Composite biological agent based on porous frame materials

Abstract

A novel composite biological agent based on a porous frame material, comprising porous frame materials and biomolecules. The porous frame materials cover a biological product, wherein the porous frame materials are metal-organic frame material (MOFs), covalent organic frame materials (COFs), and hydrogen-bonding organic frame materials (HOFs), and the biomolecules are any one or a combination of antibodies, enzymes, peptides, vaccines, nucleotides, and virus species. The composite biological agent uses the porous frame materials and biomolecules to form a porous frame material/biomolecule complex, and the biomolecules are coated to achieve the protection effect. Under the premise of remaining biomolecule activity, the system can achieve efficient separation and recovery of the porous materials and the biomolecules, so that the technical problems of synthesis, storage, release, etc. are solved, a good technical effect is achieved, and the biomolecules are effectively protected. The system is applied to the storage and transportation of biological agents and preparation of novel agents.

Claims

1. A method for preserving biomolecules, comprising the steps of: (1) mixing a metal and a ligand with the biomolecules to form a composite biological agent in which an MOF material is embedded onto the biomolecules; (2) leaving the composite biological agent, in which the MOF material encapsulates the biomolecules, at room temperature during storage; and (3) processing the composite biological agent with an EDTA solution at 20-30° C. to release the biomolecules from the composite biological agent, wherein: the MOF material is ZIF-8; the biomolecule is goat anti-BSA or Go.sub.x; and step (3) includes: processing the composite biological agent with the EDTA solution for 15 minutes; and centrifugalizing for 5 seconds a processed mixture of the composite biological agent and the EDTA solution.

Description

BRIEF DESCRIPTION OF FIGURES

(1) The following detailed descriptions, given by way of example, and not intended to limit the present invention solely thereto, will be best be understood in conjunction with the accompanying figures:

(2) FIG. 1 is an X-ray powder pattern of MOF-1/goat anti-BSA IgG complex and MOF-1/GOx complex (MOF-1 refers specifically to ZIF-8, goat anti-BSA IgG refers to goat Anti-BSA IgG);

(3) FIG. 2 is a Fourier infrared transform spectrum of MOF-1/goat anti-BSA IgG complex and MOF-1/GOx complex;

(4) FIG. 3 is a result of recovery rate of encapsulated IgG antibody and GOx enzyme released from MOF-1/goat anti-BSA IgG complex and MOF-1/GOx complex;

(5) FIGS. 4,5,6 and 7 are the comparison chart of the binding activity of goat anti-BSA IgG released from treated MOF-1/goat anti-BSA IgG complex and treated free goat anti-BSA IgG;

(6) FIG. 8,9 is a comparison chart of initial rate and conversion rate of GOx released from the MOF-1/GOx complex and free GOx;

(7) FIG. 10 is an X-ray powder pattern of MOF-2/rAd5-GFP complex (MOF-2 in this application refers specifically to ZPF-2, rAd5-GFP refers to GFP recombinant human adenovirus type 5 viral vector);

(8) FIG. 11 is a comparison of the cell infectivity of the GFP recombinant human adenovirus type 5 vectors released from the MOF-2/GFP recombinant human adenovirus type 5 virus vector complex;

(9) FIG. 12 is a result effect diagram of COF-1/peptide covalent and adsorption; and

(10) FIG. 13 is an effect diagram of MPM-1-Br adsorption of BSA.

DETAILED DESCRIPTION OF THE EMBODIMENTS

(11) Referring to FIG. 1-FIG. 13, the synthesis of the porous frame materials, the characterization test and the method for testing the activity of the active substance, the specific implementation manner is as follows.

(12) The vaccines and antibodies treated by the method in this invention, are dissolved and injected to the rat, and compare with the control group. The experiment results show that the solution system of this method is safe to organism and has no adverse reaction.

(13) If not specifically illustrated, all materials of the invention may be commercially available; or can be prepared according to conventional methods in the art. Unless otherwise defined or specified, all professional and scientific terms used herein have the same meanings as those well-known by the skilled in the art. Furthermore, any methods and materials similar or equal to those described can be used in the methods of the invention.

(14) Features mentioned in the invention and features mentioned in the examples may be combined. All of the features disclosed in this specification may be used simultaneously in any forms of combination; and each feature disclosed in the specification may be substituted by any alternative features providing same, equal or similar purpose. Therefore, unless otherwise specified, the disclosed features are only common examples of equal or similar features.

(15) Referring to FIGS. 1 to 13, the invention is described in detail in connection with the following examples, which are not construed as limiting the scope of the invention.

(16) Raw Materials

(17) All chemical reagents are commercially available products.

