding it to the solvent so that the final volume of the mixed solution is 10 to 1000 μL;

[0013] (4) The mixed solution is frozen at -20°C to -196°C for 1 min to 24 h, and then thawed naturally to room temperature. It is then centrifuged at 5000 to 10000 rpm for 1 to 60 min. The resulting precipitate is washed three times to obtain biomolecular nanoparticles.

[0014] In the present invention, there is no limitation on biological molecules, but the following are preferred: (1) nucleic acid biological molecules, mainly including one or more of single-stranded DNA, single-stranded RNA, double-stranded RNA, double-stranded DNA, circular RNA, circular DNA, oligonucleotides, and plasmids; (2) amino acid peptide biological molecules, mainly including one or more of amino acids, cyclic peptides, polypeptides, and proteins.

[0015] In the present invention, there is no limitation on whether the biological molecules are modified or not, and preferably, one or more small molecules such as: fluorescein isothiocyanate (FITC), phycoerythrin (PE), AlexaFluor series dyes, rhodamine, cyanine dyes (Cy3, Cy5, Cy5.5, Cy7), alkyl chains, etc.

[0016] Specifically, the nucleic acid biomolecule can be selected from any one of the following:

[0017] ① Single-stranded DNA: 5'-ATCCATCCCGACCTC-3';

[0018] ②Cy5-DNA: 5'-Cy5-TCCATGACGTTCCTGACGTT-3';

[0019] ③Cy5-CPG: 5'-Cy5-TCCATGACGTTCCTGACGTT-3';

[0020] ④Cy5-DNA-BHQ2: 5'-Cy5-CCGTTCTATCAACATCAGTCTGATAAGCTATAGAACGG-BHQ2-3';

[0021] ⑤siRNA: sense: 5'-GUGAAGUCAACAUGCCUGC-dTdT-3',

[0022] antisense: 5'-GCAGGCAUGUUGACUUCAC-dTdT-3';

[0023] ⑥pEGFP-C3 plasmid;

[0024] ⑦Ploy(I:C);

[0025] ⑧ mRNA of green fluorescent protein amFP486 (GFP): SEQ ID NO.11.

[0026] Specifically, the amino acid peptide biomolecule can be selected from any one of the following:

[0027] ① Polypeptide CL: N-terminus-CGTAFFINFIAIYHHASRAIPFGTMVAFRFFVLK(HHHHHH)FRSIINFEKL-C-terminus;

[0028] ② Peptide FQ-Cy5: N-terminus-Cy5-FFVLKHHHHHHKKKAVPIAQ-C-terminus;

[0029] ③ Polypeptide Obsl1: N-terminus-REGVELCPGNKYEMRRHGTTHSLVIHD-C-terminus.

[0030] In the present invention, there is no restriction on additives, and additives can be used to change freezing parameters, affect the growth of ice crystals, inhibit ice recrystallization, lower the freezing point and control the shape of ice, thereby changing the morphology of nanoparticles. Because they can affect the solvent phase diagram, the anisotropy of solid / liquid interfacial energy, the force between particles, the degree of undercooling, the viscosity of the solvent, the freezing point of the suspension and the volume expansion of the system. The promoting effect of additives on crystal growth can be through the kinetic effect of changing the growth rate, or through the thermodynamic effect of reducing the surface free energy. From a thermodynamic point of view, additives absorbed on specific surfaces of frozen crystals can adjust the anisotropy and porous structure through different modes of interfacial interaction between solid particles, additives and frozen crystals; additives are preferably: glycerol, dimethyl sulfoxide, ethylene glycol, trehalose, graphene, polymers (PVA, PVP, etc.); the final mass proportion of additives is preferably 0% to 30%.

[0031] In the present invention, preferably, the solvent can be selected from organic solvents (such as camphene, terpene, tert-butyl alcohol, dimethyl sulfoxide) and inorganic solvents (such as water, phosphate buffer, tris(hydroxymethyl)aminomethane hydrochloric acid buffer, 4-hydroxyethylpiperazineethanesulfonic acid buffer and any buffer with a pH between 5-9).

[0032] The significant advantages of the present invention are:

[0033] (1) The present invention discloses a method for preparing biomolecular nanoparticles by freezing, and for the first time proposes a method of driving biomolecular self-assembly to form nanoparticles by freezing. The water molecules are crystallized by cooling and freezing, thereby excluding biomolecules and additives from growing ice crystals, resulting in a high local concentration in non-ice areas. This concentration effect causes biomolecules to self-assemble to form nanoparticles. Compared with biomolecular nanoparticles prepared under high temperature conditions, the method for preparing nanoparticles of the present invention is simple; the equipment is low-cost and only a household refrigerator is required for preparation; it has a wider applicability and is suitable for the assembly of antigenic peptides that are unstable at high temperatures and high-temperature unstable drugs that need to be assembled and used in conjunction with biomolecules. The resulting nanostructure also has high stability.

