CN113753933B - Fucoidan/calcium carbonate hybrid nanorod and preparation method thereof - Google Patents

Abstract

The invention relates to the field of biological materials, and discloses a Fucoidan/calcium carbonate hybrid nanorod and a preparation method and application thereof. The Fucoidan/calcium carbonate hybrid nanorod is synthesized by a precipitation method, sodium carbonate and calcium chloride are used as raw materials, and Fucoidan is used as an organic regulator; the particle size of the synthesized calcium carbonate hybrid nanorod is regulated and controlled by controlling the concentration of Fucoidan. The precipitation method has simple process and convenient operation, does not need to use complex instruments and equipment, and the synthesized Fucoidan/calcium carbonate hybridized nano rod has good biocompatibility and pH response drug release characteristic. The electrostatic action and diffusion action of the medicine are loaded on the nano rod, so that the release rate of the medicine is slowed down, and the stability and the long-acting performance are improved.

Description

Fucoidan/calcium carbonate hybrid nanorod and preparation method thereof

Technical Field

The invention relates to the field of biological materials, in particular to a fucan/calcium carbonate hybrid nanorod and a preparation method thereof.

Background

Calcium carbonate (CaCO) ₃ ) Has wide and important application in various fields of coating, rubber, plastic, agriculture, medicine, pharmacy and the like. Has the inherent advantages of degradability under a certain pH value, good biocompatibility, simple chemical components, low cost, easy mass production and the like, and has wide biomedical application prospect. Several synthetic CaCO's have been developed in recent years ₃ The nanoparticle preparation method comprises a flame method, a sonochemistry method, a lime solution carbonization method in a reverse micelle system, a bionic method, a polyacrylic acid stabilization method and a micropore dispersion method. The technology based on precipitation is simple, high in yield and strong in applicability, so that the technology is the most widely applied technology. There are mainly two precipitation strategies reported in the literature. The first strategy is to form CaCO from an aqueous calcium hydroxide solution and carbon dioxide bubbles ₃ . An alternative strategy is to use a chemical combination of sodium carbonate and calcium chloride or calcium nitrate to synthesize CaCO ₃ 。

Fucoidan (Fucoidan) is a natural polysaccharide that is an interesting candidate for biomedical applications due to its good biocompatibility, ease of encapsulation of bioactive molecules, low immunogenicity, etc. In addition, it has the ability to induce apoptosis in tumor cells. Fucoidan's anticancer effect varies depending on its structure, but it can also target multiple receptors or signaling molecules of multiple cell types, such as p-selectins, including tumor cells and immune cells, demonstrating its ability to prevent or treat cancer in vivo. In recent years, there has been an increasing interest in the clinical transformation of fucose lipids. Some groups performed observational studies on fucosides for clinical trials. The method for preparing the calcium carbonate nanorods by using Fucoidan as the organic regulation and control template under the room temperature condition has the advantages of simple experimental method, mild and controllable condition, good repeatability and low production cost.

Disclosure of Invention

Aiming at the problems of strong reaction, complex operation, low biological activity of calcium carbonate and the like in the current synthesis of the calcium carbonate nanorods, the invention aims to provide a Fucoidan/calcium carbonate hybrid nanorod.

The technical scheme adopted by the invention is as follows:

a fucoidin/calcium carbonate hybrid nanorod formed by a precipitation method.

The Fucoidan/calcium carbonate hybrid nanorod has a long diameter of 500-1200 nm and a short diameter of 100-500 nm; the Fucoidan/calcium carbonate hybrid nanorods are nanorods formed by using Fucoidan as an organic matter.

Fucoidan/calcium carbonate hybrid nanorod and preparation method and application thereof, comprising the following steps:

respectively dissolving sodium carbonate and calcium chloride in ultrapure water, adding Fucoidan into a calcium chloride solution as a regulating agent, stirring for 0.5-1 h, then dropwise adding the sodium carbonate solution into the stirred calcium chloride solution, generating a Fucoidan/calcium carbonate hybrid nano rod by a precipitation method, stirring for 6-8 h, performing centrifugal separation, washing for 2-3 times, and removing impurities to obtain the product Fucoidan/calcium carbonate hybrid nano rod.

