CN112972429A - Bovine serum albumin-nano silver modified chitosan nano drug delivery system and preparation method thereof - Google Patents
Bovine serum albumin-nano silver modified chitosan nano drug delivery system and preparation method thereof Download PDFInfo
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Abstract
The invention relates to the technical field of polysaccharide, in particular to a bovine serum albumin-nano silver modified chitosan nano drug delivery system and a preparation method thereof. The nano-silver chitosan gel is prepared from the nano-silver modified chitosan, has elasticity and stability similar to those of soft tissues, can effectively prolong the circulation time of a nano drug delivery system in vivo, and controls the slow release of drugs. Bovine serum albumin is added, has excellent transmission and metabolism effects and good biocompatibility and degradability, can promote the circulation of materials in vivo and more effectively control the release of drugs; the invention adds two alkaloids drugs of berberine hydrochloride and camptothecin, camptothecin has excellent anti-tumor effect, berberine hydrochloride has the effects of improving microenvironment for survival of tumor cells and preventing migration of tumor cells, and the two drugs have synergistic effect and better treatment effect on tumors.
Description
Technical Field
The invention relates to the technical field of polysaccharide, in particular to a bovine serum albumin-nano silver modified chitosan nano drug delivery system and a preparation method thereof.
Background
Camptothecin is an alkaloid compound extracted from Camptotheca acuminata Decne, has excellent antitumor activity, and has good therapeutic effects on hepatocarcinoma, lung cancer, and colorectal cancer. However, camptothecin has very poor bioavailability, has large toxicity to normal tissue cells, and is easy to generate drug resistance. Therefore, the nano-drug delivery system has important significance for improving the anti-tumor activity of the camptothecin and reducing the toxic and side effects of the camptothecin. For example, in a method for preparing camptothecin drug nanoparticles disclosed in chinese patent CN112107690A, camptothecin drugs and copolymers having aromatic ring structures are dissolved in an organic solvent as an oil phase, and purified water is used as a water phase; slowly dropping the oil phase into the water phase dropwise under low-speed stirring to obtain blue opalescent emulsion; and then removing the organic solvent by using a rotary evaporator to obtain the camptothecin drug nanoparticles. The copolymer is an aromatic ring side chain modified amphiphilic block copolymer, and is prepared by mixing polyethylene glycol monomethyl ether serving as an initiator with an aromatic ring-containing aliphatic cyclic ester monomer and a hydrophobic block monomer and performing high-temperature ring-opening polymerization.
Chitosan is one of the most naturally occurring polysaccharides in nature, has good biocompatibility and degradability, and is used for preparing various types of antitumor drug carriers, including: microcapsules, gels, nanoparticles, microspheres, and the like. In addition, the chitosan structure contains a large amount of amino groups, and various functional groups can be introduced through structural modification, so that the treatment effect of the medicament is improved. Therefore, the chitosan has great potential in the development of nano anti-tumor drug carriers. The nano silver has various functions of good antibacterial effect, strong permeability, promotion of repair and regeneration of injured cells and the like, so that the nano silver is widely used for preparing various medical polymer materials.
The nano-silver and chitosan are used as composite materials, and the advantages of the performances of the two materials can be complemented when the nano-silver and chitosan are used in a drug delivery system, so that the nano-silver-chitosan composite material has great potential in the development of nano-drug carriers. Bovine serum albumin is globulin with high biocompatibility and biodegradability, and in the field of drug delivery, the bovine serum albumin can obviously improve the biocompatibility of a nano carrier and prolong the transportation time of the carrier in vivo. Based on the outstanding capability of nano silver, chitosan and bovine serum albumin in the field of drug delivery, the nano silver-chitosan composite nano carrier can be used for developing novel composite nano carriers, and plays an important role in solving the problems of poor absorption efficiency and large toxic and side effects of camptothecin.
Disclosure of Invention
The invention aims to solve the problems in the prior art and provides a bovine serum albumin-nano silver modified chitosan nano drug delivery system capable of improving camptothecin absorption efficiency and reducing toxic and side effects of camptothecin and a preparation method thereof.