[0034] (2) The preparation strategy of the present invention is particularly suitable for biomolecules that are unstable under high temperature conditions. For example, antigenic peptides will lose some antigens after heating, and proteins will denature after heating, while frozen assembly will not affect the properties of the biomolecules themselves.

[0035] (3) The nanoparticles provided by the present invention are non-toxic and harmless, have controllable sizes, are easy to prepare, and require mild conditions.

[0036] (4) The present invention provides a good reference for the development of the cold chain nano industry, especially the preparation of frozen nano vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 Reaction flow chart of frozen single-stranded DNA nanoparticles prepared for the example.

[0038] Figure 2 Transmission electron microscopy images of biomolecular nanoparticles prepared by freezing in water in Examples 1 and 2. (a) Single-stranded DNA nanoparticle 1; (b) Single-stranded DNA nanoparticle 2; (c) Cy5-DNA nanoparticle; (d) Cy5-DNA / DOX nanoparticle.

[0039] Figure 3 Transmission electron microscopy images of biomolecular nanoparticles prepared by freezing in water in Examples 3 to 6. (a) Peptide CL nanoparticles; (b) Peptide FQ-Cy5 nanoparticles; (c) Peptide Obsl1 / CPT nanoparticles; (d) Peptide CL / Cy5-CPG nanoparticles.

[0040] Figure 4 Characterization diagram of biomolecular nanoparticles prepared by freezing in 1×PBS buffer in Example 7. (a) TEM image of Cy5-DNA-BHQ2 nanoparticles prepared by freezing reaction at -20℃ for 24h; (b) TEM image of Cy5-DNA-BHQ2 nanoparticles prepared by freezing reaction at -80℃ for 5min; (c) TEM image of Cy5-DNA-BHQ2 nanoparticles prepared by freezing reaction at -196℃ for 5s; (d) particle size distribution of Cy5-DNA-BHQ2 nanoparticles prepared by freezing reaction at -80℃ for 5min; (e) potential change image of Cy5-DNA-BHQ2 nanoparticles prepared by freezing reaction at -80℃ for 5min.

[0041] Figure 5 The performance test results of Cy5-DNA-BHQ2 nanoparticles prepared by freezing in 1×PBS buffer at -80°C for 5 min in Example 7 are shown in Figure 1. (a) Fluorescence spectra of Cy5-DNA-BHQ2 nanoparticles incubated with different concentrations of miRNA-21; (b) Fitting curve of 665nm fluorescence peak value collected under 635nm excitation wavelength and miRNA-21 concentration after Cy5-DNA-BHQ2 nanoparticles were incubated with different concentrations of miRNA-21.

[0042] Figure 6 Characterization diagrams of biomolecular nanoparticles prepared by freezing in 1×PBS buffer in Example 8. (a) Transmission electron microscopy image of siRNA nanoparticles; (b) Particle size distribution of siRNA nanoparticles; (c) Potential change of siRNA nanoparticles.

[0043] Figure 7Transmission electron microscopy images of biomolecular nanoparticles prepared by freezing in 1×PBS buffer in Example 9. (a) pEGFP-C3 nanoparticles; (b) pEGFP-C3 / Zn 2+ Nanoparticles.

[0044] Figure 8 Fe3(CO) prepared in Example 10 12 Transmission electron microscopy image of / Cy5-Bcl2 nanoparticles.

[0045] Fig. 9 Transmission electron microscopy images of biomolecular nanoparticles prepared by freezing in 0.9% saline in Example 11. (a) Poly(I:C) nanoparticles; (b) Poly(I:C) / Obsl1 nanoparticles.

[0046] Fig.10 This is a transmission electron micrograph of the mRNA nanoparticles prepared by freezing in 0.9% saline in Example 12. DETAILED DESCRIPTION

[0047] In order to make the contents of the present invention easier to understand, the technical solution of the present invention is further described below in conjunction with specific implementation methods, but the present invention is not limited thereto.

[0048] Example 1

[0049] The biomolecule used in this example is single-stranded DNA (purchased from Shanghai Bioengineering Co., Ltd.), and its sequence is: 5'-ATCCATCCCGACCTC-3'.

