CN115282127B - Chitosan calcium alginate nanoparticle microsphere and preparation method thereof - Google Patents
Chitosan calcium alginate nanoparticle microsphere and preparation method thereof Download PDFInfo
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Abstract
The invention discloses chitosan calcium alginate nanoparticle microspheres and a preparation method thereof, wherein methotrexate solution is dripped into sulfhydrylation bovine serum albumin solution, chloroauric acid solution is added dropwise, and then sodium alginate solution is added to obtain water phase; and (3) dropping the water phase into a calcium chloride water solution through a coaxial needle under nitrogen shearing to obtain calcium alginate microspheres, cleaning with a chitosan water solution, centrifuging and freeze-drying to obtain the calcium alginate microsphere. The preparation method can realize that the bovine serum albumin methotrexate-loaded nanoparticle is simply, quickly and efficiently encapsulated in the chitosan calcium alginate microsphere, the encapsulation rate is up to 90%, and the stability of the bovine serum albumin methotrexate-loaded nanoparticle is high. Has stomach protecting function, can target intestinal tract to deliver methotrexate medicine, and can be used for effectively treating various inflammatory diseases such as intestinal inflammatory diseases, rheumatoid arthritis and the like.
Description
Technical Field
The invention belongs to the technical field of micro-nano medical treatment, and in particular relates to chitosan calcium alginate nanoparticle microspheres with gastrointestinal tract protection function and a preparation method thereof.
Background
Methotrexate (Methotrexate) is an anti-leaf acid antitumor drug, and because Methotrexate can inhibit dihydrofolate reductase to reduce formation of tetrahydrofolate and block DNA synthesis, and can effectively inhibit proliferation of immunoinflammatory cells to play a role in immunosuppression, anti-inflammatory and the like, methotrexate is a common drug for many inflammatory diseases, and has been widely used for inflammatory diseases such as rheumatoid arthritis, crohn's enteritis, systemic lupus erythematosus, vasculitis, dermatomyositis/polymyositis, polymyositis rheumatica, psoriatic arthritis and the like.
Oral methotrexate is the first treatment for many patients with medium to high disease activity inflammation, however, oral methotrexate can cause serious gastrointestinal side effects, often causes serious mucosal reactions, such as ulcerative gastritis, hemorrhagic enteritis, and even intestinal perforation to cause death, and methotrexate lacks inflammation chemotaxis, is extremely easy to cause main organ injury and even systemic toxicity. In addition, 50% -60% of methotrexate in the systemic circulation will bind to plasma proteins with a biological half-life of only about 6 hours.
Bovine serum albumin is widely studied as a nano-carrier of methotrexate, and the bovine serum albumin and the methotrexate have better affinity due to hydrophilic and hydrophobic acting force, so that the methotrexate can be efficiently loaded, the bovine serum albumin has inflammation trend ability and can be enriched at an inflammation part, in addition, the bovine serum albumin nano-particle can prolong the circulation time of the methotrexate in a body, and meanwhile, the good biological safety of the bovine serum albumin nano-particle enables the bovine serum albumin nano-carrier to have huge clinical transformation potential.
However, when the bovine serum albumin nano-drug is orally taken through the stomach, the bovine serum albumin nano-drug is influenced by physiological environments such as low pH of the stomach and abundant protease, and the bovine serum albumin nano-drug is easily damaged to cause early drug exposure, cannot safely pass through the stomach, is not timely and thoroughly controlled, and even causes a series of toxic and side effects; and free methotrexate is prone to damage gastrointestinal mucosal integrity causing gastrointestinal side effects. Therefore, the protection of the oral methotrexate bovine serum albumin nano-drug, which enables the oral methotrexate bovine serum albumin nano-drug to safely and effectively play a role in treatment, is a difficult problem which needs to be overcome.
The colon wall is easy to penetrate, the pH is close to neutral, the concentration of proteolytic enzyme is low, the stimulation of chemotherapy drugs to the gastrointestinal tract can be reduced, in addition, EPR effect exists in intestinal epithelial cells, nano-scale particles can be preferentially ingested by the intestinal cells, and therefore, the targeting of the colon is a reliable nano-drug delivery strategy.
