Dual-function type nano-particles based on natural molecules and preparation method of dual-function type nano-particles

Abstract

The invention relates to a dual-function nano-particle based on natural molecules and a preparation method of the dual-function nano-particle, and belongs to the technical field of high polymer chemistry and pharmaceutical preparations. Chitosan CS is selected as a cationic polyelectrolyte, and low-molecular-weight heparin LMWH is selected as an anionic polyelectrolyte, so that a polyelectrolyte compound PEC is formed through electrostatic interaction, and an anti-inflammatory drug is loaded, so that the nano-particles with dual functions of targeting and treatment are formed. The nanoparticle is specifically combined with an L-selectin receptor overexpressed on the surface of inflammatory neutrophil through LMWH, so that accurate targeted delivery of an inflammatory part is realized. In addition, the LMWH not only serves as a carrier and a targeting material, but also serves as an anti-inflammatory agent; the traditional Chinese medicine composition can achieve an anti-inflammatory effect through a synergistic effect with other anti-inflammatory drugs. An efficient and safe drug delivery system is provided for treatment of acute lung injury ALI and other inflammatory diseases.

Description

Technical Field

[0001] The present invention belongs to the technical fields of polymer chemistry and pharmaceutical preparations, and particularly relates to a dual-functional nanoparticle based on natural molecules and a preparation method thereof. Background Art

[0002] Acute lung injury (ALI) is a severe disease characterized by pulmonary inflammation, with high morbidity and mortality. The pathological mechanism of ALI is complex, involving infiltration of various inflammatory cells, over-release of pro-inflammatory cytokines, and enhanced oxidative stress response, ultimately leading to lung tissue damage and dysfunction. Although significant progress has been made in the research on the pathophysiological mechanism of ALI in recent years, there is currently no specific treatment method, so the development of new treatment strategies is extremely urgent.

[0003] In recent years, the nano-drug delivery system, as an emerging treatment means, has shown great potential in the treatment of ALI. By encapsulating anti-inflammatory drugs in nanoparticles, this method can not only significantly improve the stability and bioavailability of drugs, but also achieve targeted drug delivery, thereby enhancing the therapeutic effect and reducing side effects. The nano-drug delivery system can effectively overcome some limitations in the application of traditional drugs, such as poor water solubility, low bioavailability, and rapid metabolism, etc., and realize the sustained release of drugs at the inflammatory site, and then play a stronger anti-inflammatory role. In addition, nanoparticles can combine multiple treatment mechanisms, such as inhibiting specific signaling pathways, reducing oxidative stress, and inhibiting the infiltration of immune cells, providing an efficient and safe comprehensive treatment strategy for acute lung injury.

[0004] Polyelectrolyte complex (PEC) nanoparticles formed by charged natural macromolecular polymers have attracted extensive attention in the field of drug delivery. PEC nanoparticles not only improve the solubility and stability of drugs, but also show unique advantages due to their excellent biocompatibility and biodegradability. Compared with traditional drug delivery systems, the preparation of PEC nanoparticles does not require organic solvents or chemical cross-linking agents, so the preparation process is mild and safe, suitable for in vivo application. In addition, the risk of PEC nanoparticles triggering immune or allergic reactions is low, especially suitable for the treatment of inflammatory diseases.

[0005] Chitosan (CS) is a linear polysaccharide composed of β-1,4-linked D-glucosamine and N-acetyl-D-glucosamine, and is the only naturally occurring cationic polymer. It has excellent biocompatibility and biodegradability, and is widely used in drug delivery systems, wound dressings, tissue engineering, antibacterial materials, etc. Low molecular weight heparin (LMWH) is a highly negatively charged glycosaminoglycan, which is widely known for its powerful anticoagulant effect. In addition, it has been shown to have a high affinity for the L-selectin receptor, which is overexpressed on the surface of neutrophils that infiltrate the lungs during inflammation, providing the possibility for targeted drug delivery to inflamed lung tissue.