[0050] The single-stranded DNA nanoparticles were prepared by freezing in water. Figure 1 As shown, the specific steps include:

[0051] (1) Centrifuge 2.5 OD of single-stranded DNA at 4000 rpm for 60 s, collect the precipitate and dissolve it in 200 μL of sterile water, pipette and mix thoroughly at room temperature to form a 100 μM DNA stock solution, and store it at -20°C;

[0052] (2) 20 μL of the DNA stock solution of step (1) was taken and mixed evenly with 80 μL of sterile water, and then the mixture was placed in a refrigerator and frozen at -80°C for 1 hour. After the freezing was completed, the sample was taken out and thawed naturally to room temperature, and then centrifuged at 7000 rpm for 5 minutes. The resulting precipitate was washed twice with sterile water to obtain single-stranded DNA nanoparticles 1 prepared by freezing in water;

[0053] (3) At room temperature, 50 mL of 100% v / v glycerol (purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.) was added to 50 mL of secondary water to obtain a 50% v / v glycerol stock solution, which was stored at room temperature;

[0054] (4) Take 2 μL of the DNA stock solution in step (1), mix it evenly with 15 μL of 50% v / v glycerol stock solution, and then add secondary water to make the final volume of the mixed solution 100 μL, then place the mixed solution in a refrigerator and freeze it at -80°C for 1 hour. After the freezing is completed, take it out and thaw it naturally to room temperature, then centrifuge it at 7000 rpm for 5 minutes, and wash the resulting precipitate twice with secondary water to obtain single-stranded DNA nanoparticles 2 prepared by freezing in water.

[0055] Example 2

[0056] The biomolecule used in this example is Cy5-DNA (purchased from Shanghai Bioengineering Co., Ltd.), and its sequence is: 5'-Cy5-TCCATGACGTTCCTGACGTT-3'.

[0057] Single-stranded Cy5-DNA nanoparticles were prepared by freezing in water. The specific preparation steps are as follows:

[0058] (1) Centrifuge 3.5 OD of Cy5-DNA at 4000 rpm for 60 s, collect the precipitate and dissolve it in 95.5 μL of sterile water, blow and mix thoroughly at room temperature in the dark to form a 100 μM Cy5-DNA stock solution, and store it in the dark at -20°C;

[0059] (2) Disperse 57.9 mg of doxorubicin hydrochloride (DOX) in 10 mL of DMSO at room temperature and in the dark, and disperse evenly to obtain a 10 mM DOX stock solution, which is then stored in the dark.

[0060] (3) Take 4 μL of the Cy5-DNA stock solution from step (1) and mix it evenly with 96 μL of secondary water. Then place the mixture in a -80°C refrigerator for freezing reaction for 1 hour. After the freezing is completed, take it out and thaw it naturally to room temperature. Then centrifuge it at 7000 rpm for 5 minutes. The resulting precipitate is washed twice with secondary water to obtain Cy5-DNA nanoparticles prepared by freezing in water.

[0061] (4) 4 μL of 100 μM Cy5-DNA stock solution was mixed with 5 μL of 10 mM DOX stock solution and 91 μL of secondary water, and then the mixture was placed in a -80°C refrigerator for freezing reaction for 1 hour. After the freezing was completed, the mixture was taken out and thawed naturally to room temperature, and then centrifuged at 7000 rpm for 5 minutes. The resulting precipitate was washed twice with secondary water to obtain Cy5-DNA / DOX nanoparticles prepared by freezing in water.

[0062] Example 3

[0063] The biological molecule used in this example is the polypeptide CL (purchased from Shanghai Chupide Biotechnology Co., Ltd.), and its sequence is as follows: N-terminus-CGTAFFINFIAIYHHASRAIPFGTMVAFRFFVLK(HHHHHH)FRSIINFEKL-C-terminus.

[0064] The specific preparation steps of synthesizing peptide CL nanoparticles by freezing in water are as follows:

[0065] (1) Centrifuge the purchased peptide CL solution at 4000 rpm for 60 seconds, collect the precipitate and dissolve it in 159.2 μL of sterile water, pipette and mix thoroughly at room temperature to form a 2 mM peptide CL stock solution, and store it at -20°C;

[0066] (2) 0.5 μL of 2 mM polypeptide CL stock solution was added to sterile water and mixed evenly to make the final volume of the mixed solution 30 μL. The mixed solution was then placed in a -80°C refrigerator for freezing reaction for 1 h. After the freezing was completed, the mixture was taken out and thawed naturally to room temperature. The mixture was then centrifuged at 7000 rpm for 5 min. The resulting precipitate was washed twice with secondary water to obtain polypeptide CL nanoparticles synthesized by freeze-drying in water.

[0067] Example 4

[0068] The biological molecule used in this example is the polypeptide FQ-Cy5 (purchased from Shanghai Chupeptide Biotechnology Co., Ltd.), and its sequence is as follows: N-terminus-Cy5-FFVLKHHHHHHKKKAVPIAQ-C-terminus.

[0069] The peptide FQ-Cy5 nanoparticles were synthesized by freezing in water. The specific preparation steps are as follows:

[0070] (1) Centrifuge the purchased peptide FQ-Cy5 solution at 4000 rpm for 60 s, collect the precipitate and dissolve it in 330 μL of sterile water, pipette and mix thoroughly at room temperature to form a 2 mM peptide CL stock solution, and store it at -20°C;

[0071] (2) Add 0.5 μL of 2 mM peptide FQ-Cy5 stock solution into sterile water and mix evenly to make the final volume of the mixed solution 30 μL. Then place the mixed solution in a -80°C refrigerator for freezing reaction for 1 hour. After the freezing is completed, take it out and thaw it naturally to room temperature. Then centrifuge it at 7000 rpm for 5 minutes. The resulting precipitate is washed twice with secondary water to obtain peptide FQ-Cy5 nanoparticles synthesized by freeze-drying in water.