The hydrogel microsphere with the polysaccharide material source can realize stomach protection in nano drug oral administration because the nano microsphere has good biocompatibility, various functions and proper size, is widely applied to an oral drug delivery system, and polysaccharide such as chitosan, alginate and the like can be degraded by colonic enzymes, and the stomach and the small intestine lack corresponding enzymes. The low-pH stable calcium alginate hydrogel microsphere can avoid degradation and damage of gastric acid or enzyme to nano-drugs in the oral process, improve the stability of the nano-drugs, protect the nano-drugs from being released in the stomach, and dissolve in the intestinal tract with higher pH value, and avoid various gastrointestinal side reactions, so that the oral pH-dependent calcium alginate microsphere drug delivery system can provide a reliable barrier for the delivery of the nano-drugs in the gastrointestinal tract. However, the chitosan calcium alginate nanoparticle microsphere has the defects of low encapsulation rate, difficult encapsulation and easy dissociation when used for encapsulating the bovine serum albumin methotrexate-loaded nanoparticle.
Disclosure of Invention
Based on the above, one of the purposes of the invention is to provide a preparation method of chitosan calcium alginate nanoparticle microspheres, which can realize rapid and efficient encapsulation of bovine serum albumin-loaded nano-drugs inside chitosan calcium alginate nanoparticle microspheres.
The specific technical scheme for realizing the aim of the invention comprises the following steps:
a method for preparing chitosan calcium alginate nanoparticle microspheres, which comprises the following steps:
(1) Dripping the methotrexate solution into the sulfhydrylation bovine serum albumin solution, and uniformly stirring to obtain the bovine serum albumin-carried methotrexate nanoparticle solution;
(2) Dropwise adding a chloroauric acid solution with the concentration of 10-40 wt% into the bovine serum albumin methotrexate-loaded nanoparticle solution in the step (1), and stirring and centrifuging to obtain gold crosslinked methotrexate bovine serum albumin nanoparticles; the volume ratio of the chloroauric acid solution to the bovine serum albumin methotrexate-loaded nanoparticle solution is 1:900-1000;
(3) According to the volume ratio of 2: 0.8-1.2, adding the gold cross-linked methotrexate bovine serum albumin nanoparticle prepared in the step (2) into sodium alginate solution to obtain a water phase;
(4) Dropwise adding the water phase obtained in the step (3) into a calcium chloride water solution through a coaxial needle under nitrogen shearing to obtain calcium alginate nanoparticle microspheres;
(5) And (3) cleaning the calcium alginate nanoparticle microspheres obtained in the step (4) by using chitosan aqueous solution, centrifuging and freeze-drying to obtain the calcium alginate nanoparticle microsphere.
In some embodiments, the chloroauric acid solution in step (2) has a mass percent concentration of 15 wt.% to 30 wt.%.
In some embodiments, the chloroauric acid solution has a mass percent concentration of 18wt% to 25wt%.
In some embodiments, the volume ratio of chloroauric acid solution to bovine serum albumin methotrexate-loaded nanoparticle solution is 1:945-955.
In some of these embodiments, the temperature of the stirring in step (2) is from 40 ℃ to 50 ℃, the speed is from 500rpm to 1000rpm, and the time is from 4 hours to 8 hours; and/or the rotational speed of the centrifugation is 7500 rpm-8500 rpm for 20 min-30 min.
In some of these embodiments, the volume ratio of the methotrexate solution to the thiolated bovine serum albumin solution in step (1) is 1: 8-10.
In some of these embodiments, the methotrexate solution in step (1) is a methotrexate DMSO solution having a concentration of 1.5mg/mL to 2.5mg/mL; and/or the concentration of the sulfhydrylation bovine serum albumin solution is 2.0 mg/mL-3.0 mg/mL.
In some embodiments, the concentration of the sodium alginate solution in the step (3) is 4 mg/mL-6 mg/mL.
In some embodiments, the nitrogen flow rate in step (4) is 10m/s to 20m/s.
In some embodiments, the concentration of the aqueous solution of calcium chloride is 1 mg/mL-6 mg/mL.
In some embodiments, the volume ratio of the aqueous phase to the aqueous calcium chloride solution in step (4) is 1:10-20.
In some embodiments, the concentration of the aqueous chitosan solution in step (5) is 0.8 mg/mL-1.2 mg/mL.
In some embodiments, the ratio of the calcium alginate nanoparticle microsphere to the chitosan aqueous solution in the step (5) is 0.5g to 1g:40 mL-80 mL.
In some embodiments, the centrifugation in step (5) is performed at 4000rpm to 6000rpm for 20min to 30min.
The invention also provides chitosan calcium alginate nanoparticle microspheres prepared by the preparation method.
Compared with the prior art, the invention has the following beneficial effects:
1. according to the invention, the gold cross-linked bovine serum albumin methotrexate-loaded nanoparticle is prepared by dropwise adding chloroauric acid solution into the sulfhydryl bovine serum albumin methotrexate-loaded nanoparticle, and then the chitosan calcium alginate nanoparticle microsphere is prepared.
