Application of PLGA-chitosan nanoparticle delivery curcumin in PC12 cell differentiation

Abstract

The invention discloses application of curcumin delivered by PLGA (poly (lactic-co-glycolic acid))-chitosan nanoparticles in PC12 cell differentiation, and particularly relates to the cross technical field of biomedicine and nano-drug delivery. Curcumin-loaded nanoparticles are prepared through electrostatic interaction of PLGA and chitosan; the problems that curcumin is poor in stability and low in cell uptake efficiency are effectively solved. The nanoparticles are uniform in dispersion, uniform in size and reasonable in encapsulation efficiency and drug loading capacity, release follows a non-Fischer diffusion process, and slow and continuous release is realized. The anti-oxidation activity of curcumin is not less than 70%, the curcumin is nontoxic to PC12 cells under the concentration of 5-80 mu g / mL, and the survival rate exceeds 80%. Chitosan modification can enhance the cellular uptake ability, neutralize PLGA degradation acidic by-products, protect the biological activity of curcumin, and significantly promote the elongation of PC12 cell neurite and the formation of branches, provides a safe and effective novel intervention means for Alzheimer's disease and other related neurodegenerative diseases, and has outstanding clinical application potential.

Description

Technical Field

[0001] This invention relates to the interdisciplinary field of biomedicine and nanomedicine delivery, and in particular to the application of PLGA-chitosan nanoparticles in delivering curcumin during PC12 cell differentiation. Background Technology

[0002] The interdisciplinary field of biomedicine and nanomedicine delivery encompasses core technologies such as nanocarrier technology, drug delivery technology, cell differentiation regulation technology, and biomaterial preparation technology. The core of this field is the development of safe and efficient drug delivery systems to precisely deliver active ingredients to target cells and regulate cell function. This involves key steps such as the screening and synthesis of biomaterials, the preparation and characterization of nanoparticles, and the evaluation of cell biological effects. It is a multidisciplinary field integrating knowledge from biomaterials science, nanotechnology, cell biology, and analytical chemistry. It focuses on the design and preparation of biocompatible and biodegradable nanocarriers, optimizing the physicochemical properties of the carriers to improve the stability, cellular uptake efficiency, and targeting of active ingredients, thereby enabling applications in cell function regulation and intervention research related to various diseases.

[0003] The application of PLGA-chitosan nanoparticles for curcumin delivery in PC12 cell differentiation refers to the technical matter of loading curcumin with PLGA and chitosan as carrier materials, preparing nanoparticles through a specific process, and applying them to the regulation of PC12 cell differentiation. This technical matter covers the preparation steps such as the organic phase preparation of PLGA and curcumin, the aqueous phase preparation of PVA to form an emulsion to form PLGA nanoparticles, centrifugation, washing, freeze drying, and chitosan coating to form composite nanoparticles. It involves the optimization of process parameters such as raw material dosage, concentration, dissolution temperature, ultrasonic parameters, stirring conditions, and centrifugation parameters. Fourier transform infrared spectroscopy, X-ray diffraction, scanning electron microscopy, dynamic light scattering, and high performance liquid chromatography are used to characterize the structure, morphology, particle size, potential, drug loading, encapsulation rate, and in vitro release characteristics of the nanoparticles. Cell culture combined with CCK8 method, DPPH free radical scavenging method, laser confocal imaging method, and ImageJ software analysis are used to detect cytotoxicity, antioxidant activity, cell uptake capacity, and cell differentiation effect.

[0004] In existing technologies, curcumin suffers from poor stability and low bioavailability, making it difficult to fully utilize its biological activity when applied alone. While traditional PLGA nanoparticles possess biocompatibility, their negative surface charge hinders cellular uptake and is prone to burst release, preventing sustained delivery. Furthermore, the acidic byproducts generated during degradation may disrupt the biological microenvironment. Simultaneously, the lack of an effective carrier to protect curcumin makes its antioxidant activity susceptible to environmental influences. These shortcomings collectively limit the effectiveness of curcumin in promoting PC12 cell differentiation and fail to meet the technical requirements for intervention in related neurodegenerative diseases. Summary of the Invention

[0005] The main objective of this invention is to provide an application of PLGA-chitosan nanoparticles for delivering curcumin in PC12 cell differentiation, which can effectively solve the problems mentioned in the background art.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] An application of PLGA-chitosan nanoparticles for delivering curcumin in PC12 cell differentiation, characterized in that the nanoparticles are formed by the electrostatic interaction between PLGA and chitosan and are loaded with curcumin.

