A method for preparing a polymer fluorescent nanoparticle
By using waste polylactic acid to prepare polymer fluorescent nanoparticles, the problems of high cost and insufficient biocompatibility in existing technologies have been solved, and the biocompatibility and fluorescence performance have been improved, which has broad prospects for biomedical applications.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- HEFEI UNIV OF TECH
- Filing Date
- 2024-01-08
- Publication Date
- 2026-07-24
Smart Images
Figure CN117820679B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of nanomaterials technology, specifically relating to a method for preparing polymer fluorescent nanoparticles. Background Technology
[0002] With the rapid development of life science technologies, research in life sciences has penetrated to the level of cells and single molecules in organisms. Labeling, staining, and detection of cells or biomolecules have become important aspects of research in the biomedical field. Among biolabels, fluorescent labeling has attracted the most attention, but its detection sensitivity mainly depends on the fluorescence intensity and stability of the label. Traditional fluorescent labels, including organic fluorescent dyes and fluorescent proteins, have wide applications in cell imaging and biosensing. However, organic dyes and fluorescent proteins exhibit optical instability, relatively weak fluorescence signals, and short fluorescence lifetimes. These drawbacks limit their application in biomolecular detection. In contrast, fluorescent nanoparticles possess long-term photostability and a long fluorescence lifetime, making them perfect candidates for fluorescent labeling in molecular detection.
[0003] Currently, the application of fluorescent nanoparticles in the biomedical field is gaining increasing attention. However, existing preparation methods suffer from a series of problems, including high production costs and limited biocompatibility, which restricts the feasibility of large-scale applications and their widespread use in vivo. Given these challenges, there is an urgent need for a simple, economical preparation method that simultaneously possesses excellent biocompatibility and fluorescence performance. This would help promote the widespread application of fluorescent nanoparticles, particularly in areas such as cell imaging, drug delivery, and biosensing. Therefore, this invention aims to address the shortcomings of current methods by providing an innovative and feasible method for preparing fluorescent nanoparticles to meet the growing demands of biomedical applications. Summary of the Invention
[0004] To address the shortcomings of existing technologies, this invention provides a method for preparing polymer fluorescent nanoparticles to meet the needs of fluorescent labeling in cell imaging and biological detection, thereby satisfying the growing demands of biomedical applications.
[0005] The method for preparing polymer fluorescent nanoparticles of the present invention includes the following steps:
[0006] Step 1: Synthesis of polylactic acid-polyurethane-polyacrylic acid copolymer
[0007] Dihydroxyl-terminated polylactic acid (PLA) was obtained by alcoholysis of waste PLA with ethylene glycol. The dihydroxyl-terminated PLA was then reacted with 4,4'-diphenylmethane diisocyanate and polycaprolactone polyol (2000 g / mol) to obtain isocyanate-terminated PLA-polyurethane. Hydroxyethyl acrylate was then added to the system and reacted with the isocyanate-terminated PLA-polyurethane to introduce terminal double bonds. Acrylic acid monomers were then added, and polymerization yielded a PLA-polyurethane-polyacrylic acid copolymer. Finally, the PLA-polyurethane-polyacrylic acid copolymer was dissolved in butyl acetate solvent for later use.
[0008] Step 2: Prepare an aqueous solution of polyethyleneimine for later use;
[0009] Step 3: Add the polyethyleneimine aqueous solution prepared in Step 2 to the butyl acetate solvent of the polylactic acid-polyurethane-polyacrylic acid copolymer obtained in Step 1 to form a water-in-oil emulsion.
[0010] Step 4: Evaporate the water in the polylactic acid-polyurethane-polyacrylic acid emulsion obtained in Step 3 by heating with stirring to form a fluorescent solution;
[0011] Step 5: Spray dry the fluorescent solution obtained in Step 4 to obtain polymer fluorescent nanoparticles.
[0012] Furthermore:
[0013] In step 1, 5g of waste polylactic acid (PLA) granules were added to 30mL of ethylene glycol and stirred for 12h to obtain a dihydroxyl-terminated PLA product. Then, using butyl acetate as a solvent, the dihydroxyl PLA was reacted with 4,4'-diphenylmethane diisocyanate and polycaprolactone polyol (2000g / mol) in a molar ratio of 1:2.4:1 for 12h to obtain an isocyanate-terminated PLA-polyurethane product. Next, 2.5 times the molar amount of isocyanate residues of hydroxyethyl acrylate were added, and the reaction continued for 12h. Then, 7.2g of acrylic acid monomer was added, and the reaction continued for another 12h to obtain a PLA-polyurethane-polyacrylic acid copolymer solution. Finally, the product was diluted with butyl acetate to a concentration of 25-100mg / mL. In the above process, the ratio of acrylic acid monomer added has little effect; acrylic acid acts as a seed to form the copolymer.