2. The chitosan calcium alginate nanoparticle microsphere prepared by the preparation method provided by the invention has a stomach protection function (reducing gastrointestinal side effects during oral administration of methotrexate), has a drug slow release property, can deliver methotrexate drugs to intestinal tract parts in a targeting way, and can be used for effectively treating various inflammatory diseases such as intestinal inflammatory diseases and rheumatoid arthritis.
Drawings
FIG. 1 is a morphology diagram of chitosan calcium alginate nanoparticle microspheres in test example 1 of the present invention.
FIG. 2 is a graph showing the frequency of particle size distribution of chitosan calcium alginate nanoparticle microspheres in test example 1 of the present invention.
FIG. 3 is a graph showing the results of the detection of the average particle diameters of the various drug-loaded nanoparticles according to test example 2 of the present invention.
FIG. 4 is a graph showing the results of potential analysis of various drug-loaded nanoparticles and nanoparticle microspheres in test example 3 of the present invention.
FIG. 5 is a graph showing the 7-day change in particle size of various nanoparticle microspheres in test example 4 according to the present invention.
Fig. 6 is a standard curve of methotrexate in test example 5 according to the present invention.
FIG. 7 is a graph showing the encapsulation efficiency of various drug-loaded nanoparticles and nanoparticle microspheres according to test example 5 of the present invention.
FIG. 8 is a graph of the thickness of the footpad of each group of mice in test example 6 of the present invention.
FIG. 9 is a graph showing the change in body weight of each group of mice in test example 6 according to the present invention.
Detailed Description
The present invention will be described more fully hereinafter in order to facilitate an understanding of the present invention. This invention may be embodied in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and/or" as used herein includes any and all combinations of one or more of the associated listed items.
Unless otherwise indicated, the experimental methods used in the examples of the present invention were conventional experimental methods, and the various reagent consumables used in the examples were all commercially available products.
The invention firstly provides a preparation method of chitosan calcium alginate nanoparticle microspheres, which comprises the following steps:
(1) Preparation of bovine serum albumin-carried methotrexate nanoparticle
Dropping the methotrexate solution into the sulfhydrylation bovine serum albumin solution, and uniformly stirring to obtain the bovine serum albumin-carried methotrexate nanoparticle solution;
in this step, the volume ratio of the methotrexate solution to the mercaptobovine serum albumin solution is 1: 8-10. The methotrexate solution is a methotrexate DMSO solution, and the concentration of the methotrexate DMSO solution is 1.5 mg/mL-2.5 mg/mL; the concentration of the sulfhydrylation bovine serum albumin solution is 2.0 mg/mL-3.0 mg/mL.
(2) Preparation of gold-crosslinked bovine serum albumin-carried methotrexate nanoparticle
Dropwise adding a chloroauric acid solution with the concentration of 10-40 wt% into the bovine serum albumin methotrexate-loaded nanoparticle solution prepared in the step (1), stirring, and then placing into a 100KD ultrafilter tube for centrifugation to obtain gold cross-linked methotrexate bovine serum albumin nanoparticles; the chloroauric acid and sulfhydryl reaction can crosslink the nanoparticle structure by forming covalent bonds or chemical bonds;
in the step, the volume ratio of the chloroauric acid solution to the bovine serum albumin methotrexate-loaded nanoparticle solution is 1:900 to 1000, preferably 1:945 to 955; the mass percentage concentration of the chloroauric acid solution is preferably 15wt% to 30wt%, more preferably 18wt% to 25wt%, and most preferably 20wt%. The temperature of stirring is 40-50 ℃, the speed is 500-1000 rpm, and the time is 4-8 hours; the rotational speed of the centrifugation is 7500 rpm-8500 rpm, and the time is 20 min-30 min.
(3) Preparation of aqueous phase
According to the volume ratio of 2: 0.8-1.2 (preferably 2:1), adding the gold cross-linked methotrexate bovine serum albumin nanoparticle prepared in the step (2) into sodium alginate solution of 4-6 mg/mL (preferably 5 mg/mL) to obtain water phase;
(4) Preparation of calcium alginate nanoparticle microsphere by air flow control technology
Dropping the water phase in the step (3) into a calcium chloride water solution through a coaxial needle under nitrogen shearing to obtain calcium alginate nanoparticle microspheres;
in this step, the flow rate of the nitrogen gas is 10m/s to 20m/s; the concentration of the calcium chloride aqueous solution is 1 mg/mL-6 mg/mL; the volume ratio of the water phase to the calcium chloride aqueous solution is 1:10-20.