Through the technical scheme, the beneficial technical effects of the invention include:

the Fucoidan/calcium carbonate hybrid nanorod prepared by the method has controllable length-diameter ratio and good biocompatibility.

Drawings

FIG. 1 scanning electron microscope image of Fucoidan/calcium carbonate nanorods at a magnification of 10k

FIG. 2 scanning electron microscope image of Fucoidan/calcium carbonate nanorods at 30K magnification

FIG. 3 Fucoidan/calcium carbonate nanorod-supported MTO in vitro Release profile in phosphate buffer pH7.4 and 5.5

FIG. 4 haemolysis Rate of Fucoidan/calcium carbonate nanorods at different concentrations

FIG. 5L 929 cell viability after 24h co-culture with Fucoidan/calcium carbonate nanorods

FIG. 6L 929 cell viability after 48h co-culture with Fucoidan/calcium carbonate nanorods

Detailed Description

The following description of the embodiments of the present invention will be made more apparent and fully by reference to the accompanying drawings, in which some, but not all embodiments of the invention are shown.

Example 1: preparation of Fucoidan/calcium carbonate hybrid nanorods

2g of Fucoidan was weighed, placed in a 50mL centrifuge tube, 40mL of ultrapure water was weighed by a cartridge, poured into the centrifuge tube, and after ultrasonic dissolution, filtered through a 0.22 μm filter membrane to obtain 50mg/mL of Fucoidan solution. Then CaCl of 0.02mol/L was prepared with ultrapure water ₂ Solution and Na ₂ CO ₃ A solution. Respectively sucking 10, 8, 6, 4, 2, 1, 0.6, 0.4, 0.2, and 0mL of 50mg/mL Fucoidan solution, adding into 8 solutions containing CaCl ₂ Adding 0, 2, 4, 6, 8, 9, 9.4, 9.6, 9.8 and 10mL of ultrapure water into the beaker of the solution, and stirring for 0.5 to 1 hour. Taking 10mL of Na ₂ CO ₃ The solution was added to CaCl, which had been stirred for half an hour ₂ And Fucoidan, wherein the stirring speed is adjusted to be more than 1400rpm, and the mouth of the beaker is sealed by a preservative film. After stirring for 24 hours, centrifugingWashing for 2-3 times after 5 min.

Example 2: fucoidan/calcium carbonate hybrid nanorod structure

From the scanning electron microscope images (FIGS. 1 and 2) of the product of example 1, it can be seen that the Fucoidan/calcium carbonate hybrid nanorods have a uniform rod-like structure, and the dimension distribution of the nanorods is statistically determined to have a long diameter of about 570nm and a short diameter of about 120nm.

Example 3: drug loading and release

The experimental drug model was mitoxantrone (Mitoxantrone hydrochloride, MTO), the following experimental steps: weighing 2mg of calcium carbonate nanorods, placing the nanorods in a 25mL beaker, sucking 5mL of PBS (phosphate buffer solution) by using a pipetting gun, adding the PBS into the beaker, firstly performing ultrasonic dispersion by using an ultrasonic machine, then placing the mixture on a stirrer for stirring, and adjusting the rotating speed to 800rpm; weighing 4mg of MTO, adding 20mL of PBS, dissolving by ultrasonic, then adding into a calcium carbonate solution, wrapping a beaker by using tin foil paper, keeping the beaker away from light, and magnetically stirring for one night; centrifuging and washing with PBS for 2-3 times to obtain the product. The absorbance of the supernatant was measured, and the concentration was calculated from the standard curve, while the drug loading and the encapsulation efficiency were calculated to be 43.7% and 34.5%, respectively. Drug release studies were performed in phosphate buffers at pH7.4 and 5.5 at 37 ℃.1mL of supernatant is taken out at the wavelength of 660nm to measure absorbance at 1h, 2h, 4h, 6h, 9h, 12h, 24h, 36h, 2d, 3d and 5d, and a new phosphate buffer solution with corresponding pH is added to measure that the release rate of MTO is obviously influenced by the pH. The results show that: the calcium carbonate nanorods have pH responsiveness to drug release.