The technical scheme of the invention is as follows:
a preparation method of a bovine serum albumin-nano silver modified chitosan nano drug delivery system comprises the following steps:
preparing nano-silver modified chitosan: adding silver nitrate into a first solvent, heating to 50-70 ℃, dropwise adding a sodium ascorbate aqueous solution with the mass fraction of 5-10% at the dropping speed of 1-3 drops/second, stirring for 2-3 hours after the dropwise adding is finished, cooling to 20-40 ℃, adding chitosan, dropwise adding an acetic acid aqueous solution with the mass fraction of 2.5-10%, stirring for 1-2 hours, dialyzing for 1-3 days by using a dialysis bag with the molecular weight of 3500, and freeze-drying to obtain nano-silver modified chitosan;
preparing a bovine serum albumin-nano silver modified chitosan nano drug-loading system: adding the nano-silver modified chitosan prepared in the step into a solvent II, heating to 20-50 ℃, stirring for 10-30 minutes, adding a berberine hydrochloride methanol solution, stirring for 0.5-1.5 hours, adding a camptothecin methanol solution, stirring for 10-30 minutes, adding a folic acid modified bovine serum albumin conjugate and graphene oxide, stirring for 1-2 hours, dialyzing for 1-3 days by using a dialysis bag with the molecular weight of 3500, and freeze-drying to obtain a bovine serum albumin-nano-silver modified chitosan nano drug loading system;
the first solvent is water;
the second solvent is water;
the mass fraction of the berberine hydrochloride in the berberine hydrochloride methanol solution is 5-15%;
the mass fraction of camptothecin in the camptothecin methanol solution is 2-6%;
wherein the mass ratio of silver nitrate, a first solvent, an aqueous solution of sodium ascorbate, chitosan, an aqueous solution of acetic acid, a second solvent, a methanol solution of berberine hydrochloride, a methanol solution of camptothecin, a conjugate of folic acid modified bovine serum albumin and graphene oxide is 0.1-0.5: 100-130: 2-6: 1-2: 5-15: 150-200: 3-6: 5-15: 0.1-0.3: 0.2 to 0.4.
Preferably, the mass fraction of sodium ascorbate in the aqueous solution of sodium ascorbate is 7.5%.
Preferably, the chitosan has a molecular weight of 20 ten thousand and a degree of deacetylation of greater than 95%.
Preferably, the mass fraction of acetic acid in the aqueous acetic acid solution is 6%.
Preferably, the mass fraction of the berberine hydrochloride in the berberine hydrochloride methanol solution is 10%.
Preferably, the mass fraction of camptothecin in the methanol solution of camptothecin is 4%.
The bovine serum albumin-nano silver modified chitosan nano drug delivery system is of a two-layer spherical structure, wherein the inner layer is a nano silver modified chitosan combined graphene oxide, and the outer layer is a folic acid modified bovine serum albumin conjugate.
The invention has the beneficial effects that: the nano-silver chitosan gel is prepared from the nano-silver modified chitosan, the obtained gel has elasticity and stability similar to those of soft tissues, the circulation time of a nano drug delivery system in vivo can be effectively prolonged, the slow release of the drug is controlled, in addition, the chitosan contains a large amount of amino groups, and the amino groups can be protonated under the acidic environment of tumor cells, so that the rapid release of the drug around the tumor tissue is realized.
According to the preparation method, the bovine serum albumin is added in the preparation process, is a protein contained in blood, has excellent transmission and metabolism effects and good biocompatibility and degradability, and can promote the circulation of materials in vivo and more effectively control the release of the medicine in a nano medicine carrying system; the invention adds two alkaloids drugs of berberine hydrochloride and camptothecin, camptothecin has excellent anti-tumor effect, berberine hydrochloride has the functions of improving microenvironment for survival of tumor cells and preventing migration of tumor cells, and the two drugs have synergistic effect and better treatment effect on tumors; according to the preparation method disclosed by the invention, the graphene oxide is added in the preparation process, the graphene oxide has a large conjugated plane system, the molecular structures of the camptothecin and the berberine hydrochloride belong to conjugated plane structures, and the camptothecin and the berberine hydrochloride can be combined with the graphene oxide in a pi-pi accumulation mode, so that the drug loading efficiency of the gel is higher.