[0014] In step 2, 2g of polyethyleneimine is added to distilled water and stirred for 30 minutes to form an aqueous solution of polyethyleneimine with a concentration of 15-25mg / mL.
[0015] In step 3, 10g of the polyethyleneimine aqueous solution prepared in step 2 is added dropwise to the butyl acetate solution of polylactic acid-polyurethane-polyacrylic acid copolymer at a certain rate, and stirred at high speed for 2 hours to form a water-in-oil emulsion.
[0016] In step 3, the dropping rate is controlled at 1–10 mL / min; the stirring speed is controlled at 500–2000 rpm.
[0017] In step 3, the mass ratio of the polyethyleneimine aqueous solution to the polylactic acid-polyurethane-polyacrylic acid copolymer butyl acetate solution is 1:5 to 1:10.
[0018] In step 5, the fluorescent solution is spray-dried using nitrogen as the drying carrier. The drying temperature is 100–200℃, and the feed rate is 5–20 mL / min.
[0019] The polymer fluorescent nanoparticles prepared by this invention can emit bright fluorescence under 365nm ultraviolet light irradiation.
[0020] Furthermore, the size and fluorescence intensity of nanoparticles can be adjusted by regulating the mass ratio of polyethyleneimine aqueous solution to polylactic acid-polyurethane-polyacrylic acid copolymer butyl acetate emulsion, the dropping rate, and the rotation speed under high-speed stirring conditions.
[0021] The present invention has the following beneficial effects:
[0022] 1. This invention uses environmentally friendly waste polylactic acid for alcoholysis, realizing resource reuse and reducing production costs.
[0023] 2. The polymer fluorescent nanoparticles of this invention have excellent biocompatibility and no biotoxicity.
[0024] 3. This invention introduces a design that uses hydroxyethyl acrylate and acrylic monomers to form a polylactic acid-polyurethane-polyacrylic acid copolymer. The nanoparticles have a negative charge on their surface and possess potential tumor marker functions. Attached Figure Description
[0025] Figure 1 The figure shows the fluorescence emission spectrum of the fluorescent nanoparticles prepared in Example 1 of this invention. As can be seen from the figure, the fluorescent nanoparticles have excellent luminescence properties, with the fluorescence emission peak at the maximum excitation wavelength located at 460 nm.
[0026] Figure 2 The figure shows the particle size distribution of the fluorescent nanoparticles prepared in Example 1 of this invention. As can be seen from the figure, the particle size distribution of the nanoparticles is at about 40 nm, and the nanoparticle size is uniform.
[0027] Figure 3 The fluorescent nanoparticles prepared in this invention exhibit excellent biocompatibility in their cytotoxicity evaluation.
[0028] Figure 4The relationship between the size and fluorescence intensity of the fluorescent nanoparticles prepared in this invention is determined by adjusting the mass ratio of polyethyleneimine aqueous solution to butyl acetate emulsion of polylactic acid-polyurethane-polyacrylic acid copolymer.
[0029] Figure 5 The fluorescent nanoparticles prepared in this invention are related to the drop rate of the polyethyleneimine aqueous solution and the butyl acetate emulsion of polylactic acid-polyurethane-polyacrylic acid copolymer, as well as the changes in the size and fluorescence intensity of the fluorescent nanoparticles.
[0030] Figure 6 The relationship between the size and fluorescence intensity of the fluorescent nanoparticles prepared in this invention was investigated by adjusting the stirring speed of the polyethyleneimine aqueous solution and the butyl acetate emulsion of polylactic acid-polyurethane-polyacrylic acid copolymer under high-speed stirring.
[0031] Figure 7 The relationship between the size and fluorescence intensity of the fluorescent nanoparticles prepared in this invention was investigated by adjusting the dropping rate of an equal mass of polyethyleneimine aqueous solution and a polylactic acid-polyurethane-polyacrylic acid copolymer butyl acetate emulsion, as well as the rotation speed under high-speed stirring conditions. Detailed Implementation
[0032] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention are described clearly and completely below. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without inventive step are within the scope of protection of this invention.