[0072] Example 5

[0073] The biological molecule used in this example is the polypeptide Obsl1 (purchased from Shanghai Chupeptide Biotechnology Co., Ltd.), and its sequence is as follows: N-terminus-REGVELCPGNKYEMRRHGTTHSLVIHD-C-terminus.

[0074] The peptide Obsl1 / CPT / glycerol nanoparticles were frozen and synthesized in water. The specific preparation steps are as follows:

[0075] (1) The purchased polypeptide Obsl1 solution was centrifuged at 4000 rpm for 60 s, the precipitate was collected and dissolved in 303 μL of sterile water, and the solution was pipetted at room temperature and fully shaken to form a 2 mM polypeptide Obsl1 stock solution, which was then stored at -20°C;

[0076] (2) Disperse 85.39 mg of camptothecin (CPT) in 10 mL of DMSO at room temperature and in the dark, and disperse evenly to obtain a 10 mM CPT stock solution, which is then stored in the dark.

[0077] (3) At room temperature, 50 mL of 100% v / v glycerol (purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.) was added to 50 mL of secondary water to obtain a 50% v / v glycerol stock solution, which was stored at room temperature;

[0078] (4) 0.5 μL of 2 mM polypeptide Obsl1 stock solution, 1.5 μL of 10 mM CPT stock solution and 3 μL of 50% v / v glycerol stock solution were added into secondary water to make the final volume of the mixed solution 30 μL. The mixed solution was then placed in a -80°C refrigerator for freezing reaction for 1 hour. After the freezing was completed, the mixture was taken out and thawed naturally to room temperature. The mixture was then centrifuged at 7000 rpm for 5 minutes. The resulting precipitate was washed twice with secondary water to obtain polypeptide Obsl1 / CPT nanoparticles synthesized by freeze-drying in water.

[0079] Example 6

[0080] The biomolecule used in this example is Cy5-CPG (purchased from Shanghai Chupeptide Biotechnology Co., Ltd.), and its sequence is as follows: 5'-Cy5-TCCATGACGTTCCTGACGTT-3'.

[0081] The peptide CL / Cy5-CPG nanoparticles were synthesized by freezing in water. The specific preparation steps are as follows:

[0082] (1) Centrifuge 3.5 OD Cy5-CPG at 4000 rpm for 60 s, collect the precipitate and dissolve it in 98.2 μL sterile water, blow and mix thoroughly at room temperature in the dark to form a 100 μM Cy5-CPG stock solution, and store in the dark at -20°C;

[0083] (2) The purchased peptide CL solution was centrifuged at 4000 rpm for 60 s, and the precipitate was collected and dissolved in 159.2 μL of sterile water. The solution was pipetted at room temperature and fully shaken to form a 2 mM peptide CL stock solution, which was stored at -20°C.

[0084] (3) 0.5 μL of 2 mM peptide CL stock solution and 3 μL of 100 μM Cy5-CPG stock solution were added to secondary water to make the final volume of the mixed solution 30 μL. The mixed solution was then placed in a -80°C refrigerator for freezing reaction for 1 hour. After the freezing was completed, the mixture was taken out and thawed naturally to room temperature. It was then centrifuged at 7000 rpm for 5 minutes. The resulting precipitate was washed twice with secondary water to obtain peptide CL / Cy5-CPG nanoparticles synthesized by freeze-drying in water.

[0085] Example 7

[0086] The biomolecule used in this example is Cy5-DNA-BHQ2 (purchased from Shanghai Bioengineering Co., Ltd.), and its sequence is as follows: 5'-Cy5-CCGTTCTATCAACATCAGTCTGATAAGCTATAGAACGG-BHQ2-3'.

[0087] Single-stranded Cy5-DNA-BHQ2 nanoparticles were prepared by freezing in 1× PBS buffer. The specific preparation steps are as follows:

[0088] (1) Centrifuge 3.5 OD of Cy5-DNA-BHQ2 at 4000 rpm for 60 s, collect the precipitate and dissolve it in 89.2 μL of sterile water, blow and mix thoroughly at room temperature in the dark to form a 100 μM Cy5-DNA-BHQ2 stock solution, and store in the dark at -20°C;

[0089] (2) At room temperature, 50 mL of 100% v / v glycerol (purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.) was added to 50 mL of secondary water to obtain a 50% v / v glycerol stock solution, which was stored at room temperature;