Drawings
Fig. 1 shows the external morphology of the nano drug delivery system prepared in example 1 determined by scanning electron microscopy;
FIG. 2 is the external morphology of the nano drug delivery system prepared in example 2 determined by scanning electron microscopy;
FIG. 3 is the external morphology of the nano drug delivery system prepared in example 3 determined by scanning electron microscopy;
FIG. 4 shows the microstructure of the Nanocarrier system prepared in example 1 as measured by transmission electron microscopy;
FIG. 5 shows the microstructure of the Nanocarrier system prepared in example 2 as measured by transmission electron microscopy;
fig. 6 shows the microstructure of the nano drug delivery system prepared in example 3 measured by transmission electron microscopy.
Detailed Description
In order to make the technical means, technical features, objects and technical effects of the present invention easily understandable, the present invention is further described below with reference to the specific drawings.
The folic acid modified bovine serum albumin conjugate of the embodiment of the invention is purchased from sienna ruixi biotechnology limited.
Example 1:
preparing nano-silver modified chitosan: adding 1g of silver nitrate into 1000g of water, heating to 50 ℃, dropwise adding 20g of sodium ascorbate water solution with the mass fraction of 5%, dropwise adding at the speed of 1 drop/second, stirring for 2 hours after dropwise adding, cooling to 20 ℃, adding 10g of chitosan, dropwise adding 50g of acetic acid water solution with the mass fraction of 2.5%, dropwise adding at the speed of 2 drops/second, stirring for 1 hour, dialyzing for 1 day by using a dialysis bag with the molecular weight of 3500, and freeze-drying to obtain nano-silver modified chitosan;
preparing a bovine serum albumin-nano silver modified chitosan nano drug-loading system: adding the nano-silver modified chitosan prepared in the step into 1500g of water, heating to 20 ℃, stirring for 10 minutes, adding 30g of berberine hydrochloride methanol solution (the mass fraction of berberine hydrochloride is 5%), stirring for 0.5 hour, adding 50g of camptothecin methanol solution (the mass fraction of camptothecin is 2%), stirring for 10 minutes, adding 1g of folic acid modified bovine serum albumin conjugate and 2g of graphene oxide, stirring for 1 hour, dialyzing for 1 day by using a dialysis bag with the molecular weight of 3500, and freeze-drying to obtain the bovine serum albumin-nano-silver modified chitosan nano drug delivery system.
Example 2:
preparing nano-silver modified chitosan: adding 5g of silver nitrate into 1300g of water, heating to 70 ℃, dropwise adding 60g of sodium ascorbate water solution with the mass fraction of 10%, dropwise adding at the speed of 3 drops/second, stirring for 3 hours after dropwise adding, cooling to 40 ℃, adding 20g of chitosan, dropwise adding 150g of acetic acid water solution with the mass fraction of 10%, dropwise adding at the speed of 4 drops/second, stirring for 2 hours, dialyzing for 3 days by using a dialysis bag with the molecular weight of 3500, and freeze-drying to obtain nano-silver modified chitosan;
preparing a bovine serum albumin-nano silver modified chitosan nano drug-loading system: adding the nano-silver modified chitosan prepared in the step into 2000g of water, heating to 50 ℃, stirring for 30 minutes, adding 60g of berberine hydrochloride methanol solution (the mass fraction of berberine hydrochloride is 15%), stirring for 1.5 hours, adding 150g of camptothecin methanol solution (the mass fraction of camptothecin is 6%), stirring for 30 minutes, adding 3g of folic acid modified bovine serum albumin conjugate and 4g of graphene oxide, stirring for 2 hours, dialyzing for 3 days by using a dialysis bag with the molecular weight of 3500, and freeze-drying to obtain the bovine serum albumin-nano-silver modified chitosan nano drug delivery system.