(5) Preparation of chitosan calcium alginate nanoparticle microsphere
And (3) cleaning the calcium alginate nanoparticle microspheres in the step (4) by using chitosan aqueous solution, centrifuging and freeze-drying to obtain the calcium alginate nanoparticle microspheres.
In this step, the concentration of the aqueous chitosan solution is 0.8mg/mL to 1.2mg/mL. The dosage ratio of the calcium alginate nanoparticle microspheres to the chitosan aqueous solution is 0.5 g-1 g:40 mL-80 mL. The rotational speed of the centrifugation is 4000 rpm-6000 rpm, and the time is 20 min-30 min.
The invention is described in detail below with reference to the drawings and the specific embodiments.
Example 1 preparation method of Chitosan calcium alginate nanoparticle microsphere
The preparation method of the chitosan calcium alginate nanoparticle microsphere comprises the following steps:
(1) Preparation of bovine serum albumin-carried methotrexate nanoparticle solution
5mg of sulfhydrylation bovine serum albumin is added into 2mL of 10mM PBS solution, and the mixture is added into a brown penicillin bottle after being uniformly mixed; taking 250 mu L of methotrexate DMSO solution (1 mg of methotrexate is dissolved in 500 mu L of DMSO solution), dropwise adding the solution into the bovine serum albumin solution, uniformly stirring the solution on a magnetic stirrer, and stopping the reaction after 30min to obtain the bovine serum albumin-carried methotrexate nanoparticle solution; placing in a refrigerator at 4 ℃ for standby;
(2) Preparation of gold-crosslinked bovine serum albumin-carried methotrexate nanoparticle
Dropwise adding 10 mu L of 20% chloroauric acid solution into 10mL of the prepared bovine serum albumin methotrexate-loaded nanoparticle solution prepared in the step (1), stirring for 6 hours at 500rpm, transferring the solution into a 100KD ultrafilter tube, centrifuging at 8000rpm for 30min, purifying to obtain gold cross-linked methotrexate bovine serum albumin nanoparticle, and placing the gold cross-linked methotrexate bovine serum albumin nanoparticle in a refrigerator at 4 ℃ for later use;
(3) Preparation of aqueous phase
Adding the gold crosslinked methotrexate bovine serum albumin nanoparticle prepared in the step (2) into sodium alginate solution (weighing 500mg of sodium alginate powder, adding 100mL of ultrapure water for dissolution to obtain sodium alginate aqueous solution) according to the volume ratio of 2:1, and premixing to obtain aqueous phase;
(4) Preparation of calcium alginate nanoparticle microsphere by air flow control technology
Dropwise adding 20mL of the water phase obtained in the step (3) into 200mL of calcium chloride aqueous solution (500 mg of anhydrous calcium chloride is weighed and dissolved in 100mL of ultrapure water to obtain calcium chloride aqueous solution) through a coaxial needle under nitrogen shearing at a flow rate of 20m/s to obtain calcium alginate nanoparticle microspheres;
(5) Preparation of chitosan calcium alginate nanoparticle microsphere
And (3) washing 1g of the calcium alginate nanoparticle microsphere obtained in the step (4) with 50mL of chitosan aqueous solution (100 mg of chitosan is weighed, dissolved in 100mL of PBS aqueous solution, and placed in a refrigerator at 4 ℃ for standby), centrifuging at 5000rpm for 20min, removing the supernatant, and freeze-drying by a freeze dryer to obtain the chitosan calcium alginate nanoparticle microsphere.