Example 1

(18) Preparation of MOF-1/Goat Anti-BSA IgG Complex:

(19) Preparation of MOF-1/goat anti-BSA IgG complex: The PBS solution containing 50 μL and 10 mg/mL Goat anti BSA IgG (Goat anti bovine serum albumin IgG), mixed with 200 μL aqueous solution of 200 mM imidazole compounds, and then 40 mM 250 μL zinc ion aqueous solution was added into the protein-imidazole aqueous solution. μL These two solutions were mixed and placed at room temperature for 10 min. Subsequently, the as-synthesized products were collected by centrifugation (6,000 rmp) for 10 min, washed with excess D.I. water, and then pre-frozen at −80° C., and then lyophilized for 8 hours. (FIG. 1, X-ray powder pattern, FIG. 2, Fourier infrared transform spectrum). The experimental results are shown that the anti-BSA IgG antibody is encapsulated into MOF-1.

(20) Preparation of MOF-1/GOx Complex:

(21) Preparation of MOF-1/GOx complex: Antibody of 0.5 mg was added into a solution of imidazoles (1600 mM, pH 10.3, 250 μL). Zinc ion solution was also prepared in DI water (40 mM, 250 μL). These two solutions were mixed and placed at 4° C. for 12 h. Subsequently, the as-synthesized products were collected by centrifugation (6,000 rmp) for 10 min, washed with excess D.I. water, and then pre-frozen at −80° C., and then lyophilized for 8 hours. (FIG. 1, X-ray powder pattern, FIG. 2, Fourier infrared transform spectrum). The experimental results are shown that the anti-BSA IgG antibody is encapsulated into MOF-1.

Example 2

(22) Experiment of recovering encapsulated goat anti-BSA IgG antibody and GOx enzyme from MOF-1/goat anti-BSA IgG complex and MOF-1/GOx complex: The recovered and lyophilized MOF-1/goat anti-BSA complex and MOF-1/GOx complex were mixed with the same 20 mM EDTA solution as the reaction system, and after reacting for 15 minutes, the mixture was briefly centrifuged for 5 seconds using a centrifuge. And then ultrafiltration was performed for 10-20 minutes at 10000 g (The specific time depends on the concentration and molecular weight of the protein biologic and the pore size of the ultrafiltration membrane filter used). After the ultrafiltration, the antibody was recovered by washing the ultrafiltration membrane with 1× PBS. The concentration of the recovered antibody was measured using the A280 method to obtain recovery amount data. After calculation, it is compared with the total amount of antibody originally added. The final recovery rate is not less than 80%. Recovery rate=the amount of biologics recovered/the amount of biologics initially added to the system ×100%. The recovery rate experimental results are shown in FIG. 3. As can be seen from FIG. 3, the method developed by this application has a good recovery rate for both IgG antibodies and enzymes.

(23) (FIG. 3, recovery rate of encapsulated IgG antibody and GOx enzyme released from MOF-1/goat anti-BSA IgG complex and MOF-1/GOx complex).

Example 3

(24) Antibody Protection Testing.

(25) Goat anti-BSA IgG solution (0.5 mg/ml) and goat anti-BSA IgG/MOF-1 complex generate were treated under three different conditions. Heating test: high temperature 50-75° C. in 1-5 ml reaction tube, temperature change test: 4-60° C. (20° C./min temperature gradient change) and −8° C. room temperature repeated freezing and thawing experimental conditions to carry out the heat stress test of the protected antibody and protein. Metal oxidation test: the antibody samples and the ascorbic acid and 0.08 mM CuCl2 incubated at room temperature for 3 h. Mechanical test: The mechanical pressure treatment for goat anti-BSA IgG/MOF-1 complex is conducted by a tablet machine with 20 MPa pressure.

Example 4

(26) Size-Exclusion Chromatography HPLC

(27) Size exclusion chromatography HPLC was used to detect the molecular weight of the antibody and the antibody in the complex after several treatments in Example 3. SEC was performed on an HPLC using a pre-prepared column (300 mm×7.8 mm). Each sample (Free IgGs or IgGs recovered from antibody@MOFs) (300 μL) was injected and separation was performed at a flow rate of 0.5 mL/min. The elution buffer was composed of 100 mM sodium phosphate and 100 mM sodium sulfate at pH 7.1. UV detection was performed at 280 nm, while multi-angle laser light scattering (MALLS) detection is performed at 658 nm using an 18-angle detector operating with a 50-nW solid-state laser. The extinction coefficient is 1.69 (mL mg.sup.−1 cm.sup.−1), the second dimensional coefficient dn/dc of 0.185 (mL/g) is 0. The AUC of the UV signal was used to calculate the percentage of fragments, monomers, oligomers, protein recovery and total aggregation. For relative protein recovery, the total AUC of the stressed samples with the total AUC of the unstressed samples (it is set to 100%) is compared. The total aggregation percentage considers the oligomer, and the percentage of protein that has not been recovered, which usually contains aggregates that are too large to enter the column. The experimental results are shown in Table 1