[0008] Preferably, the encapsulation rate of curcumin in the nanoparticles is 14.33±0.08% to 19.85±0.04%, and the drug loading is 4.09±0.02% to 5.67±0.01%.

[0009] Preferably, the release of curcumin from the nanoparticles follows a non-Fick diffusion process (diffusion index n=0.53), achieving slow and continuous release. Furthermore, the nanoparticles are non-toxic to PC12 cells, with a cell survival rate ≥80% in the concentration range of 5-80 μg / mL and an antioxidant activity ≥70% (detected by DPPH free radical scavenging method).

[0010] Preferably, the nanoparticles promote neurite elongation and neuronal branching in PC12 cells by enhancing cellular uptake of curcumin, protecting the bioactivity of curcumin, and neutralizing acidic byproducts of PLGA degradation.

[0011] In addition, the present invention also includes a method for preparing PLGA-chitosan nanoparticles loaded with curcumin, comprising the following steps:

[0012] S1. Dissolve PLGA and curcumin in dichloromethane to form an organic phase, and dissolve PVA in water to form an aqueous phase;

[0013] S2. The organic phase is added dropwise to the aqueous phase, and the emulsion is formed by ultrasonic treatment. After stirring to evaporate the solvent, the emulsion is centrifuged, washed, and freeze-dried to obtain PLGA nanoparticles.

[0014] S3. Disperse PLGA nanoparticles in chitosan solution, stir and centrifuge to collect the mixture, and obtain PLGA-chitosan nanoparticles loaded with curcumin.

[0015] Preferably, in step S1, the amount of dichloromethane is 2-20 ml, the concentration of the mixed solution of PLGA and curcumin is 1-10 mg / ml, the amount of PLGA added is 0-100 mg, the amount of curcumin added is 5-50 mg, the concentration of PVA solution is 1-10 mg / ml, the dissolution temperature is 60-100℃, the concentration of chitosan solution is 1-10 mg / ml, dissolved in 1%-5% acetic acid solution, and the dissolution temperature is 25-30℃.

[0016] Preferably, in step S2, the ultrasonic treatment time is 2-10 min with a cycle of 5 seconds on and 3 seconds off, the stirring temperature is 25-30℃ for 2-10 hours, the centrifugation speed is 9000-12000 rpm for 10-30 min, and 1% w / v of polyoxyethylene-polyoxypropylene copolymer 188 or F127 is added to the aqueous phase as a stabilizer in an amount of 10 ml.

[0017] Preferably, the curcumin-loaded PLGA-chitosan nanoparticles are used to prepare a neuroprotective agent suitable for intervention in neurodegenerative diseases associated with PC12 cell differentiation.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] This invention prepares curcumin-loaded nanoparticles through the electrostatic interaction between PLGA and chitosan, effectively solving the problems of poor curcumin stability and low cellular uptake efficiency. The nanoparticles are uniformly dispersed and sized, with curcumin encapsulation efficiency and drug loading within a reasonable range. The release follows a non-Fick diffusion process, achieving slow and sustained release and avoiding the side effects of sudden drug release. They not only retain ≥70% of the antioxidant activity of curcumin but also exhibit no significant toxicity to PC12 cells within a concentration range of 5-80 μg / mL, with cell viability exceeding 80%. Simultaneously, chitosan modification enhances the cells' ability to uptake curcumin, neutralizes acidic byproducts from PLGA degradation, protects the biological activity of curcumin, and significantly promotes neurite elongation and neuronal branching in PC12 cells. This provides a safe and effective novel intervention for neurodegenerative diseases related to PC12 cell differentiation, such as Alzheimer's disease, and possesses significant clinical application potential. Attached Figure Description

[0020] Figure 1 The Fourier transform infrared spectrum of the nanoparticles of this invention is shown below.

