Nanoparticle composition as well as preparation method and application thereof

Abstract

The invention provides a nanoparticle composition as well as a preparation method and application thereof. The nano-particle composition comprises the charged nano-particles and the loaded drug, and the drug loading efficiency is remarkably improved by treating a mixed solution of the nano-particles and the drug through low-temperature plasma jet. According to the method, the electrical property of the nano-particles is changed by utilizing low-temperature plasma, so that the adsorption capacity of the nano-particles on charged drugs is enhanced, and the method is suitable for various drug types such as small molecule drugs, gene drugs and proteins. Experimental results show that the drug loading capacity is remarkably improved in multiple drug loading systems, the method has the advantages of being easy to operate, non-toxic, low in cost and the like, and the method is suitable for industrial production and promotes application of drug delivery carriers in the medical field.

Description

Technical Field

[0001] The invention belongs to the field of nano materials and relates to a nano particle composition and a preparation method and application thereof. Background Art

[0002] In recent years, the application of nanomaterials in medicine is attracting widespread attention due to their many unique advantages, including high surface area, tunable morphology, and biocompatibility.

[0003] Nanomaterials are currently widely used in the development of drug delivery systems. These nanoparticles can achieve targeted drug delivery, improve drug bioavailability, reduce adverse effects on healthy tissues, improve therapeutic effects and reduce side effects by regulating their size, shape and surface properties.

[0004] Therefore, the application of nanomaterials in the medical field provides new solutions for disease diagnosis, treatment and tissue engineering, which is expected to promote the development of medical technology and improve the quality of life of patients. However, the drug loading efficiency of nanomaterials is still an issue that needs to be further studied to ensure their safety and effectiveness in clinical applications. Summary of the invention

[0005] In response to the challenge of low drug loading rate of nanomaterials, the present invention proposes a method for improving drug loading efficiency based on low-temperature plasma. Its core innovation lies in changing the electrical properties of nanoparticles through a simple and low-cost low-temperature plasma strategy, so as to encapsulate more drug molecules, including genes, plasmids, proteins, and ionizable chemicals, by electrostatic adsorption.

[0006] In order to meet the necessary conditions for the nanoparticles to be charged, the present invention selects ionizable lipids as an important component of the membrane material. Due to its ionizable property, ionizable lipids are common amphiphilic materials in the current gene delivery field. Based on this, the inventors found that by applying low-temperature plasma to the dispersed solution of ionizable lipids, the electrical properties of the lipid solution can be significantly improved. Further, in order to meet the needs of in vivo delivery, the present invention limits the nanoparticles to have a particle size range of 100-800nm.

[0007] Preferably, the ionizable lipids selected in the present invention are cationic lipids or anionic lipids, and the phase transition temperatures are less than 70°C.

[0008] Preferably, the cationic lipid is 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), and the anionic lipid is 1,2-distearoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DSPG).

[0009] In order to meet the demand for drug loading, the target drugs selected in the present invention include one or more of small interfering RNA (siRNA), small hairpin RNA (shRNA), microRNA (miRNA), messenger RNA (mRNA), plasmid DNA (Plasmid), protein and ionizable drugs. The main characteristic of the above drugs is that they are all electrically charged. The reason why the above drugs are charged is mainly because they contain groups that can be charged in their chemical structure. RNA and DNA molecules usually connect their nucleotide units through a phosphate backbone, and the phosphate group itself carries a negative charge in aqueous solution, which makes these molecules negatively charged in aqueous solution. Although the bases of the nucleotides themselves are not charged, they are connected together through phosphate groups, making the entire molecule negatively charged. Specifically, nucleic acid molecules such as siRNA, shRNA, miRNA and mRNA all have similar negative charge characteristics. Plasmid DNA also exhibits negative charge in aqueous solution due to the presence of its phosphate backbone. Unlike nucleic acid molecules, the charge state of proteins depends on the side chains of their amino acids. Some amino acid residues (such as aspartic acid, glutamic acid, lysine and arginine) have groups that can be positively or negatively charged, which makes the protein appear to be positively or negatively charged under different pH conditions. Similarly, ionizable drugs (such as doxorubicin hydrochloride, etc.) also have positively or negatively charged groups and can show different electrical properties in solvents of specific pH.