Example 3:
preparing nano-silver modified chitosan: adding 2g of silver nitrate into 1100g of water, heating to 60 ℃, dropwise adding 40g of sodium ascorbate water solution with the mass fraction of 7.5%, dropwise adding at the speed of 2 drops/second, stirring for 2.5 hours after dropwise adding, cooling to 30 ℃, adding 15g of chitosan, dropwise adding 100g of acetic acid water solution with the mass fraction of 6%, dropwise adding at the speed of 3 drops/second, stirring for 1.5 hours, dialyzing for 2 days by using a dialysis bag with the molecular weight of 3500, and freeze-drying to obtain nano-silver modified chitosan;
preparing a bovine serum albumin-nano silver modified chitosan nano drug-loading system: adding the nano-silver modified chitosan prepared in the step into 1700g of water, heating to 35 ℃, stirring for 20 minutes, adding 50g of berberine hydrochloride methanol solution (the mass fraction of berberine hydrochloride is 10%), stirring for 1 hour, adding 90g of camptothecin methanol solution (the mass fraction of camptothecin is 4%), stirring for 20 minutes, adding 2g of folic acid modified bovine serum albumin conjugate and 3g of graphene oxide, stirring for 1.5 hours, dialyzing for 2 days by using a dialysis bag with the molecular weight of 3500, and freeze-drying to obtain the bovine serum albumin-nano-silver modified chitosan nano drug delivery system.
Example 4:
encapsulation efficiency of nano drug-loading system to camptothecin and berberine hydrochloride
Firstly, respectively making standard curves of camptothecin and berberine hydrochloride by using a high performance liquid chromatography, weighing a certain amount of camptothecin or berberine hydrochloride, dissolving the camptothecin or berberine hydrochloride in dimethyl sulfoxide, and fixing the volume to different concentrations. Measuring peak areas of camptothecin or berberine hydrochloride with different concentrations on a high performance liquid chromatograph, and making a standard curve according to the concentration and the peak areas.
The encapsulation efficiency of the nano drug-carrying system to camptothecin and berberine hydrochloride is measured by adopting a high performance liquid chromatography, a certain amount of the nano drug-carrying system prepared by the invention is weighed, DMSO is added, ultrasonic crushing and centrifugation are carried out, supernatant is taken, the peak area of the camptothecin or berberine hydrochloride is measured on a high performance liquid chromatograph, and the content of the camptothecin or berberine hydrochloride in the nano drug-carrying system is calculated according to the standard curve made in the last step. The encapsulation rate of camptothecin or berberine hydrochloride is calculated according to the following formula:
the encapsulation efficiency is equal to the content of the tested medicine in the nano medicine carrying system/the adding amount of the tested medicine multiplied by 100 percent
The test results are shown in Table 1.
TABLE 1 encapsulation efficiency of nano drug delivery system for camptothecin and berberine hydrochloride
| Examples samples | Example 1 | Example 2 | Example 3 |
| Encapsulation efficiency of camptothecin | 95.6% | 95.9% | 96.3% |
| Encapsulation rate of berberine hydrochloride | 90.8% | 92.8% | 92.3% |
The results in table 1 show that the nano drug delivery system prepared by the invention has higher encapsulation efficiency on camptothecin and berberine hydrochloride. This is probably because camptothecin and berberine hydrochloride have plane conjugated structures, and pi electrons on the camptothecin and berberine hydrochloride are easily combined with graphene oxide in a pi-pi stacking mode and are well stabilized in the material.
External form of nano drug-loading system determined by scanning electron microscope
And (3) determining the external form of the nano drug-carrying system by using a scanning electron microscope. As shown in fig. 1 to 3, the nano drug delivery systems prepared in examples 1 to 3 are all spherical structures and have uniform size distribution, as measured by a scanning electron microscope, the nano drug delivery systems prepared in examples 1 to 3 have a large diameter of about 300 to 400nm, and the nano drug delivery systems prepared in examples 2 and 3 have a small diameter of 100 to 200 nm.
Microstructure for measuring nano drug-loading system by transmission electron microscope
The microstructure of the nano drug-loading system is further tested by a transmission electron microscope, as shown in fig. 4-6, the microstructure of the nano drug-loading system prepared in examples 1-3 determined by the transmission electron microscope is similar to the result of a scanning electron microscope, and the nano drug-loading system prepared in example 1 has a larger particle size and is spherical. The nano drug delivery systems prepared in examples 2 and 3 have smaller particle size and are also spherical structures.
Evaluation of antitumor Activity in vitro
The MTT method is adopted to measure the cytotoxicity of the compound on a plurality of tumor cell lines such as HepG2, HeLa, A549, MCF7 and the like, and the inhibition rate of the compound on each tumor cell line under different concentrations is calculated. The concentrations tested in this experiment were 0, 1, 2, 4, 8, 16 μmol/L, calculated as camptothecin concentration, and the time for treating tumor cells with the drug was 24 hours. As shown in Table 2, the nano drug delivery system prepared by the embodiment of the invention has significantly better inhibitory activity on tumor cells than camptothecin, and the survival rate of each tumor cell line is about 10 when the concentration of camptothecin is 8 μmol/L. The result shows that the drug carrier has obvious enhancement effect on the anti-tumor activity of the camptothecin.