Example 2 preparation method of chitosan calcium alginate nanoparticle microsphere
The preparation method of the chitosan calcium alginate nanoparticle microsphere comprises the following steps:
(1) Preparation of bovine serum albumin-carried methotrexate nanoparticle solution
5mg of sulfhydrylation bovine serum albumin is added into 2mL of 10mM PBS solution, and the mixture is added into a brown penicillin bottle after being uniformly mixed; taking 250 mu L of methotrexate DMSO solution (1 mg of methotrexate is dissolved in 500 mu L of DMSO solution), dropwise adding the solution into the bovine serum albumin solution, uniformly stirring the solution on a magnetic stirrer, and stopping the reaction after 30min to obtain the bovine serum albumin-carried methotrexate nanoparticle solution; placing in a refrigerator at 4 ℃ for standby;
(2) Preparation of gold-crosslinked bovine serum albumin-carried methotrexate nanoparticle
Dropwise adding 10 mu L of 25% chloroauric acid solution into 9mL of the bovine serum albumin methotrexate-loaded nanoparticle solution prepared in the step (1), stirring for 6 hours at 500rpm, transferring the solution into a 100KD ultrafilter tube, centrifuging at 8000rpm for 30min, purifying to obtain gold cross-linked methotrexate bovine serum albumin nanoparticle, and placing the gold cross-linked methotrexate bovine serum albumin nanoparticle in a refrigerator at 4 ℃ for later use;
(3) Preparation of aqueous phase
Adding the gold crosslinked methotrexate bovine serum albumin nanoparticle prepared in the step (2) into sodium alginate solution (weighing 500mg of sodium alginate powder, adding 100mL of ultrapure water for dissolution to obtain sodium alginate aqueous solution) according to the volume ratio of 2:1, and premixing to obtain aqueous phase;
(4) Preparation of calcium alginate nanoparticle microsphere by air flow control technology
Dropwise adding 15mL of the water phase obtained in the step (3) into 200mL of calcium chloride aqueous solution (500 mg of anhydrous calcium chloride is weighed and dissolved in 100mL of ultrapure water to obtain calcium chloride aqueous solution) through a coaxial needle under nitrogen shearing at a flow rate of 20m/s to obtain calcium alginate nanoparticle microspheres;
(5) Preparation of chitosan calcium alginate nanoparticle microsphere
And (3) washing 0.8g of the calcium alginate nanoparticle microsphere obtained in the step (4) with 40mL of chitosan aqueous solution (100 mg of chitosan is weighed, dissolved in 100mL of PBS aqueous solution, and placed in a refrigerator at 4 ℃ for standby), centrifuging at 5000rpm for 20min, removing the supernatant, and freeze-drying by a freeze dryer to obtain the chitosan calcium alginate nanoparticle microsphere.
Example 3 preparation method of Chitosan calcium alginate nanoparticle microsphere
The preparation method of the chitosan calcium alginate nanoparticle microsphere comprises the following steps:
(1) Preparation of bovine serum albumin-carried methotrexate nanoparticle solution
5mg of sulfhydrylation bovine serum albumin is added into 2mL of 10mM PBS solution, and the mixture is added into a brown penicillin bottle after being uniformly mixed; taking 250 mu L of methotrexate DMSO solution (1 mg of methotrexate is dissolved in 500 mu L of DMSO solution), dropwise adding the solution into the bovine serum albumin solution, uniformly stirring the solution on a magnetic stirrer, and stopping the reaction after 30min to obtain the bovine serum albumin-carried methotrexate nanoparticle solution; placing in a refrigerator at 4 ℃ for standby;
(2) Preparation of gold-crosslinked bovine serum albumin-carried methotrexate nanoparticle
Dropwise adding 10 mu L of 15% chloroauric acid solution into 10mL of the bovine serum albumin methotrexate-loaded nanoparticle solution prepared in the step (1), stirring for 6 hours at 500rpm, transferring the solution into a 100KD ultrafilter tube, centrifuging at 8000rpm for 30min, purifying to obtain gold cross-linked methotrexate bovine serum albumin nanoparticle, and placing the gold cross-linked methotrexate bovine serum albumin nanoparticle in a refrigerator at 4 ℃ for later use;
(3) Preparation of aqueous phase
Adding the gold crosslinked methotrexate bovine serum albumin nanoparticle prepared in the step (2) into sodium alginate solution (weighing 500mg of sodium alginate powder, adding 100mL of ultrapure water for dissolution to obtain sodium alginate aqueous solution) according to the volume ratio of 2:1, and premixing to obtain aqueous phase;
(4) Preparation of calcium alginate nanoparticle microsphere by air flow control technology
Dropwise adding 10mL of the water phase obtained in the step (3) into 200mL of calcium chloride aqueous solution (500 mg of anhydrous calcium chloride is weighed and dissolved in 100mL of ultrapure water to obtain calcium chloride aqueous solution) through a coaxial needle under nitrogen shearing at a flow rate of 20m/s to obtain calcium alginate nanoparticle microspheres;
(5) Preparation of chitosan calcium alginate nanoparticle microsphere
And (3) washing 0.5g of chitosan calcium alginate nanoparticle microspheres obtained in the step (4) with 40mL of chitosan aqueous solution (weighing 100mg of chitosan, dissolving the chitosan with 100mLPBS aqueous solution, placing the chitosan aqueous solution in a refrigerator at the temperature of 4 ℃ for standby), centrifuging at 5000rpm for 20min, removing the supernatant, and freeze-drying by a freeze dryer to obtain the chitosan calcium alginate nanoparticle microspheres. Test example 1 the morphology and particle size distribution frequency of the chitosan calcium alginate nanoparticle microspheres in example 1 of the present invention
1. Morphology of
The chitosan calcium alginate nanoparticle microspheres in example 1 were resuspended in PBS, and then placed on a glass slide, and the morphology was observed using a fluorescence microscope, and the results are shown in fig. 1.