[0010] To achieve the above object, the technical solution of the present invention is as follows: 1) Mix the nanoparticles with the target drug and transfer them into deionized water at pH 7 to obtain solution A; 2) using a low-temperature plasma generator with a hollow electrode dielectric barrier structure to treat solution A; 3) Centrifuging the solution obtained in step 2) to obtain nanoparticles with high drug loading rate.

[0011] Preferably, in step 1, the concentration of the nanoparticles is 0-100 mg / ml.

[0012] Preferably, in step 2, the low-temperature plasma device selected in the present invention refers to patent 201510313424.8.

[0013] Preferably, in step 2, the parameters of the low-temperature plasma generating device are set to a working voltage of 600-1200 V, a working current of 5-10 mA, an argon or nitrogen working gas, and a flow rate of 400-800 sccm.

[0014] Preferably, based on the parameter selection of step 2, an electron density of 10 15 / cm 3 Low-temperature plasma jet of orders of magnitude.

[0015] Preferably, in step 2, the low-temperature plasma jet treatment time is 5-15 minutes.

[0016] Through the above technical solution, the present invention has at least the following advantages over the prior art: the processing device of the present invention is simple, no toxic chemical reagents or expensive biological reagents are required, and the electrical properties of the nanoparticles can be changed only by low-temperature plasma treatment, thereby enhancing their loading efficiency for charged drugs. The technology can be further used for the industrial production of drug-loaded nanoparticles. DETAILED DESCRIPTION

[0017] The embodiments described in the present invention are only a part of the embodiments of the present invention, not all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the protection scope of the present invention.

[0018] Unless otherwise specified, the materials used in the following examples were of analytical grade.

[0019] Example 1.

[0020] 1) Mix 50 mg of charged lipid nanoparticles (DSPG liposomes) with a particle size of 400 nm with 10 mg of the anticancer drug doxorubicin and add them to 5 mL of deionized water with a pH of 7 to form a mixed solution A.

[0021] 2) The mixed liquid A is placed in a low-temperature plasma treatment device, and a low-temperature plasma generator with a hollow electrode dielectric barrier structure is used for treatment at a working voltage of 1200 V and a working current of 10 mA. The working gas is nitrogen with a flow rate of 800 sccm and the treatment time is 10 minutes.

[0022] 3) After treatment, the solution is centrifuged and the supernatant is removed to obtain drug-loaded nanoparticles.

[0023] 4) Determination of drug loading by high performance liquid chromatography.

[0024] Example 2.

[0025] 1) Mix 30 mg of 300 nm charged lipid nanoparticles (DOTAP liposomes) with 1 µg / µl plasmid DNA and add to 5 mL of deionized water at pH 7 to form mixed solution B.

[0026] 2) The mixed liquid B is placed in a low-temperature plasma treatment device and treated under the conditions of a working voltage of 600 V, a working current of 5 mA, an argon working gas, a flow rate of 400 sccm, and a treatment time of 5 minutes.

[0027] 3) After treatment, the drug-loaded nanoparticles were collected by centrifugation.

[0028] 4) qPCR experiment to determine the loading efficiency of plasmid.

[0029] Example 3.

[0030] 1) Mix 100 mg of charged lipid nanoparticles (DOTAP liposomes) with a particle size of 400 nm and 20 mg of glucose oxidase, and add them to 5 mL of deionized water with a pH of 7 to form a mixed solution C.

[0031] 2) The mixed liquid C was placed in a low-temperature plasma treatment device and treated with a low-temperature plasma jet with a hollow electrode dielectric barrier structure. The parameters were set as an operating voltage of 800 V, an operating current of 8 mA, an argon gas flow rate of 600 sccm, and a treatment time of 8 minutes.

[0032] 3) After treatment, the drug-loaded nanoparticles were collected by centrifugation.

[0033] 4) In vitro enzymatic oxidation was used to determine the loading efficiency of the oxidase.

[0034] Comparative example 1.