(28) TABLE-US-00001 fragment monomer oligomer untreated goat anti BSA IgG samples 1.5 97 1.5 Heated MOF-1 encapsulated samples 1.5 94.4 4 Heated samples 70.1 25.4 4 Metal oxidation MOF-1 encapsulated 4.3 72.4 4 samples Metal oxidation samples 99 1 — Mechanical force treatment MOF-1 1.5 95 3.5 encapsulated samples

(29) The results illustrated that antibodies@MOFs exhibited good resistance against heated treatment, metal oxidation, mechanical pressure.

Example 5

(30) The Binding Ability Experiment of the Antibody Released from the MOF-1/Goat Anti-BSA IgG Complex:

(31) 96-well plates were coated overnight with 100 ng/mL BSA in PBS (100 μL/well). Removed the BSA solution, then the residual binding sites were blocked by incubation step with 5% skim milk solution (200 μL/well) for 2 h at 37° C. Removed the skim milk solution, following by 4 times of washing with 0.05% PBST. Free goat anti-BSA IgG and goat anti-BSA IgG released from MOF-1/goat anti-BSA IgG was diluted at various concentrations (initial concentration was 18 μg/mL, 3-fold dilution) in 1% BSA, and then added 50 μL/well to the plate and incubated for 1 h at 37° C. Samples were then removed and followed with thorough washing. HRP conjugated rabbit anti-goat IgG antibody was diluted to 1:2000 using 1% BSA, and 50 μL/well was added to the plate. Subsequently, incubation was performed for 45 min at 37° C. After removing the solution, all wells were washed with PBST. Next, TMB (100 μL/well) were added to each well and incubated for 15 min in the dark at room temperature. The reaction was stopped by adding 2 N H2SO4 (50 μL/well), and absorbance was measured at 450 nm using microplate reader.

(32) Bingding capacity assay showed that goat anti-BSA IgG released from MOF-1 (after heating at 75° C. for 20 minutes) possessed similar binding abilities as their original antigen of untreated G-IgG (>90%), whereas the unprotected G-IgG almost lost all binding activity, (FIG. 4, binding activity of goat anti-BSA IgG released from treated MOF-1/goat anti-BSA IgG complex and treated free goat anti-BSA IgG after heating at 75° C. for 20 minutes)

(33) Bingding capacity assay showed that goat anti-BSA IgG released from MOF-1 (after metal oxidation for 3 h) possessed similar binding abilities as their original antigen of untreated G-IgG (>90%), whereas the unprotected G-IgG almost lost all binding activity, (FIG. 5, binding activity of goat anti-BSA IgG released from treated MOF-1/goat anti-BSA IgG complex and treated free goat anti-BSA IgG after metal oxidation for 3 h)

(34) Bingding capacity assay showed that goat anti-BSA IgG released from MOF-1 (after treated at a pressure of 20 MPa) possessed similar binding abilities as their original antigen of untreated G-IgG (>90%), whereas the unprotected G-IgG almost lost all binding activity, (FIG. 6, binding activity of goat anti-BSA IgG released from treated MOF-1/goat anti-BSA IgG complex and treated free goat anti-BSA IgG after treated at a pressure of 20 MPa)

(35) Bingding capacity assay showed that goat anti-BSA IgG released from MOF-1 (after repeated freezing and thawing) possessed similar binding abilities as their original antigen of untreated G-IgG (>90%), whereas the unprotected G-IgG almost lost all binding activity, (FIG. 7, binding activity of goat anti-BSA IgG released from treated MOF-1/goat anti-BSA IgG complex and treated free goat anti-BSA IgG after repeated freezing and thawing)

Example 6

(36) Comparing the Activity of GOx Released from the MOF-1/GOx Complex with GOx not Encapsulated by MOF-1:

(37) The GOx from the MOF-1/GOx Complex is Released by the Method in Example 2. To quantify GOx and free GOx in the complex, 10 μg of free GOx and GOx from the MOF-1/GOx are added into the reaction solution (100 μL 10% glucose, volume solution, horseradish peroxidase, 30 pL 5 mM resorcinol solution) and PBS added to 3 mL, and then measured the absorption value at 420 nm. Compare the initial rate of the reaction 60 s before the reaction and the total conversion amount after the reaction is stopped. The results are shown in FIGS. 8 that the initial reaction rate of GOx released from porous frame materials is slightly lower than that of unencapsulated GOx. It can be seen that the process of forming a complex with the porous frame material and recovering the complex after decomposition has a slight effect on the initial reaction rate of the enzyme GOx.