[0021] Figure 2 The X-ray diffraction pattern of the nanoparticles of this invention is shown below.

[0022] Figure 3 The image shows a scanning electron microscope image of the nanoparticles of this invention.

[0023] Figure 4 The diagram shows the physical size, polydispersity index (PDI), and zeta potential of different nanoparticles in this invention.

[0024] Figure 5 This is a cumulative release curve of curcumin in the nanoparticles of the present invention;

[0025] Figure 6 This is a graph showing the DPPH free radical scavenging rate of the nanoparticles of the present invention;

[0026] Figure 7 This is a graph showing the effect of different concentrations of nanoparticles of the present invention on the survival rate of PC12 cells;

[0027] Figure 8 Laser confocal microscopy image of HCT116 cells taking up coumarin-6 nanoparticles according to the present invention;

[0028] Figure 9 This diagram illustrates the effect of different nanoparticles of the present invention on the growth of neurites in PC12 cells. Detailed Implementation

[0029] To make the technical means, creative features, objectives and effects of this invention easier to understand, the invention will be further described below in conjunction with specific embodiments.

[0030] Example 1: Preparation of PLGA-chitosan nanoparticles loaded with curcumin.

[0031] The experimental materials included: polylactic acid-glycolic acid copolymer (PLGA), curcumin, polyvinyl alcohol (PVA), polyoxyethylene-polyoxypropylene copolymer 188 (P188), polyoxyethylene-polyoxypropylene copolymer 127 (F127), chitosan (CS), dichloromethane, acetic acid, and distilled water, all of which were of analytical grade; electronic balance, ultrasonic homogenizer, centrifuge, freeze dryer, constant temperature water bath, and magnetic stirrer.

[0032] Preparation steps:

[0033] Organic phase preparation: Accurately weigh 50 mg PLGA and 40 mg curcumin, dissolve them in 5 mL dichloromethane, and stir magnetically at 25 °C for 30 min to prepare a PLGA-curcumin mixed solution with a concentration of 5 mg / mL as the organic phase;

[0034] Aqueous phase preparation: Weigh 20 mg PVA, dissolve it in 20 mL of deionized water, stir in an 80 °C constant temperature water bath until completely dissolved, prepare a PVA solution with a concentration of 1 mg / mL, cool to room temperature, add 10 mL of 1% w / v P188 or F127 as a stabilizer, stir evenly, and use as the aqueous phase.

[0035] Emulsion preparation: The organic phase is slowly added dropwise to the aqueous phase, the ultrasonic disruptor is turned on, and the ultrasonic parameters are set as follows: ultrasonic time 2 minutes, cycle 5 seconds on and 3 seconds off, power 200W, to form a uniform emulsion;

[0036] Preparation of PLGA nanoparticles: The emulsion was placed on a magnetic stirrer at 25°C and stirred for 24 hours to promote the volatilization of dichloromethane. Then, it was centrifuged at 12,000 rpm for 30 minutes, the precipitate was collected, washed three times with distilled water, and freeze-dried to obtain PLGA nanoparticles.

[0037] Preparation of PLGA-chitosan nanoparticles: Weigh 500 mg of chitosan and dissolve it in 1% acetic acid solution. Stir at 25 °C until completely dissolved to prepare a chitosan solution with a concentration of 5 mg / ml. Take 10 mg of PLGA nanoparticles and disperse them in 1 ml of chitosan solution (PLGA nanoparticle concentration of 10 mg / ml). Stir magnetically at 25 °C for 6 hours. Centrifuge to collect the precipitate and obtain PLGA-chitosan nanoparticles loaded with curcumin (named PCPC188 and PCPC127, respectively).

[0038] Results: Two types of PLGA-chitosan nanoparticles loaded with curcumin were successfully prepared. They appeared as off-white powders with good flowability and no obvious agglomeration.

[0039] Example 2, as Figure 1-5 Physical properties characterization of PLGA-chitosan nanoparticles loaded with curcumin.

[0040] Experimental materials included: nanoparticles (PCPC127, PCPC188, PCP127, PCP188, PPC127, PPC188, PP127, PP188) prepared in Example 1; Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), scanning electron microscopy (SEM), dynamic light scattering (DLS), and high performance liquid chromatography (HPLC).