[0090] (3) Add 1 μL of 100 μM Cy5-DNA-BHQ2 stock solution and 5 μL of 50% v / v glycerol stock solution to 1× PBS buffer (137 mM NaCl, 2.68 mM KCl, 8.1 mM Na2HPO4, 1.76 mM KH2PO4; pH = 7.2) and mix well to make the final volume of the mixed solution 100 μL, and then perform a freezing reaction. The freezing reaction conditions are selected from any one of the following: ① Reaction at -20°C for 24 hours; ② Reaction at -80°C for 5 minutes; ③ Reaction in liquid nitrogen at -196°C for 5 seconds. After freezing, the pellet was taken out and thawed naturally to room temperature, and then centrifuged at 7000 rpm for 5 min. The precipitate was washed twice with 1×PBS buffer (137 mM NaCl, 2.68 mM KCl, 8.1 mM Na2HPO4, 1.76 mM KH2PO4; pH = 7.2) to obtain single-stranded Cy5-DNA-BHQ2 nanoparticles synthesized at different freezing temperatures.

[0091] Example 8

[0092] The biomolecule used in this example is double-stranded siRNA (purchased from Suzhou Genewise Biotechnology Co., Ltd.), and its sequence is as follows:

[0093] sense: 5'-GUGAAGUCAACAUGCCUGC-dTdT-3',

[0094] antisense: 5'-GCAGGCAUGUUGACUUCAC-dTdT-3'; where U represents uracil.

[0095] Double-stranded siRNA nanoparticles were prepared by freezing in 1× PBS buffer as follows:

[0096] (1) 3.18 OD of sense and antisense were centrifuged at 4000 rpm for 60 s, and the precipitates were collected and dissolved in 150 μL of sterile water. The precipitates were pipetted down and mixed in a clean bench at room temperature to form 100 μM sense stock solutions and 100 μM antisense stock solutions, which were stored at -20°C.

[0097] (2) At room temperature, 50 mL of 100% v / v glycerol (purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.) was added to 50 mL of secondary water to obtain a 50% v / v glycerol stock solution, which was stored at room temperature;

[0098] (3) 2 μL of 100 μM sense stock solution, 2 μL of 100 μM antisense stock solution and 15 μL of 50% v / v glycerol stock solution were added to 1×PBS buffer (137 mM NaCl, 2.68 mM KCl, 8.1 mM Na2HPO4, 1.76 mM KH2PO4; pH = 7.2) and mixed evenly to make the final volume of the mixed solution 100 μL. The mixed solution was first reacted at 37°C for 2 h, and then placed in a -80°C refrigerator for 1 h. After the freezing, it was taken out and naturally thawed to room temperature, and then centrifuged at 7000 rpm for 5 min. The resulting precipitate was washed twice with 1×PBS buffer (137 mM NaCl, 2.68 mM KCl, 8.1 mM Na2HPO4, 1.76 mM KH2PO4; pH = 7.2) to obtain a double-stranded siRNA nanoparticle sample prepared by freezing in 1×PBS buffer.

[0099] Example 9

[0100] The biomolecule used in this example is plasmid pEGFP-C3 (purchased from Shanghai Bioengineering Co., Ltd.), product number B540191.

[0101] Biomolecular nanoparticles were prepared by freezing in 1× PBS buffer as follows:

[0102] (1) The purchased plasmid pEGFP-C3 was centrifuged at 4000 rpm for 60 s, the precipitate was collected and dissolved in 1 mL of sterile water, and the solution was pipetted at room temperature and thoroughly shaken to form a 10 μg / ml plasmid pEGFP-C3 stock solution, which was stored at -20°C;

[0103] (2) At room temperature, add 50 mL of 100% v / v glycerol into 50 mL of secondary water to obtain a 50% v / v glycerol stock solution, which is then stored at room temperature;

[0104] (3) At room temperature, 0.1487 g of zinc nitrate (purchased from Sigma-Aldrich Shanghai Trading Co., Ltd.) was added to 50 mL of secondary water, shaken and mixed, and then ultrasonicated for 15 min (40 kHz, 100 W) to obtain a 10 mM zinc nitrate stock solution, which was stored at room temperature;

[0105] (3) Add 6 μL of 10 μg / ml plasmid pEGFP-C3 stock solution and 5 μL of 50% v / v glycerol stock solution to 1×PBS buffer (137 mM NaCl, 2.68 mM KCl, 8.1 mM Na2HPO4, 1.76 mM KH2PO4; pH = 7.2) and mix well to make the final volume of the mixed solution 100 μL, then place the mixed solution in a -80°C refrigerator and freeze for 4 h. After the freezing period, take it out and thaw it naturally to room temperature, then centrifuge it at 7000 rpm for 5 min. The resulting precipitate is washed twice with 1×PBS buffer (137 mM NaCl, 2.68 mM KCl, 8.1 mM Na2HPO4, 1.76 mM KH2PO4; pH = 7.2) to obtain plasmid pEGFP-C3 nanoparticles prepared by freezing in 1×PBS buffer.