[0006] Therefore, the development of a PEC nanoparticle based on CS and LMWH as a drug delivery system has important clinical significance and application prospects. Summary of the Invention

[0007] The object of the present invention is to provide a dual-functional nanoparticle drug delivery system that can accurately target the inflammatory site and synergistically exert an anti-inflammatory effect. By using polyelectrolyte complexes formed by charged natural macromolecular polymers to load the targeting moiety and anti-inflammatory drugs, the disadvantages of low bioavailability and poor stability of anti-inflammatory drugs are solved, providing a new efficient and safe strategy for the treatment of ALI diseases.

[0008] The technical solution adopted by the present invention is:

[0009] A dual-functional nanoparticle based on natural molecules, which forms a polyelectrolyte complex by electrostatic interaction between a cationic polyelectrolyte and an anionic polyelectrolyte with opposite charges, and loads an anti-inflammatory drug.

[0010] Preferably, the cationic polyelectrolyte is chitosan (CS), and the anionic polyelectrolyte is low molecular weight heparin (LMWH).

[0011] More preferably, the relative molecular weight of CS is 50 kDa - 200 kDa; the relative molecular weight of LMWH is 4 kDa - 20 kDa.

[0012] Preferably, the anti-inflammatory drug is curcumin (Cur), aspirin, indomethacin, dexamethasone or prednisone.

[0013] The preparation method of the dual-functional nanoparticle based on natural molecules described in any one of the above, includes the following steps:

[0014] 1) Dissolve the anionic polyelectrolyte and sodium tripolyphosphate (TPP) in pure water;

[0015] 2) Add the mixed ethanol solution of the anti-inflammatory drug and surfactant S100 to the cationic polyelectrolyte solution;

[0016] 3) Mix the aqueous solution containing anionic polyelectrolyte and TPP obtained in step 1) with the cationic polyelectrolyte solution containing anti-inflammatory drug and S100 obtained in step 2), and stir magnetically. Through electrostatic interaction, drug-loaded nanoparticles are self-assembled.

[0017] Furthermore, in the above preparation method, in step 1), the concentrations of the anionic polyelectrolyte and TPP in pure water are 0.8 - 3.2 mg / mL and 0.05 mg / mL respectively.

[0018] Furthermore, in the above preparation method, in step 2), the mass ratio of the anti-inflammatory drug to S100 is 1:1.

[0019] Furthermore, in the above preparation method, in step 2), the preparation method of the cationic polyelectrolyte solution is as follows: Add the cationic polyelectrolyte into pure water, and adjust the pH value to 4.9 with acetic acid solution. Continuously stir until the cationic polyelectrolyte is completely dissolved, and finally prepare a cationic polyelectrolyte solution with a concentration range of 0.8 - 3.2 mg / mL.

[0020] Furthermore, in the above preparation method, in step 3), the mass ratio of the cationic polyelectrolyte to the anionic polyelectrolyte is 1:4 - 4:1.

[0021] Furthermore, in the above preparation method, in step 3), the molar ratio of the anti-inflammatory drug to the anionic polyelectrolyte is 1:5 - 5:1.

[0022] The beneficial effects of the present invention are as follows:

[0023] 1. The design of the nanoparticles of the present invention fully exerts the dual functions of LMWH: LMWH not only acts as a carrier and targeting material, but also as an anti-inflammatory agent. By specifically binding to the L-selectin receptor overexpressed on the surface of inflammatory neutrophils, LMWH can accurately target the inflammatory site; at the same time, LMWH significantly reduces the inflammatory response by inhibiting neutrophil infiltration and synergistically anti-inflammates with other anti-inflammatory drugs.

[0024] 2. The nanoparticles of the present invention have pH-sensitive drug release characteristics, can rapidly release drugs in the acidic environment with pH = 5.5 at the inflammatory site, while release slowly in the neutral environment with pH = 7.4 of normal tissues, realizing the precise release of drugs. In addition, the nanoparticles have a high encapsulation rate, can effectively load and protect drugs, and significantly improve the bioavailability of drugs.

[0025] 3. The nanoparticles are prepared from natural polymer materials. The nanoparticles composed of CS and LMWH can form a hydrophobic region, have good biocompatibility and biodegradability, and are safe and non-toxic.