[0035] 1) Mix 50 mg of charged lipid nanoparticles (DSPG liposomes) with a particle size of 400 nm with 10 mg of the anticancer drug doxorubicin and add them to 5 mL of deionized water with a pH of 7 to form a mixed solution A.

[0036] 2) Without low-temperature plasma treatment, the mixed solution A was mechanically stirred for 10 minutes.

[0037] 3) Centrifuge and remove the supernatant to obtain drug-loaded nanoparticles.

[0038] 4) Determination of drug loading by spectrophotometer.

[0039] Comparative example 2.

[0040] 1) Mix 30 mg of 300 nm charged lipid nanoparticles (DOTAP liposomes) with 1 µg / µl plasmid DNA and add to 5 mL of deionized water at pH 7 to form mixed solution B.

[0041] 2) Without low-temperature plasma treatment, the mixed solution B was mechanically stirred for 10 minutes.

[0042] 3) After treatment, the drug-loaded nanoparticles were collected by centrifugation.

[0043] 4) qPCR determination of plasmid loading amount.

[0044] Comparative example 3.

[0045] 1) Mix 100 mg of charged lipid nanoparticles (DOTAP liposomes) with a particle size of 400 nm and 20 mg of glucose oxidase, and add them to 5 mL of deionized water with a pH of 7 to form a mixed solution C.

[0046] 2) Without low-temperature plasma treatment, the mixed solution C was mechanically stirred for 10 minutes.

[0047] 3) Collect the drug-loaded lipid nanoparticles by centrifugation.

[0048] 4) The loading amount of oxidase was determined by in vitro enzymatic oxidation method.

[0049] Test Example 1: Determination of drug loading efficiency by spectrophotometer.

[0050] 1. Preparation of standard curve: 1) Prepare free drug solutions of different concentrations as standards; 2) Select the appropriate wavelength on the spectrophotometer (usually determined by the maximum absorption wavelength of the drug); 3) Adjust to zero with PBS buffer and measure the absorbance of standard solutions of different concentrations; 4) Draw a concentration-absorbance standard curve.

[0051] 2. Sample processing: 1) Take an appropriate amount of nanoparticle suspension sample, transfer it to a centrifuge tube, and use a specific organic solvent to destroy its structure so that the loaded drug is completely released; 2) Centrifuge at high speed for 10-15 minutes to separate the nanoparticles and loaded drugs; 3) Carefully collect the supernatant to avoid disturbing the pellet.

[0052] 3. Determination of total drug loading: 1) Dilute the supernatant to an appropriate concentration within the measurable range of the spectrophotometer; 2) Measure the absorbance of the supernatant with a spectrophotometer and calculate the drug concentration against the standard curve; 3) Total amount of drug loaded = drug concentration × sample volume.

[0053] Test Example 2: qPCR determination of plasmid loading efficiency.

[0054] 1. Preparation of standard curve: 1) Prepare a pure plasmid standard solution of known concentration; 2) Use a qPCR instrument to amplify standards of different concentrations and determine the cycle threshold (Ct value) of each concentration; 3) Draw a standard curve: the horizontal axis is the logarithmic concentration of the standard, and the vertical axis is the corresponding Ct value. This standard curve will be used to calculate the plasmid content in the sample.

[0055] 2. Sample processing: 1) Take an appropriate amount of nanoparticle suspension sample for plasmid loading efficiency determination; 2) Removal of free plasmids: Add an appropriate amount of DNase enzyme (according to the recommended amount of enzyme), incubate for a period of time (usually 37°C, 30 minutes) to degrade free plasmids, and add DNase inhibitors (such as EDTA) to terminate the reaction; 3) After sample preparation is completed, proceed directly to the next step of qPCR detection.

[0056] 3.qPCR reaction: 1) Place the processed samples and standards into PCR tubes respectively; 2) Prepare the qPCR reaction system, including the following components: qPCR primers (specific primers for plasmid), qPCR reaction reagents (such as SYBR Green or TaqMan probes), an appropriate amount of treated nanoparticle sample, and adjust the final volume to a suitable reaction volume; 3) Perform amplification on a qPCR instrument and record the Ct value.