(38) The results are shown in FIG. 9 that the conversion rate of GOx released from porous frame materials is slightly lower than that of unencapsulated GOx. It can be seen that the process of forming a complex with the porous frame material and recovering the complex after decomposition has a slight effect on the conversion rate of the enzyme GOx.

Example 7

(39) Preparation of MOF-2/GFP Recombinant Human Adenovirus Type 5 Virus Vector Complex

(40) Add desalted rAd5-GFP (GFP recombinant human adenovirus type 5 vector) solution that contains 1 mg virus particles, followed by 2500 μL of the 50-500 mM pyrimidines aqueous solution and 2500 μL of the 50-500 mM zinc ion aqueous solution. Then the reaction mixture will turn turbid immediately. Put the mixture at 4° C. and leave it for 0.5-1 hour. After the reaction, the reaction mixture is centrifuged at 6000 rpm, and the supernatant is discarded to obtain a white solid, which is pre-frozen by −80° C., and then lyophilized for 8 h. (FIG. 10, X-ray powder pattern of MOF-2/rAd5-GFP complex). The experimental results shown that the GFP recombinant human adenovirus type 5 virus vector is encapsulated into MOF-2.

Example 8

(41) Detection of the Protective Effect of Recombinant Adenovirus.

(42) Take 293T cells in the logarithmic growth phase, trypsinize, count, and put into a 24-well plate, 500 μL per well (5×104 cells/mL). Add 50 μL virus samples to each well, then at 37° C., 5% CO2 incubator culture 48 h. Directly observe the number of fluorescent cells in the field of view under a fluorescent microscope. (FIG. 11, comparison of the cell infectivity of the GFP recombinant human adenovirus type 5 vector released from the MOF-2/GFP recombinant human adenovirus type 5 virus vector complex). The experimental results are shown that rAd5-GFP released from MOF-2/rAd5-GFP complex has the same infectivity as free rAd5-GFP.

Example 9

(43) Peptide Covalent and Adsorption Fixation Experiments.

(44) To covalently immobilize biomolecules, the same molar amount of EDC and NHS was first dissolved in 0.1 M MES buffer (pH 6.0), and COF-1 was added into this solution and mixed for 1 h. The NHS-functionalized material was then dispersed in 0.1 M MES buffer (pH 7.0) containing 10.0 mg/mL of peptide. The supernatants were then scanned by UV-Vis (A280 nm, A227.5 nm, and quantified by standard curve for peptide to obtain the amount of peptide covalent immobilized in COF-1. The material COF-1 is directly added to 0.1 MES buffer (pH 7.0) with 10 mg/mL peptide. The supernatants are then scanned by UV-Vis (A227.5 nm, quantified by standard curve for peptide to obtain the amount of peptide adsorption in COF-1. (FIG. 12, effect diagram of COF-1/peptide covalent and adsorption).

Example 10

(45) Adsorption of Vancomycin by Hydrogen Bonding Organic Framework Materials.

(46) Synthesis of hydrogen-bonded organic framework materials: 11 mg of adenine is added into 12 mL methanol, 8.8 mg of copper bromide is added into 12 mL of isopropanol, and then mix them. The isopropyl alcohol solution of copper bromide was added dropwise to cover the methanol solution, and the mixture incubate for one week at room temperature. The product is obtained and washed with methanol to obtain MPM-1-Br. 5 mg MPM-1-Br was add into 2 mL of 5 mg/mL BSA water solution, shaken under 37° C. The supernatants are then scanned by UV-Vis to calculate the amount of BSA adsorbed. (FIG. 13, effect diagram of MPM-1-Br adsorption of BSA). The experimental results are shown that that BSA can be adsorbed into the interior of MPM-1-Br.

(47) Having described at least one of the embodiments of the claimed invention with reference to the accompanying drawings, it will be apparent to those skills that the invention is not limited to those precise embodiments, and that various modifications and variations can be made in the presently disclosed system without departing from the scope or spirit of the invention. Thus, it is intended that the present disclosure cover modifications and variations of this disclosure provided they come within the scope of the appended claims and their equivalents. Specifically, one or more limitations recited throughout the specification can be combined in any level of details to the extent they are described to improve the present invention. g. 13