[0106] (4) 6 μL of 10 μg / ml plasmid pEGFP-C3 stock solution, 5 μL of 50% v / v glycerol stock solution and 2 μL of 10 mM zinc nitrate stock solution were added to 1×PBS buffer (137 mM NaCl, 2.68 mM KCl, 8.1 mM Na2HPO4, 1.76 mM KH2PO4; pH = 7.2) and mixed evenly to make the final volume of the mixed solution 100 μL, and then the mixed solution was placed in a -80°C refrigerator for 24 h. After the freezing was completed, it was taken out and thawed naturally to room temperature, and then centrifuged at 7000 rpm for 5 min. The resulting precipitate was washed twice with 1×PBS buffer (137 mM NaCl, 2.68 mM KCl, 8.1 mM Na2HPO4, 1.76 mM KH2PO4; pH = 7.2) to obtain pEGFP-C3 / Zn nitrate prepared by freezing in 1×PBS buffer. 2+ Nanoparticles.

[0107] Example 10

[0108] The biomolecule used in this example is Cy5-Bcl2, and its sequence is: 5'-Cy5-GCAGGCATGTTGACTTCACTT-3'.

[0109] The steps for preparing biomolecular nanoparticles that are unstable under high temperature conditions are as follows:

[0110] (1) Centrifuge 3.5 OD of Cy5-Bcl2 at 4000 rpm for 60 s, collect the precipitate and dissolve it in 950.5 μL of sterile water, blow and mix thoroughly under light-proof and room temperature conditions to form a 10 μM Cy5-Bcl2 stock solution, and store it at -20°C in a dark place;

[0111] (2) Under nitrogen flow, triiron dodecacarbonyl Fe3(CO) 12Dissolve in pre-deoxygenated DMSO to a final concentration of 4 mM Fe3(CO) 12 Original solution;

[0112] (3) Under nitrogen flow, 3 μL of 10 μM Cy5-Bcl2 stock solution was mixed with 6 μL of 4 mM Fe3(CO) 12 The stock solution was added to 21 μL of 1×PBS buffer (137 mM NaCl, 2.68 mM KCl, 8.1 mM Na2HPO4, 1.76 mM KH2PO4; pH = 7.2), and then the mixture was placed in a -80°C refrigerator for freezing reaction for 30 min. After the freezing was completed, the sample was taken out, thawed naturally to room temperature, and then centrifuged at 7000 rpm for 5 min. The resulting precipitate was washed twice with sterile water to obtain biomolecular nanoparticles Fe3(CO) 12 / Cy5-Bcl2.

[0113] Embodiment 11

[0114] The biomolecule used in this example is Poly(I:C) (purchased from InvivoGen), product number Tlrl-picw.

[0115] Biomolecular nanoparticles were prepared by freezing in 0.9% saline as follows:

[0116] (1) Dissolve 25 mg of commercial adjuvant Poly(I:C) in 12.5 mL of sterile 0.9% saline, mix by blowing in a biological operation table to form a 2 mg / ml Poly(I:C) stock solution, and store at -20°C;

[0117] (2) At room temperature, add 50 mL of 100% v / v glycerol into 50 mL of secondary water to obtain a 50% v / v glycerol stock solution, which is then stored at room temperature;

[0118] (3) The purchased polypeptide Obsl1 solution was centrifuged at 4000 rpm for 60 s, and the precipitate was collected and dissolved in 303 μL of sterile 0.9% saline. The solution was pipetted at room temperature and fully shaken to form a 2 mM polypeptide Obsl1 stock solution, which was then stored at -20°C;

[0119] (4) 10 μL of 2 mg / ml Poly(I:C) stock solution and 3 μL of 50% v / v glycerol stock solution were added to sterile 0.9% saline and mixed to make the final volume of the mixture 60 μL. The mixture was then placed in a -80°C refrigerator and frozen for 1 hour. After the freezing, the mixture was taken out and thawed naturally to room temperature. The mixture was then centrifuged at 7000 rpm for 5 minutes. The resulting precipitate was washed twice with sterile 0.9% saline to obtain Poly(I:C) nanoparticles prepared by freezing in sterile 0.9% saline.

[0120] (5) Add 10 μL of 2 mg / ml Poly(I:C) stock solution, 3 μL of 50% v / v glycerol stock solution and 12 μL of 2 mM polypeptide Obsl1 stock solution to sterile 0.9% saline and mix well to make the final volume of the mixed solution 60 μL. Then place the mixed solution in a -80°C refrigerator and freeze it for 1 hour. After freezing, take it out and thaw it naturally to room temperature. Then centrifuge it at 7000 rpm for 5 minutes. Wash the resulting precipitate twice with sterile 0.9% saline to obtain Poly(I:C) / Obsl1 nanoparticles prepared by freezing in sterile 0.9% saline.