As can be seen from fig. 1, the chitosan calcium alginate nanoparticle microsphere of example 1 has a spherical shape with uniform particle size and good dispersibility.
2. Frequency of particle size distribution
The chitosan calcium alginate nanoparticle microspheres in example 1 were subjected to particle size observation under a microscope, and the approximate particle size was recorded, and the recorded data were counted.
The particle size distribution frequency of the chitosan calcium alginate nanoparticle microspheres in example 1 is shown in fig. 2, and total 84 microsphere particle sizes are counted, and the average particle size is about 226+ -13 μm.
Test example 2 detection of the average particle size of different nanoparticles
The test example uses a laser particle size analyzer to detect the average particle size of different nanoparticles. Wherein the nanoparticle comprises:
1. bovine serum albumin-supported methotrexate (bovine serum albumin-supported methotrexate nanoparticle of example 1 step 1)
2. Gold cross-linked bovine serum albumin-loaded methotrexate (bovine serum albumin-loaded methotrexate nanoparticle of example 1 step 2)
3. Gold cross-linked bovine serum albumin (preparation method comprises adding 5mg of sulfhydrylation bovine serum albumin into 2mL 10mM PBS solution, mixing, adding into brown penicillin bottle, dropwise adding 1mL 20% chloroauric acid solution into 900mL bovine serum albumin solution, stirring at 500rpm for 6 hr, transferring into 100KD ultrafilter tube, centrifuging at 8000rpm for 30min to obtain gold cross-linked bovine serum albumin nanoparticle)
The average particle size of the different nanoparticles is shown in FIG. 3.
As can be seen from fig. 3, the average particle diameters of the gold-crosslinked bovine serum albumin nanoparticle, the bovine serum albumin-carried methotrexate and the gold-crosslinked bovine serum albumin-carried methotrexate nanoparticle are 205, 225 and 270 respectively, and no statistical difference exists, which indicates that the prepared nanoparticles all accord with the nano administration size, and the addition of gold ions does not damage the nano size.
Test example 3 potential analysis of different drug-loaded nanoparticles and nanoparticle microspheres
The experimental example uses a malvern particle sizer to analyze the potential of different drug-loaded nanoparticles and nanoparticle microspheres. Wherein the different drug-loaded nanoparticles and nanoparticle microspheres comprise:
1. gold cross-linked bovine serum albumin-loaded methotrexate (bovine serum albumin-loaded methotrexate nanoparticle of example 1 step 2)
2. Calcium alginate microsphere
The preparation method comprises the following steps: the aqueous phase (20 mL of aqueous sodium alginate solution) was dropped into 200mL of aqueous calcium chloride solution (500 mg of anhydrous calcium chloride was weighed and dissolved in 100mL of ultrapure water to obtain an aqueous calcium chloride solution) through a coaxial needle under nitrogen shear at a flow rate of 20m/s, to obtain calcium alginate microspheres.
3. Calcium alginate gold Niu Jiana granule microsphere (namely calcium alginate gold cross-linked bovine serum albumin methotrexate nanoparticle microsphere, step 4 in example 1)
4. Chitosan calcium alginate nanoparticle-carried gold Niu Jiana nanoparticle microsphere (namely chitosan calcium alginate-carried gold cross-linked bovine serum albumin-carried methotrexate nanoparticle microsphere, the final product prepared in example 1)
The results of the potential analysis of the different drug-loaded nanoparticles and nanoparticle microspheres are shown in figure 4.
As can be seen from FIG. 4, the sodium alginate microsphere has stronger negative potential, the potential is neutralized after the nanoparticle is encapsulated, and the positive charge is enhanced by the addition of the external chitosan, which shows that the chitosan calcium alginate nanoparticle microsphere of the invention is in stomach H + May be more stable under rich conditions.
Test example 4 7 day particle size variation of different nanoparticles
This test example compares the particle size change over 7 days of a bovine serum albumin-loaded methotrexate nanoparticle (i.e., the bovine serum albumin-loaded methotrexate nanoparticle of example 1, step 1) and a gold-crosslinked bovine serum albumin-loaded methotrexate nanoparticle (i.e., the gold-crosslinked bovine serum albumin-loaded methotrexate nanoparticle of example 1, step 2).
As shown in fig. 5, the particle size of the bovine serum albumin-carried methotrexate nanoparticle gradually increased within one week, while the particle size of the gold-crosslinked bovine serum albumin-carried methotrexate nanoparticle remained substantially stable within one week, indicating that the addition of chloroauric acid solution helped the particle size of the nanoparticle to be stable.