[0057] 4. Calculation of plasmid copy number: 1) Use the standard curve to convert the Ct value of the sample into the plasmid copy number; 2) The copy number of the plasmid in the sample (the amount of encapsulated plasmid) is the amount of plasmid loaded in the nanoparticles.

[0058] Test Example 3: Determination of the loading amount of oxidase by in vitro enzymatic oxidation method.

[0059] 1. Preparation of standard curve: 1) Prepare oxidase standard solutions of different concentrations; 2) Each concentration of standard solution is reacted with a fixed concentration of substrate for a period of time (usually 5-10 minutes) to generate a product; 3) Immediately detect the absorbance of the reaction product using a spectrophotometer or microplate reader; 4) Draw a standard curve of absorbance-enzyme concentration to facilitate the subsequent calculation of the enzyme loading in the sample.

[0060] 2. Sample processing: 1) Take an appropriate amount of enzyme-containing nanoparticle suspension sample and add it to a centrifuge tube; 2) Separate the nanoparticles by centrifugation and discard the supernatant to remove unbound free enzyme; 3) Resuspend the nanoparticles with an appropriate amount of buffer to ensure that the particles are intact.

[0061] 3. Enzyme activity determination: 1) Mix the treated nanoparticle sample with an appropriate amount of enzyme substrate; 2) Maintain appropriate reaction conditions (e.g., 37°C, reaction time 5-10 min) to allow the enzyme to catalyze the substrate to generate product; 3) Measure the absorbance of the product immediately after the reaction is completed; 4) If the reaction product is generated slowly or the substrate reaction rate is fast, the assay can be stabilized by terminating the reaction (e.g., adding a stop solution).

[0062] 4. Calculate the oxidase loading: 1) Calculate the enzyme loading of the nanoparticles in the sample based on the absorbance of the sample against the standard curve.

[0063] Table 1.

[0064]

[0065] The above embodiments illustrate the application of the present invention in different types of nanoparticles and drugs, and show the wide applicability and significant advantages of the present invention in improving the drug loading efficiency of nanoparticles.

Claims

1. A nanoparticle composition, characterized in that: The nanoparticle composition comprises nanoparticles and loaded drugs; wherein both the nanoparticles and the target drugs are electrically conductive.

2. The nanoparticle composition according to claim 1, characterized in that The nanoparticles are charged organic nanoparticles.

3. The nanoparticle composition according to claim 1, characterized in that Preferably, the charged organic nanoparticles include one or more of liposomes, lipid nanoparticles, polymer vesicles, dendrimers, polymer micelles.

4. The nanoparticle composition according to claim 1, characterized in that Preferably, the average particle size of the organic nanoparticles is 100-800 nm.

5. The nanoparticle composition according to claim 1, characterized in that Preferably, the target drug includes one or more of small interfering RNA (siRNA), small hairpin RNA (shRNA), micro RNA (miRNA), messenger RNA (mRNA), plasmid DNA (Plasmid), protein and ionizable drug.

6. A method for preparing nanoparticles, characterized in that: The nanoparticle and target drug mixture is treated by low-temperature plasma jet to increase the drug loading of the nanoparticles; the loading method comprises the following steps: 1) Mix the nanoparticles with the target drug and disperse them in deionized water at pH 7; 2) treating the mixed solution in step 1) using low-temperature plasma; 3) Centrifuging the solution obtained in step 2) to obtain drug-loaded nanoparticles.

7. The method according to claim 4, characterized in that The low-temperature plasma generating device is a hollow electrode dielectric barrier structure.

8. The method according to claim 4, characterized in that The parameters of the low-temperature plasma emission device are set as follows: working voltage 600-1200V, working current 5-10mA, working gas argon or nitrogen, and flow rate 400-800sccm.

9. The method according to claim 4, characterized in that With an electron density of 10 15 / cm 3 The mixed solution is treated with a low-temperature plasma jet of the order of magnitude for 5-15 minutes.

10. Use of the nanoparticle composition according to any one of claims 1 to 5 in preparing a drug delivery carrier.

Images