[0121] Example 12

[0122] The biomolecule used in this example is the mRNA of green fluorescent protein amFP486 (GFP) (SEQ ID NO. 11), gene symbol: LOC107331116.

[0123] mRNA nanoparticles were prepared by freezing in 0.9% saline as follows:

[0124] (1) mRNA of green fluorescent protein amFP486 (GFP) was dispersed in sterile 0.9% physiological saline and mixed by blowing in a biological operation table to form a 1 mg / ml mRNA stock solution, which was stored at -20°C;

[0125] (2) At room temperature, add 50 mL of 100% v / v glycerol into 50 mL of secondary water to obtain a 50% v / v glycerol stock solution, which is then stored at room temperature;

[0126] (3) Add 10 μL of 1 mg / ml mRNA stock solution and 3 μL of 50% v / v glycerol stock solution to sterile 0.9% saline and mix them so that the final volume of the mixture is 60 μL. Then place the mixture in a -80°C refrigerator and freeze it for 1 hour. After freezing, take it out and thaw it naturally to room temperature. Then centrifuge it at 7000 rpm for 5 minutes. Wash the resulting precipitate twice with sterile 0.9% saline to obtain mRNA nanoparticles prepared by freezing in sterile 0.9% saline.

[0127] Figure 2The transmission electron micrographs of biomolecule nanoparticles prepared by freezing in water according to Examples 1 and 2 are shown in FIG. 1 ; wherein (a) is a transmission electron micrograph of single-stranded DNA nanoparticle 1; (b) is a transmission electron micrograph of single-stranded DNA nanoparticle 2; (c) is a transmission electron micrograph of Cy5-DNA nanoparticles; and (d) is a transmission electron micrograph of Cy5-DNA / DOX nanoparticles. It can be observed from the figure that, without the addition of the additive glycerol, the yield of single-stranded DNA nanoparticles prepared by freezing is relatively low, but nanoparticles can still be formed. After the addition of the additive glycerol, the yield of nanoparticles is high and the size is uniform. In addition, the yield of nanoparticles is also increased after the functional molecule Cy5 is modified or DOX is added, indicating that the functional molecule and the auxiliary raw material increase the interaction between the biomolecules, thereby improving the efficiency of freezing.

[0128] Figure 3 The TEM images of biomolecule nanoparticles prepared by freezing in water in Examples 3 to 6 are shown in Figures 3 to 6; (a) is a TEM image of polypeptide CL nanoparticles; (b) is a TEM image of polypeptide FQ-Cy5 nanoparticles; (c) is a TEM image of polypeptide Obsl1 / CPT nanoparticles; (d) is a TEM image of polypeptide CL / Cy5-CPG nanoparticles. It can be observed from the figures that freezing polypeptides can also form nanoparticles, and modifying the functional molecule Cy5 and adding auxiliary materials do not affect the formation of nanoparticles.

[0129] Figure 4 The figure is a characterization diagram of the biomolecular nanoparticles prepared by freezing in 1×PBS buffer in Example 7; wherein, (a) is a transmission electron micrograph of the Cy5-DNA-BHQ2 nanoparticles prepared by freezing reaction at -20℃ for 24h; (b) is a transmission electron micrograph of the Cy5-DNA-BHQ2 nanoparticles prepared by freezing reaction at -80℃ for 5min; (c) is a transmission electron micrograph of the Cy5-DNA-BHQ2 nanoparticles prepared by freezing reaction at -196℃ for 5s; (d) is a particle size distribution diagram of the Cy5-DNA-BHQ2 nanoparticles prepared by freezing reaction at -80℃ for 5min; (e) is a potential change diagram of the Cy5-DNA-BHQ2 nanoparticles prepared by freezing reaction at -80℃ for 5min. It can be observed from the figure that nanoparticles can be formed at different freezing temperatures, wherein the nanoparticles formed under -80℃ condition have uniform size, with the particle size concentrated at 800nm, and the surface charge of the nanoparticles is -43.6mV.

[0130] Take the Cy5-DNA-BHQ2 nanoparticles prepared by freezing at -80°C (wherein the concentration of Cy5-DNA-BHQ2 is 300nM). Mix them evenly with different concentrations of miRNA-21 (0, 5, 10, 20, 40, 60, 80, 100, 150, 300nM) in 1×PBS buffer (137mMNaCl, 2.68mMKCl, 8.1mMNa2HPO4, 1.76mMKH2PO4; pH=7.2), incubate at 37°C in the dark for 1h, and then centrifuge at 7000rpm for 5min. Take 160μL of the supernatant and collect the fluorescence spectrum at 650nm-800nm ​​under the condition of 635nm excitation wavelength. The results are shown in Figure 5 shown. Figure 5 In the figure, (a) is the fluorescence spectra of Cy5-DNA-BHQ2 nanoparticles incubated with different concentrations of miRNA-21; (b) is the fitting curve of the 665nm fluorescence peak value collected under the condition of 635nm excitation wavelength and miRNA-21 concentration after Cy5-DNA-BHQ2 nanoparticles were incubated with different concentrations of miRNA-21. It can be observed from the figure that the prepared Cy5-DNA-BHQ2 nanoparticles can detect miRNA-21.