[0041] Test methods and results:

[0042] Infrared spectroscopy analysis: KBr pellet method was used, with spectra taken at 4000-500 cm⁻¹. -1 FTIR detection of the nanoparticles was performed within the wavenumber range. The results showed that PCP127 and PCP188 nanoparticles were detected at 3509 cm⁻¹. -1 3508cm -1 The characteristic absorption peak of curcumin appears at 1627 cm⁻¹. -1 and 1511cm -1 C=O and benzene ring stretching vibration peaks appeared at 3500 cm⁻¹; PCPC127 and PCPC188 nanoparticles showed peaks at 3500 cm⁻¹.-1 The absorption peaks nearby broadened significantly, confirming that chitosan and PLGA nanoparticles are bound together through electrostatic interactions.

[0043] XRD analysis: XRD analysis was performed on the nanoparticles within the range of 2θ = 5°–40°. The results showed that PLGA exhibited an amorphous diffraction peak at 2θ = 20.9°, while F127 and P188 showed crystalline diffraction peaks at 2θ = 19° and 23°, respectively. After the addition of curcumin, PCP127 and PCP188 showed characteristic curcumin diffraction peaks (2θ = 8.8°, 12.2°, etc.). After being coated with chitosan, PCPC127 and PCPC188 still retained the crystalline structure of curcumin, and chitosan did not change the overall crystalline properties of the nanoparticles.

[0044] SEM and particle size and potential analysis: The microstructure of nanoparticles was observed by SEM, and particle size, polydispersity index (PDI), and zeta potential were detected by DLS. The results showed that the uncoated chitosan nanoparticles tended to aggregate, while the PCPC127 and PCPC188 nanoparticles were uniformly dispersed and had smooth surfaces, with particle sizes of 325 nm and 348 nm, respectively, and PDI less than 0.3. Zeta potential detection showed that the potential of PLGA nanoparticles was -20.2 to -28.1 mV, and the potential changed to +12.2 to +14.7 mV after chitosan coating, confirming that chitosan was successfully modified on the particle surface.

[0045] Drug loading (LC) and encapsulation efficiency (EE) were determined by HPLC with methanol as the mobile phase and a detection wavelength of 425 nm. The results showed that the drug loading of PCPC127 was 4.72±0.03% and the encapsulation efficiency was 16.53±0.11%; the drug loading of PCPC188 was 4.74±0.01% and the encapsulation efficiency was 16.58±0.01%; the drug loading of PCP127 was 5.67±0.01% and the encapsulation efficiency was 19.85±0.04%; and the drug loading of PCP188 was 4.09±0.02% and the encapsulation efficiency was 14.33±0.08%.

[0046] In vitro release and kinetics studies: Nanoparticles were placed in PBS buffer (pH=7.4) and oscillated at 37℃. Samples were taken at different time points, and the curcumin release was detected by HPLC. The results showed that the cumulative curcumin release of PLGA nanoparticles was approximately 41% within 4 hours, while that of PCPC127 and PCPC188 was only 36%. Fitting the data using the Rittger-Peppers model, the diffusion index n=0.53, indicating that curcumin release follows a non-Fick diffusion process, achieving a slow and continuous release.

[0047] Example 3, as Figure 6-8 The antioxidant activity, cytotoxicity, and cellular uptake of nanoparticles were detected.

[0048] Experimental materials included: various nanoparticles prepared in Example 1; DPPH free radical reagent, PC12 cells, HCT116 cells; cell culture medium (DMEM), fetal bovine serum, horse serum, CCK-8 kit, coumarin-6, DAPI staining solution, DID cell membrane staining solution; ELISA reader, laser confocal microscope, and cell culture incubator.

[0049] Experimental steps and results:

[0050] Antioxidant activity assay (DPPH free radical scavenging method): Different nanoparticles were prepared into suspensions of the same concentration, mixed with DPPH solution, and reacted at room temperature in the dark for 30 min. The absorbance at 517 nm was measured. The results showed that the antioxidant activity of nanoparticles without curcumin (PP127, PP188, etc.) was less than 20%, while the antioxidant activity of nanoparticles loaded with curcumin (PCPC127, PCPC188, etc.) all exceeded 70%, confirming that nanoparticles can effectively retain the antioxidant properties of curcumin.