[0026] 4. The entire preparation of the nanoparticles of the present invention is a physical process, and the preparation process is simple and easy to implement. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Graph showing the level of IL-1β released by neutrophils after treatment with different doses of Cur and LMWH for 12 hours.

[0028] Figure 2 A is a graph showing the measurement of the particle size and PDI of different ratios of CS / LMWH.

[0029] Figure 2 B is a graph showing the measurement of the Zeta potential of different ratios of CS / LMWH.

[0030] Figure 2 C is a graph showing the measurement of the particle size and PDI of CS / LMWH / Cur and CS / LMWH / S100 / Cur.

[0031] Figure 2 D is a graph showing the measurement of the Zeta potential of CS / LMWH / Cur and CS / LMWH / S100 / Cur.

[0032] Figure 3 Graph showing the measurement of the encapsulation efficiency of LWMH and Cur in CS / LMWH / S100 / Cur.

[0033] Figure 4 Graph showing the transmission electron micrographs of CS / LMWH (A), CS / LMWH / Cur (B), and CS / LWMH / S100 / Cur (C).

[0034] Figure 5 Graph showing the in vitro release curves of Cur and LWMH in CS / LWMH / S100 / Cur. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0035] The technical solution of the present invention will be further described below through specific embodiments. Those skilled in the art should understand that the embodiments are only for helping to understand the present invention and should not be regarded as specific limitations of the present invention.

[0036] Example 1 Preparation of drug-loaded nanoparticles (I) The preparation method is as follows:

[0037] 1. Preparation of CS / LWMH

[0038] 1) Dissolve low molecular weight heparin (LMWH) and sodium tripolyphosphate (TPP) in pure water to prepare an aqueous solution mixture with an LMWH concentration of 0.8 - 3.2 mg / mL and a TPP concentration of 0.05 mg / mL.

[0039] 2) Chitosan (CS) was added to pure water, and the pH value was adjusted to 4.9 with acetic acid solution. Stir continuously until CS was completely dissolved, and finally a CS solution with a concentration range of 0.8 - 3.2 mg / mL was prepared.

[0040] 3) 3 mL of an aqueous solution mixture containing LMWH and TPP was dropped into 3 mL of the CS solution, and magnetically stirred at 25 °C for 1 hour to form CS-LMWH polyelectrolyte complex (PEC) nanoparticles through electrostatic interaction.

[0041] As Figure 2 A, Figure 2 B shows, when the mass ratio of CS / LWMH is 6:4, the particle size is the smallest, and the zeta potential is -18.34 ± 4.93 mV.

[0042] 2. Preparation of CS / LWMH / Cur

[0043] 1) The ratio of Cur and LMWH was screened by measuring the level of pro-inflammatory cytokine IL-1β secreted under the action of different concentrations of Cur and LMWH through ELISA experiment. As Figure 1 shown, when the molar ratio of Cur and LMWH is 3:1, it can most effectively inhibit the secretion of IL-1β by inflammatory neutrophils, achieving the best synergistic anti-inflammatory effect.

[0044] 2) LMWH and TPP were respectively dissolved in water to prepare an aqueous solution with a LMWH concentration of 1.6 mg / mL and a TPP concentration of 0.05 mg / mL.

[0045] 3) CS was added to pure water, and the pH value was adjusted to 4.9 with acetic acid solution. Stir continuously until CS was completely dissolved, and finally a CS solution with a concentration range of 2.4 mg / mL was prepared.

[0046] 4) The ethanol solution of Cur was slowly dropped into the CS solution to prepare a CS mixed solution with a LMWH molar concentration 3 times that in step 2).

[0047] 5) 3 mL of an aqueous solution containing LMWH and TPP was dropped into 3 mL of the CS solution obtained in step 3) to prepare a mixed solution with a molar ratio of Cur and LMWH of 3:1, and magnetically stirred at 25 °C for 1 hour to form drug-loaded CS-LMWH polyelectrolyte complex PEC nanoparticles through electrostatic interaction. The nanoparticles were washed with distilled water at least three times to completely remove free LMWH and anti-inflammatory drugs. Finally, the PEC nanoparticles were dispersed in water, freeze-dried and stored for further use.