[0131] Figure 6 The following are characterization diagrams of biomolecular nanoparticles prepared by freezing in 1×PBS buffer in Example 8; (a) is a transmission electron microscope image of siRNA nanoparticles; (b) is a particle size distribution diagram of siRNA nanoparticles; and (c) is a potential change diagram of siRNA nanoparticles. It can be observed from the figure that the double-stranded siRNA nanoparticles are uniform in size, with a particle size concentrated at 200 nm, and the surface charge of the nanoparticles is -34.6 mV.

[0132] Figure 7 Transmission electron microscopy images of biomolecular nanoparticles prepared by freezing in 1×PBS buffer in Example 9. (a) pEGFP-C3 nanoparticles; (b) pEGFP-C3 / Zn 2+ Nanoparticles. It can be observed from the figure that pEGFP-C3 nanoparticles and pEGFP-C3 / Zn 2+ The nanoparticles are uniform in size.

[0133] Figure 8 Fe3(CO) prepared in Example 10 12 / Cy5-Bcl2 nanoparticles. It can be observed from the figure that Fe3(CO) 12 The uniform size of the Cy5-Bcl2 nanoparticles indicates that our method is suitable for the assembly of high-temperature unstable drugs and nucleic acid drugs.

[0134] Fig. 9 Transmission electron micrographs of biomolecular nanoparticles prepared by freezing in 0.9% saline in Example 11. (a) Poly(I:C) nanoparticles; (b) Poly(I:C) / Obsl1 nanoparticles. We found that the commercial adjuvant - Poly(I:C) can also form nanoparticles by freezing. In addition, after adding the antigen peptide, the size of the nanoparticles becomes 50-80nm, which provides a good size for the nanoparticles to target the draining lymph nodes, so this freezing method has the potential to promote the development of nanovaccines.

[0135] Fig.10 This is a transmission electron micrograph of mRNA nanoparticles prepared by freezing in 0.9% saline in Example 12. We found that mRNA can also be formed into nanoparticles by freezing. This provides a very good delivery method for mRNA. This can also be used for the delivery of mRNA vaccines.

[0136] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.

Claims

1. A method for preparing biomolecular nanoparticles by freezing, characterized in that: The freezing method is used to drive the self-assembly of biomolecules to form nanoparticles; specifically, biomolecules are used as raw materials, and after the solution is cooled and frozen, water molecules crystallize, thereby repelling biomolecules and additives from growing ice crystals, resulting in an increase in local concentration in non-ice areas, thereby enhancing the attachment dynamics of biomolecules. This concentration effect strengthens the intramolecular and intermolecular electrostatic attraction, π-π stacking, and hydrophilic and hydrophobic interactions between biomolecules or between biomolecules and additives, leading to the self-assembly of biomolecules to form nanoparticles; The method specifically comprises the following steps: (1) dissolving a biomolecule raw material in a solvent to obtain a biomolecule stock solution with a concentration of 50 μM to 20 mM; the biomolecule is selected from nucleic acid biomolecules and amino acid peptide biomolecules, the biomolecule raw material is modified with a functional small molecule, and the functional small molecule is selected from fluorescein isothiocyanate, phycoerythrin, AlexaFluor series dyes, rhodamine, cyanine dyes, and alkyl chains; the solvent is selected from sterile water and sterile 0.9% saline; (2) dissolving the additive raw material in a solvent to obtain an additive stock solution with a concentration of 10wt% to 100wt%; the additive is glycerol; and the solvent is secondary water; (3) taking 0.1-500 μL of the biomolecule stock solution, mixing it evenly with 0-500 μL of the additive stock solution, and then adding it to the solvent so that the final volume of the mixed solution is 10-1000 μL; wherein the solvent is selected from sterile water, secondary water, 1× PBS buffer, and sterile 0.9% saline; (4) The mixture is frozen at -80°C for 5s to 24h, thawed naturally to room temperature, and then centrifuged at 5000 to 10000 rpm for 1 to 60 min. The precipitate is washed twice to obtain biomolecular nanoparticles.

2. The method for preparing biomolecular nanoparticles by freezing according to claim 1, characterized in that: The nucleic acid biological molecules include one or more of single-stranded DNA, single-stranded RNA, double-stranded RNA, double-stranded DNA, circular RNA, circular DNA, oligonucleotides, and plasmids.

3. The method for preparing biomolecular nanoparticles by freezing according to claim 1, characterized in that: The amino acid peptide biomolecules include one or more of amino acids, cyclic peptides, polypeptides, and proteins.

Images