[0033] Example 1:
[0034] The preparation method of fluorescent nanoparticles in this embodiment includes the following specific steps:
[0035] Step 1: Synthesis of polylactic acid-polyurethane-polyacrylic acid copolymer
[0036] First, 5g of waste polylactic acid (PLA) granules were accurately weighed and added to 30mL of ethylene glycol. The mixture was stirred in a magnetically stirred water bath at 80℃ for 12 hours to induce an alcoholysis reaction, yielding a dihydroxyl-terminated PLA product. The molecular weight was determined by GPC to be 3500g / mol. Next, the dihydroxyl-terminated PLA was added to a three-necked flask with 4,4'-diphenylmethane diisocyanate and polycaprolactone polyol (2000g / mol) in a ratio of 1:2.4:1. Using butyl acetate as the solvent, the mixture was reacted at 60℃ under a nitrogen atmosphere for 12 hours to obtain an isocyanate-terminated PLA-polyurethane product. The number of residual isocyanate end groups was determined by titration, and the reaction was stopped when the number of isocyanate residues fell below 3%. Next, hydroxyethyl acrylate (2.5 times the molar amount of isocyanate residues) was added to a three-necked flask, and the mixture was reacted at 70°C for 12 hours. Then, 7.2 g of acrylic acid monomer was added, and the reaction was continued for another 12 hours to obtain a polylactic acid-polyurethane-polyacrylic acid copolymer solution. The product was further diluted with butyl acetate to a solid content of 10%.
[0037] Step 2: Add 2g of polyethyleneimine to 100mL of distilled water and stir at room temperature for 30min under magnetic stirring to dissolve it in the water to form an aqueous polyethyleneimine solution, thus forming an aqueous phase.
[0038] Step 3: Weigh 5g of polyethyleneimine aqueous solution and gradually add it dropwise to 25g of polylactic acid-polyurethane-polyacrylic acid copolymer butyl acetate solution at a rate of 1mL / min, while stirring at 25℃ for 2h at a high speed of 2000rpm to form a water-in-oil emulsion.
[0039] Step 4: Heat the prepared water-in-oil emulsion to 70°C while stirring to evaporate the water, ensuring that the resulting fluorescent solution is more uniform.
[0040] Step 5: The fluorescent solution was spray-dried using nitrogen as the drying carrier. The drying temperature was 120℃, the feed rate was 5 mL / min, and the nitrogen flow rate was 2 L / min, ultimately yielding the prepared fluorescent nanoparticles.
[0041] When the fluorescent nanoparticles are placed under 365nm ultraviolet light, they turn blue and exhibit a certain degree of brightness.
[0042] Example 2:
[0043] The mass ratio of the polyethyleneimine aqueous solution and the polylactic acid-polyurethane-polyacrylic acid copolymer butyl acetate solution in Example 1 was adjusted to 1:6, while other components and operations remained unchanged. At this point, under 365nm ultraviolet light irradiation, the nanoparticles appeared blue, the nanoparticle size increased, and the fluorescence intensity decreased compared to Example 1.
[0044] Example 3:
[0045] The mass ratio of the polyethyleneimine aqueous solution and the polylactic acid-polyurethane-polyacrylic acid copolymer butyl acetate solution in Example 1 was adjusted to 1:8, while other components and operations remained unchanged. At this point, under 365nm ultraviolet light irradiation, the particles appeared blue, the nanoparticle size further increased, and the fluorescence intensity decreased compared to Example 1.
[0046] Example 4:
[0047] The mass ratio of the polyethyleneimine aqueous solution and the polylactic acid-polyurethane-polyacrylic acid copolymer butyl acetate solution in Example 1 was adjusted to 1:10, while other components and operations remained unchanged. At this point, under 365nm ultraviolet light irradiation, it appeared blue, and the fluorescence intensity decreased compared to that of Example 1.
[0048] Example 5:
[0049] The dropping rate of the equal mass of polyethyleneimine aqueous solution and polylactic acid-polyurethane-polyacrylic acid copolymer butyl acetate solution in Example 1 was adjusted to 4 mL / min, while other components and operations remained unchanged. The resulting fluorescent nanoparticles were larger in size and had poorer distribution uniformity than those in Example 1. Under 365 nm ultraviolet light irradiation, their fluorescence intensity was reduced.