Example 4: biocompatibility study of Fucoidan/calcium carbonate hybrid nanorods

(1) Hemolysis test

1mL of the sample was withdrawn from the post-aural vein of the rabbit using a blood-taking needle and placed in a heparin anticoagulation tube. 0.8mL of fresh anticoagulated rabbit blood is added into 1mL of normal saline to prepare mother liquor. Calcium carbonate nanorod suspensions of different concentrations were prepared with physiological saline at concentrations of 10, 20, 50, 100, 200 μg/mL. The negative control group is normal saline, and the positive control group is ultrapure water. The five groups of solutions were taken 5mL in a 10mL centrifuge tube and water-bath at 37℃for 30min. Then 0.1mL of anticoagulated rabbit blood mother liquor is added into each group respectively, and the mixture is placed in a water bath at 37 ℃ for 60min again after uniform mixing. The sample was taken out and centrifuged at 2500rpm for 5min, and the supernatant was measured for absorbance at the maximum absorption peak wavelength and the data was recorded. The results show that: the concentration range is between 0 and 200 mug/mL, and the hemolysis rate of Fucoidan/calcium carbonate nano rod is lower than 5 percent, and the product is qualified.

(2) Cytotoxicity of cells

L929 cells in logarithmic growth phase were inoculated into 96-well cell culture plates at a density of 6X 10 without wells of 100. Mu.L ⁴ At a concentration of one ml, placed in CO ₂ Culturing in an incubator for 12h. Fucoidan/calcium carbonate nanorod solutions with different concentrations of 10, 20, 50, 100 and 200 mug/mL are added. And a blank group and a negative control group were set, each group being provided with 6 parallel duplicate wells. After incubation for 24h and 48h, old media was discarded from the plates and rinsed 2 times with fresh PBS. 10. Mu.L of MTT reagent and 100. Mu.L of fresh medium were added to each well, and the wells were incubated in an incubator for 3 hours. The MTT reagent and the medium were then removed from the 96-well plate, and 150. Mu.L of DMSO solution was added thereto, followed by shaking to promote crystallization and dissolution. The absorbance was measured at 570nm using an enzyme-labeled instrument and the cell viability was calculated according to the formula. The results show that: cell viability was over 90% and cytotoxicity of the material was assessed according to national standard GB/T16886, and Fucoidan/calcium carbonate nanorods were rated as 1 for toxicity, and were considered acceptable.

The above is only a preferred embodiment of the present invention and not intended to limit the scope thereof, and any modifications which are not creatively made by those skilled in the art under the principle of the present invention should be considered as being within the scope of the present invention.

Claims (2)

1. A Fucoidan/calcium carbonate hybrid nanorod, wherein the hybrid nanorod is a nanorod formed by a precipitation method, and the Fucoidan is fucan;

wherein the length of the Fucoidan/calcium carbonate hybridized nano rod is 500-1200 nm, and the length of the Fucoidan/calcium carbonate hybridized nano rod is 100-500 nm, and the Fucoidan/calcium carbonate hybridized nano rod is a nano rod formed by taking Fucoidan as an organic matter.

2. Preparing a fucoidin/calcium carbonate hybrid nanorod according to claim 1, comprising the steps of:

respectively dissolving sodium carbonate and calcium chloride in ultrapure water, adding Fucoidan into a calcium chloride solution as a regulating agent, stirring for 0.5-1 h, then dropwise adding the sodium carbonate solution into the stirred calcium chloride solution, generating a Fucoidan/calcium carbonate hybrid nano rod by a precipitation method, stirring for 6-8 h, performing centrifugal separation, washing for 2-3 times, and removing impurities to obtain the product Fucoidan/calcium carbonate hybrid nano rod.