[0048] 3. Preparation of CS / LWMH / S100 / Cur

[0049] 1) Dissolve LMWH and TPP in water respectively to prepare an aqueous solution containing LMWH at a concentration of 1.6 mg / mL and TPP at a concentration of 0.05 mg / mL.

[0050] 2) Add CS to pure water and adjust the pH value to 4.9 with acetic acid solution. Continuously stir until CS is completely dissolved, and finally prepare a CS solution with a concentration range of 2.4 mg / mL.

[0051] 3) Slowly add a mixed ethanol solution of Cur and surfactant S100 with a mass ratio of 1:1 dropwise to the CS solution.

[0052] 4) Add 3 mL of the aqueous solution containing LMWH and TPP to 3 mL of the CS solution obtained in step 3) to prepare a mixture with a molar ratio of Cur to LWMH of 3:1. Magnetically stir at 25 °C for 1 hour to form drug-loaded CS-LMWH polyelectrolyte complex (PEC) nanoparticles through electrostatic interaction. The nanoparticles are washed with distilled water at least three times to completely remove free LMWH and anti-inflammatory drugs. Finally, disperse the PEC nanoparticles in water, freeze-dry and store for further use.

[0053] As Figure 2 C, Figure 2 D shows, after adding surfactant S100 and Cur, the particle size and PDI of the PEC nanoparticles are further reduced, while the Zeta potential remains unchanged.

[0054] (II) Detection and Characterization

[0055] 1. Determination of particle size and Zeta potential

[0056] Take CS / LWMH, CS / LWMH / Cur, and CS / LWMH / S100 / Cur, dilute them with sterile injection water and transfer them to the sample cell. Use a Zetasizer Nano S90 dynamic light scattering particle size analyzer to measure the particle size and polydispersity coefficient of the samples at a wavelength of 632.8 nm. Each sample is operated in parallel 3 times to obtain the particle size, Zeta potential, and PDI value of the sample respectively.

[0057] As Figure 2 shown, the particle size of the prepared CS / LWMH / S100 / Cur is about 100 - 200 nm, and the particle size distribution is relatively uniform. The Zeta potential is -18.34 ± 4.93 mV.

[0058] 2. Determination of the encapsulation efficiency of Cur and LWMH

[0059] Precisely pipette 2 portions of the prepared CS / LWMH / S100 / Cur, 0.1 mL for each portion. Transfer one portion to a 10 mL volumetric flask, add 1.2 mL of sterile injection water, dilute to the mark with the demulsifier methanol, mix well, and measure the total drug absorbance A0 using a UV / Visible spectrophotometer. Place the other portion at the top of a Sephadex gel column, centrifuge at 2000 rpm for 4 minutes, then add 400 μL of sterile injection water at the top of the column and centrifuge at 2000 rpm for 4 minutes. Repeat this operation 3 times, combine the eluates, transfer to a 10 mL volumetric flask, dilute to the mark with the demulsifier methanol, mix well, and measure its absorbance value using a UV / Visible spectrophotometer. Measure the absorbance A1 of the encapsulated drug, and calculate the encapsulation efficiency EE% of LWMH and Cur according to the following formula. The results are as shown in Figure 3 .

[0060] EE% = A1 / A0 × 100%

[0061] It can be seen from Figure 3 that the encapsulation efficiencies of Cur and LMWH in CS / LMWH / S100 / Cur are 90.67 ± 2.08% and 90.67 ± 2.65% respectively, both of which can meet the subsequent in vitro and in vivo experiments.

[0062] 3. Transmission Electron Microscopy Test

[0063] Use a CM200-FEG TEM, Philips transmission electron microscope to examine the morphology and size of the PEC nanoparticles. For TEM analysis, place the PEC preparation on a 300-mesh copper grid coated with a thin film. The sample is negatively stained with a 1.0% phosphotungstic acid solution, air-dried at 25 °C, and observed and imaged under the TEM. The results are as shown in Figure 4 .

[0064] As shown in Figure 4 , CS / LMWH, CS / LMWH / Cur, and CS / LMWH / S100 / Cur all exhibit a uniform solid spherical structure with consistent sizes, which is in good agreement with the data of the particle size analyzer.