[0050] Example 6:
[0051] The dropping rate of the equal mass of polyethyleneimine aqueous solution and polylactic acid-polyurethane-polyacrylic acid copolymer butyl acetate solution in Example 1 was adjusted to 7 mL / min, while other components and operations remained unchanged. The resulting fluorescent nanoparticles were larger in size and had poorer distribution uniformity than those in Example 1. Under 365 nm ultraviolet light irradiation, their fluorescence intensity was reduced.
[0052] Example 7:
[0053] The dropping rate of the equal mass of polyethyleneimine aqueous solution and polylactic acid-polyurethane-polyacrylic acid copolymer butyl acetate solution in Example 1 was adjusted to 10 mL / min, while other components and operations remained unchanged. The resulting fluorescent nanoparticles were larger in size and had poorer distribution uniformity than those in Example 1. Under 365 nm ultraviolet light irradiation, their fluorescence intensity was reduced.
[0054] Example 8:
[0055] When an aqueous solution of polyethyleneimine of medium mass was added dropwise to a polylactic acid-polyurethane-polyacrylic acid copolymer butyl acetate solution in Example 1, the stirring speed was adjusted to 1500 rpm under high-speed stirring conditions, while other components and operations remained unchanged. The resulting fluorescent nanoparticles were larger in size, and their fluorescence intensity decreased under 365 nm ultraviolet light irradiation.
[0056] Example 9:
[0057] When an aqueous solution of polyethyleneimine of medium mass was added dropwise to a butyl acetate solution of polylactic acid-polyurethane-polyacrylic acid copolymer in Example 1, the stirring speed was adjusted to 1000 rpm while other components and operations remained unchanged. The resulting fluorescent nanoparticles were larger in size and their fluorescence intensity decreased by about half under 365 nm ultraviolet light irradiation.
[0058] Example 10:
[0059] When an aqueous solution of polyethyleneimine of medium mass was added dropwise to a butyl acetate solution of polylactic acid-polyurethane-polyacrylic acid copolymer in Example 1, the stirring speed was adjusted to 500 rpm under high-speed stirring conditions, while other components and operations remained unchanged. The size of the resulting fluorescent nanoparticles was further increased, and their fluorescence intensity decreased under irradiation with a 365 nm ultraviolet lamp.
[0060] In summary, this invention provides a novel method for preparing fluorescent nanoparticles. The method involves dropwise adding a polyethyleneimine aqueous solution to a butyl acetate solution of a polylactic acid-polyurethane-polyacrylic acid copolymer under high-speed stirring, followed by spray drying to obtain the prepared fluorescent nanoparticles. The size, uniformity, and fluorescence intensity of the fluorescent nanoparticles can be controlled by varying the amounts of the polyethyleneimine aqueous solution and the butyl acetate solution, as well as the stirring speed, thereby enabling the fluorescent nanoparticles to possess fluorescent properties and better application prospects.
[0061] Figure 1 The figure shows the fluorescence emission spectrum of the fluorescent nanoparticles prepared in Example 1 of this invention. As can be seen from the figure, the fluorescent nanoparticles have excellent luminescence properties, with the fluorescence emission peak at the maximum excitation wavelength located at 460 nm.
[0062] Figure 2 The figure shows the particle size distribution of the fluorescent nanoparticles prepared in Example 1 of this invention. As can be seen from the figure, the particle size distribution of the nanoparticles is at about 40 nm, and the nanoparticle size is uniform.
[0063] Figure 3 The fluorescent nanoparticles prepared in this invention exhibit excellent biocompatibility in their cytotoxicity evaluation.
[0064] Figure 4The relationship between the size and fluorescence intensity of the fluorescent nanoparticles prepared in this invention is determined by adjusting the mass ratio of polyethyleneimine aqueous solution to butyl acetate emulsion of polylactic acid-polyurethane-polyacrylic acid copolymer.
[0065] Figure 5 The fluorescent nanoparticles prepared in this invention are related to the drop rate of the polyethyleneimine aqueous solution and the butyl acetate emulsion of polylactic acid-polyurethane-polyacrylic acid copolymer, as well as the changes in the size and fluorescence intensity of the fluorescent nanoparticles.