Test example 5 encapsulation efficiency of different drug-loaded nanoparticles and nanoparticle microspheres
The test example detects the encapsulation rate of different drug-loaded nanoparticles and nanoparticle microspheres.
Preparation of 0mg/mL, 000005mg/mL, 0.0005mg/mL, 0.0025mg/mL, 0.025mg/mL, 0.05mg/mL, 0.00125mg/mL of methotrexate solution, 1mL of which absorbance values are detected at 303nm respectively by using an ultraviolet-visible spectrophotometer, and then a straight line graph is drawn with the concentration as an abscissa and the absorbance as an ordinate, and a standard curve is shown in FIG. 6, so as to obtain a standard curve equation y= 42.681x-0.006; r is R 2 ＝0.9998。
The absorption values of methotrexate in the different drug-loaded nanoparticles and nanoparticle microspheres were detected by the same method as above, and the corresponding methotrexate concentrations were calculated according to the standard curve equation, and the encapsulation efficiency was calculated, and the results are shown in fig. 7.
Wherein, drug-loaded nanoparticles and nanoparticle microspheres comprise:
1. bovine serum albumin-loaded methotrexate (i.e., bovine serum albumin-loaded methotrexate nanoparticle in example 1, step 1)
2. Gold cross-linked bovine serum albumin-loaded methotrexate (i.e., gold cross-linked bovine serum albumin-loaded methotrexate nanoparticle in example 1, step 2)
3. Chitosan calcium alginate nanoparticle-supported bovine methotrexate nanoparticle (i.e., chitosan calcium alginate microsphere-supported bovine serum albumin-supported methotrexate nanoparticle, example 1 omitted the product of step 2)
4. Gold Niu Jia nanoparticle carried by chitosan calcium alginate nanoparticle (namely, gold-carried crosslinked bovine serum albumin-carried methotrexate nanoparticle carried by chitosan calcium alginate nanoparticle, example 1 finally prepared product)
As shown in fig. 7, the encapsulation efficiency of the bovine serum albumin-supported methotrexate nanoparticle (No. 1) was about 80%, while the encapsulation efficiency of the gold-crosslinked bovine serum albumin-supported methotrexate nanoparticle (No. 2) was about 90%, and the encapsulation efficiency was slightly increased.
Compared with the chitosan calcium alginate nanoparticle-carried bovine methyl nanoparticle (sequence number 3), the chitosan calcium alginate nanoparticle-carried gold Niu Jia nanoparticle (sequence number 4) has obviously improved encapsulation efficiency, which means that the addition of gold ions improves the stability of the nanoparticle-carried albumin nanoparticle, so that the albumin-carried nanoparticle is not easy to depolymerize in the microsphere preparation process.
Test example 6 therapeutic Effect of the chitosan calcium alginate nanoparticle microspheres of the present invention on rheumatoid arthritis
The test example verifies the treatment effect of the chitosan calcium alginate nanoparticle microsphere in the invention on rheumatoid arthritis.
The experimental method comprises the following steps:
1. 200ul Freund's complete adjuvant was injected into the right plantar of SD rats and 300ug was administered daily by gavage, starting on day 7, in the following groups.
2. Grouping
(1) Normal control group
(2) Non-therapeutic group for moulding
(3) Methotrexate group
(4) Bovine serum albumin-loaded methotrexate group (product of step 1, example 1)
(5) Gold cross-linked bovine serum albumin-loaded methotrexate group (product of step 2 of example 1)
(6) Chitosan calcium alginate microsphere supported ox-nail nanoparticle group (example 1 omitting the 2 step product)
(7) Gold Niu Jia nanoparticle group of chitosan calcium alginate microsphere (final product of example 1)
3. After 30 days of treatment, the thickness of the rat footpad was measured and the weight of the rat was weighed.
The results are shown in fig. 8 and 9.
The results show that: compared with other groups except normal mice, the thickness of the foot pad of the chitosan calcium alginate microsphere gold-loaded Niu Jia nanoparticle group (namely the 7 th group, the final product chitosan calcium alginate drug-loaded microsphere of the example 1) is obviously reduced, and the treatment effect is better. The chitosan calcium alginate microsphere-loaded gold Niu Jia nanoparticle group (i.e., group 7) also showed a significant reduction in the rat footpad thickness compared to the chitosan calcium alginate microsphere-loaded bovine nail nanoparticle group (i.e., group 6) (fig. 8).