[0065] 4. Investigation of Release Behavior

[0066] Prepare 100 mL of phosphate buffer containing 5% Tween 80 with a pH of 7.4 or 5.5. Precisely pipette 1 mL of Cur, LMWH, and CS / LMWH / S100 / Cur each into a dialysis bag with a molecular weight cut-off of 10 kDa, immerse it in the release medium, and continuously stir at 37 °C at 100 rpm for 24 hours. Collect 1 mL of samples at specific time points of 0.5, 1, 2, 4, 6, 8, 12, and 24 hours, and measure the concentrations of Cur and LMWH using ultraviolet spectrophotometry. Replace the sampled volume with an equal volume of fresh release medium. The results are as shown inFigure 5 。

[0067] Figure 5 It was shown that CS / LMWH / S100 / Cur continuously released LMWH and Cur within 24 hours, and no burst release of anti-inflammatory drugs was observed. This indicates that CS / LMWH / S100 / Cur PEC nanoparticles have good stability under physiological conditions. At pH 5.5, LMWH and Cur were gradually released from the PEC nanoparticles within 24 hours, and the cumulative release percentages were 43% and 57% respectively. In contrast, at pH 7.4, only about 10% of LMWH and 19% of Cur were released within 24 hours. Therefore, CS / LMWH / S100 / Cu is pH-dependent and can achieve intracellular targeted therapy.

Claims

1. A dual-functional nanoparticle based on natural molecules, characterized in that, The nanoparticles are formed by electrostatic interaction of cationic polyelectrolytes and anionic polyelectrolytes with opposite charges to form a polyelectrolyte complex, and are loaded with anti-inflammatory drugs.

2. The dual-functional nanoparticle based on natural molecules according to claim 1, wherein, The cationic polyelectrolyte is chitosan CS, and the anionic polyelectrolyte is low molecular weight heparin LMWH.

3. The dual-functional nanoparticle based on natural molecules according to claim 2, characterized in that, The relative molecular weight of CS is 50 kDa - 200 kDa; the relative molecular weight of LMWH is 4 kDa - 20 kDa.

4. A dual-functional nanoparticle based on natural molecules according to claim 1, characterized in that, The anti-inflammatory drug is curcumin Cur, aspirin, indomethacin, dexamethasone or prednisone.

5. A method for preparing a dual-functional nanoparticle based on natural molecules according to any one of claims 1-4, characterized in that, It includes the following steps: 1) Dissolve the anionic polyelectrolyte and sodium tripolyphosphate TPP in pure water; 2) Add the mixed ethanol solution of the anti-inflammatory drug and surfactant S100 to the cationic polyelectrolyte solution; 3) Mix the aqueous solution containing the anionic polyelectrolyte and TPP obtained in step 1) with the cationic polyelectrolyte solution containing the anti-inflammatory drug and S100 obtained in step 2), and stir magnetically. Through electrostatic interaction, self-assembly forms drug-loaded nanoparticles.

6. The preparation method according to claim 5, characterized in that, In step 1), the concentrations of the anionic polyelectrolyte and TPP in pure water are respectively 0.8 - 3.2 mg / mL and 0.05 mg / mL.

7. The preparation method according to claim 5, characterized in that, In step 2), the mass ratio of the anti-inflammatory drug to S100 is 1:

1.

8. The preparation method according to claim 5, characterized in that, In step 2), the preparation method of the cationic polyelectrolyte solution is: add the cationic polyelectrolyte to pure water, and adjust the pH value to 4.9 with acetic acid solution, and continuously stir until the cationic polyelectrolyte is completely dissolved, and finally prepare a cationic polyelectrolyte solution with a concentration range of 0.8 - 3.2 mg / mL.

9. The preparation method according to claim 5, characterized in that, In step 3), the mass ratio of the cationic polyelectrolyte to the anionic polyelectrolyte is 1:4 - 4:

1.

10. The preparation method according to claim 5, characterized in that, In step 3), the molar ratio of the anti-inflammatory drug to the anionic polyelectrolyte is 1:5 - 5:1.

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