TABLE 2 survival of various tumor cell lines under the action of different concentrations of camptothecin
In summary, the embodiments of the present invention are merely exemplary and should not be construed as limiting the scope of the invention. All equivalent changes and modifications made according to the content of the claims of the present invention should fall within the technical scope of the present invention.
Claims (7)
1. A preparation method of a bovine serum albumin-nano silver modified chitosan nano drug delivery system is characterized by comprising the following steps: the method comprises the following steps:
preparing nano-silver modified chitosan: adding silver nitrate into a first solvent, heating to 50-70 ℃, dropwise adding a sodium ascorbate aqueous solution with the mass fraction of 5-10% at the dropping speed of 1-3 drops/second, stirring for 2-3 hours after the dropwise adding is finished, cooling to 20-40 ℃, adding chitosan, dropwise adding an acetic acid aqueous solution with the mass fraction of 2.5-10%, stirring for 1-2 hours, dialyzing for 1-3 days by using a dialysis bag with the molecular weight of 3500, and freeze-drying to obtain nano-silver modified chitosan;
preparing a bovine serum albumin-nano silver modified chitosan nano drug-loading system: adding the nano-silver modified chitosan prepared in the step into a solvent II, heating to 20-50 ℃, stirring for 10-30 minutes, adding a berberine hydrochloride methanol solution, stirring for 0.5-1.5 hours, adding a camptothecin methanol solution, stirring for 10-30 minutes, adding a folic acid modified bovine serum albumin conjugate and graphene oxide, stirring for 1-2 hours, dialyzing for 1-3 days by using a dialysis bag with the molecular weight of 3500, and freeze-drying to obtain a bovine serum albumin-nano-silver modified chitosan nano drug loading system;
the first solvent is water;
the second solvent is water;
the mass fraction of the berberine hydrochloride in the berberine hydrochloride methanol solution is 5-15%;
the mass fraction of camptothecin in the camptothecin methanol solution is 2-6%;
wherein the mass ratio of silver nitrate, a first solvent, an aqueous solution of sodium ascorbate, chitosan, an aqueous solution of acetic acid, a second solvent, a methanol solution of berberine hydrochloride, a methanol solution of camptothecin, a conjugate of folic acid modified bovine serum albumin and graphene oxide is 0.1-0.5: 100-130: 2-6: 1-2: 5-15: 150-200: 3-6: 5-15: 0.1-0.3: 0.2 to 0.4.
2. The preparation method of the bovine serum albumin-nano silver modified chitosan nano drug delivery system according to claim 1, which is characterized in that: the mass fraction of sodium ascorbate in the sodium ascorbate water solution is 7.5%.
3. The preparation method of the bovine serum albumin-nano silver modified chitosan nano drug delivery system according to claim 1, which is characterized in that: the molecular weight of the chitosan is 20 ten thousand, and the degree of deacetylation is more than 95%.
4. The preparation method of the bovine serum albumin-nano silver modified chitosan nano drug delivery system according to claim 1, which is characterized in that: the mass fraction of acetic acid in the acetic acid aqueous solution is 6%.
5. The preparation method of the bovine serum albumin-nano silver modified chitosan nano drug delivery system according to claim 1, which is characterized in that: the mass fraction of the berberine hydrochloride in the berberine hydrochloride methanol solution is 10 percent.
6. The preparation method of the bovine serum albumin-nano silver modified chitosan nano drug delivery system according to claim 1, which is characterized in that: the mass fraction of camptothecin in the camptothecin methanol solution is 4%.
7. A bovine serum albumin-nano silver modified chitosan nano drug delivery system is characterized in that: the preparation method of any one of claims 1 to 6, wherein the bovine serum albumin-nano silver modified chitosan nano drug delivery system is of a two-layer spherical structure, the inner layer is nano silver modified chitosan combined graphene oxide, and the outer layer is a folic acid modified bovine serum albumin conjugate.
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