Compared with other groups except normal mice, the weight change of the chitosan calcium alginate microsphere gold-loaded Niu Jia nanoparticle group (namely the 7 th group, the final product chitosan calcium alginate drug-loaded microsphere of the example 1) is closer to the weight change of the normal mice, so that diarrhea gastrointestinal side effects of the mice are presumed to be lower, and toxic side effects are relieved on the premise of ensuring the treatment effect. The chitosan calcium alginate microsphere-loaded gold Niu Jia nanoparticle group (i.e., group 7) also showed less weight change in rats compared to the chitosan calcium alginate microsphere-loaded bovine nail nanoparticle group (i.e., group 6) (fig. 9).
The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.
The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.
Claims (10)
1. The preparation method of the chitosan calcium alginate nanoparticle microsphere is characterized by comprising the following steps of:
(1) Dripping the methotrexate solution into the sulfhydrylation bovine serum albumin solution, and uniformly stirring to obtain the bovine serum albumin-carried methotrexate nanoparticle solution;
(2) Dropwise adding a chloroauric acid solution with the concentration of 10-40 wt% into the bovine serum albumin methotrexate-loaded nanoparticle solution in the step (1), and stirring and centrifuging to obtain gold crosslinked methotrexate bovine serum albumin nanoparticles; the volume ratio of the chloroauric acid solution to the bovine serum albumin methotrexate-loaded nanoparticle solution is 1:900-1000;
(3) According to the volume ratio of 2: 0.8-1.2, adding the gold cross-linked methotrexate bovine serum albumin nanoparticle prepared in the step (2) into sodium alginate solution to obtain a water phase;
(4) Dropwise adding the water phase obtained in the step (3) into a calcium chloride water solution through a coaxial needle under nitrogen shearing to obtain calcium alginate nanoparticle microspheres;
(5) And (3) cleaning the calcium alginate nanoparticle microspheres obtained in the step (4) by using chitosan aqueous solution, centrifuging and freeze-drying to obtain the calcium alginate nanoparticle microsphere.
2. The method for preparing chitosan calcium alginate nanoparticle microspheres according to claim 1, wherein the mass percentage concentration of the chloroauric acid solution in the step (2) is 15wt% to 30wt%.
3. The method for preparing chitosan calcium alginate nanoparticle microspheres according to claim 2, wherein the mass percentage concentration of the chloroauric acid solution is 18-25 wt%.
4. The method for preparing chitosan calcium alginate nanoparticle microspheres according to claim 1, wherein the volume ratio of chloroauric acid solution to bovine serum albumin methotrexate-loaded nanoparticle solution is 1:945-955.
5. The method for preparing chitosan calcium alginate nanoparticle microspheres according to any one of claims 1 to 4, wherein the stirring temperature in step (2) is 40 to 50 ℃, the speed is 500 to 1000rpm, and the time is 4 to 8 hours; and/or the rotational speed of the centrifugation is 7500 rpm-8500 rpm for 20 min-30 min.
6. The method for preparing chitosan calcium alginate nanoparticle microspheres according to any one of claims 1 to 4, wherein the volume ratio of the methotrexate solution to the thiolated bovine serum albumin solution in step (1) is 1: 8-10.
7. The method for preparing chitosan calcium alginate nanoparticle microspheres according to any one of claims 1 to 4, wherein the methotrexate solution in step (1) is a methotrexate DMSO solution, and the concentration of the methotrexate DMSO solution is 1.5mg/mL to 2.5mg/mL; and/or the concentration of the sulfhydrylation bovine serum albumin solution is 2.0 mg/mL-3.0 mg/mL.
8. The method for preparing chitosan calcium alginate nanoparticle microspheres according to any one of claims 1 to 4, wherein the flow rate of the nitrogen gas in the step (4) is 10m/s to 20m/s; and/or the concentration of the calcium chloride aqueous solution is 1 mg/mL-6 mg/mL; and/or the volume ratio of the water phase to the calcium chloride aqueous solution in the step (4) is 1:10-20.
9. The method for preparing chitosan calcium alginate nanoparticle microspheres according to any one of claims 1 to 4, wherein the concentration of the sodium alginate solution in the step (3) is 4mg/mL to 6mg/mL; and/or the concentration of the chitosan aqueous solution in the step (5) is 0.8 mg/mL-1.2 mg/mL; and/or the dosage ratio of the calcium alginate nanoparticle microsphere to the chitosan aqueous solution is 0.5 g-1 g:40 mL-80 mL; and/or the rotational speed of the centrifugation is 4000 rpm-6000 rpm for 20 min-30 min.
10. The chitosan calcium alginate nanoparticle microsphere prepared by the preparation method of any one of claims 1 to 9.
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