[0066] Figure 6 The relationship between the size and fluorescence intensity of the fluorescent nanoparticles prepared in this invention was investigated by adjusting the stirring speed of the polyethyleneimine aqueous solution and the butyl acetate emulsion of polylactic acid-polyurethane-polyacrylic acid copolymer under high-speed stirring.
[0067] Figure 7 The relationship between the size and fluorescence intensity of the fluorescent nanoparticles prepared in this invention was investigated by adjusting the dropping rate of an equal mass of polyethyleneimine aqueous solution and a polylactic acid-polyurethane-polyacrylic acid copolymer butyl acetate emulsion, as well as the rotation speed under high-speed stirring conditions.
[0068] Note: The above examples are only used to illustrate the present invention and are not intended to limit the technical solutions described in the present invention. Therefore, although the present invention has been described in detail with reference to the above examples, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the present invention. All technical solutions and improvements that do not depart from the spirit and scope of the present invention should be covered within the scope of the claims of the present invention.
Claims
1. A method for preparing polymer fluorescent nanoparticles, characterized in that... Includes the following steps: Step 1: Synthesis of polylactic acid-polyurethane-polyacrylic acid copolymer Dihydroxyl-terminated polylactic acid (PLA) was obtained by alcoholysis of waste PLA with ethylene glycol. The dihydroxyl-terminated PLA was then reacted with 4,4′-diphenylmethane diisocyanate and polycaprolactone polyol to obtain isocyanate-terminated PLA-polyurethane. Hydroxyethyl acrylate was then added to the system and reacted with the isocyanate-terminated PLA-polyurethane to introduce terminal double bonds. Acrylic acid monomers were then added, and polymerization was carried out to obtain PLA-polyurethane-polyacrylic acid copolymer. Finally, the PLA-polyurethane-polyacrylic acid copolymer was dissolved in butyl acetate solvent for later use at a concentration of 25-100 mg / mL. Step 2: Prepare an aqueous solution of polyethyleneimine with a concentration of 15-25 mg / mL; Step 3: Add the polyethyleneimine aqueous solution prepared in Step 2 to the polylactic acid-polyurethane-polyacrylic acid copolymer butyl acetate solution obtained in Step 1 to form a water-in-oil emulsion. The mass ratio of the polyethyleneimine aqueous solution to the polylactic acid-polyurethane-polyacrylic acid copolymer butyl acetate solution is 1:5 to 1:
10. Step 4: Evaporate the water in the polylactic acid-polyurethane-polyacrylic acid emulsion obtained in Step 3 by heating with stirring to form a fluorescent solution; Step 5: Spray dry the fluorescent solution obtained in Step 4 to obtain polymer fluorescent nanoparticles.
2. The preparation method according to claim 1, characterized in that: In step 1, 5 g of waste polylactic acid granules were added to 30 mL of ethylene glycol and stirred for 12 h to obtain a dihydroxyl-terminated polylactic acid product. Then, using butyl acetate as a solvent, the dihydroxyl-terminated polylactic acid was reacted with 4,4′-diphenylmethane diisocyanate and polycaprolactone polyol in a molar ratio of 1:2.4:1 for 12 h to obtain an isocyanate-terminated polylactic acid-polyurethane product. Then, 2.5 times the molar amount of isocyanate residues of hydroxyethyl acrylate were added, and the reaction was continued for 12 h. Subsequently, 7.2 g of acrylic acid monomer was added, and the reaction was continued for 12 h to obtain a polylactic acid-polyurethane-polyacrylic acid copolymer solution. Finally, the product was diluted with butyl acetate.
3. The preparation method according to claim 1, characterized in that: In step 2, 2 g of polyethyleneimine is added to distilled water and stirred for 30 min to form an aqueous solution of polyethyleneimine.
4. The preparation method according to claim 1, characterized in that: In step 3, 10g of the polyethyleneimine aqueous solution prepared in step 2 is added dropwise to the butyl acetate solution of polylactic acid-polyurethane-polyacrylic acid copolymer at a certain rate, and stirred at high speed for 2 hours to form a water-in-oil emulsion.
5. The preparation method according to claim 1, characterized in that: In step 3, the dropping rate is controlled at 1~10 mL / min; the stirring speed is controlled at 500~2000 rpm.
6. The preparation method according to claim 1, characterized in that: In step 5, the fluorescent solution is spray-dried using nitrogen as the drying carrier.
7. The preparation method according to claim 6, characterized in that: The drying temperature is 100~200℃, and the feeding rate is 5~20 mL / min.