[0051] Cytotoxicity assay (CCK-8 assay): PC12 cells were cultured at 1 × 10⁶ cells per well. 4 PC12 cells were seeded at a density of 5-80 μg / mL in 96-well plates and cultured for 24 hours. Then, different concentrations (5, 10, 20, 40, 80 μg / mL) of nanoparticles were added, and the cells were cultured for another 24 hours. Fresh medium was then added to each well, and the cells were incubated at 37°C for 1 hour. The absorbance at 450 nm was measured using a microplate reader. The results showed that PC12 cell viability remained above 80% within the concentration range of 5-80 μg / mL, indicating that the nanoparticles had no significant cytotoxicity.

[0052] Cellular uptake assay (laser confocal imaging): HCT116 cells were seeded on coverslips of 6-well plates and cultured at 37°C in a 5% CO2 incubator for 24 hours. Drug-loaded nanoparticles were prepared using coumarin-6 instead of curcumin and added to the cell culture system, incubated for 4 hours. The coverslips were then removed, washed with PBS at 4°C for 5 minutes, and the cell nuclei were stained with DAPI and the cell membranes with DID staining. The results showed that cells treated with PCPC127 and PCPC188 exhibited significantly higher intracellular green fluorescence intensity than those treated with uncoated chitosan-based PLGA nanoparticles, confirming that chitosan coating enhances the cells' ability to uptake nanoparticles.

[0053] Example 4, as Figure 9 The effect of curcumin-loaded PLGA-chitosan nanoparticles on PC12 cell differentiation.

[0054] Experimental materials included: nanoparticles (PCPC127, PCPC188, PCP127, PCP188) prepared in Example 1; PC12 cells, cell culture media (high serum medium: DMEM + 10% fetal bovine serum + 5% horse serum; low serum medium: DMEM + 1% fetal bovine serum + 0.5% horse serum); ImageJ image analysis software, cell culture incubator, and inverted microscope.

[0055] Experimental steps:

[0056] Cell seeding and treatment: PC12 cells were seeded at a rate of 1 × 10⁶ cells per well. 5 The nanoparticles were seeded at a density of 1000 μM each in a 6-well plate and cultured in high-serum medium for 24 hours. The medium was then replaced with low-serum medium and each nanoparticle suspension containing 20 μM curcumin was added. A blank control group (without nanoparticles) and a curcumin control group (with 20 μM curcumin added directly) were set up.

[0057] Culture and observation: The culture was carried out at 37℃ and 5% CO2 for 7 days, and the low serum culture medium containing the corresponding nanoparticles was replaced every 2 days during the period.

[0058] Differentiation index detection: After culture, cell morphology was observed under an inverted microscope, and ImageJ software was used to quantitatively analyze neurite length, neurite number and neuronal branching status.

[0059] Results: In the blank control group, PC12 cells showed no significant differentiation, with short and few neurites; in the curcumin control group, cells showed partial differentiation, but the length and number of neurites were limited; while the PCPC127 and PCPC188 treatment groups showed significant differentiation, with significantly longer neurites and a significantly increased number of branches, which were superior to the uncoated chitosan PCPC127 and PCPC188 treatment groups. These results confirm that curcumin-loaded PLGA-chitosan nanoparticles can effectively promote PC12 cell differentiation.

[0060] In summary, through systematic preparation, characterization, and biological function experiments, this invention fully demonstrates the technical advantages of PLGA-chitosan nanoparticles as a curcumin delivery system. It not only solves the problems of poor stability and low delivery efficiency of curcumin, but also significantly enhances its biological activity in promoting PC12 cell differentiation. This provides a novel technical means and product support for the intervention of neurodegenerative diseases such as Alzheimer's disease, and has significant potential for clinical application.

[0061] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents.

Claims

1. An application of PLGA-chitosan nanoparticles for delivering curcumin in PC12 cell differentiation, characterized in that, The nanoparticles are formed through the electrostatic interaction between PLGA and chitosan and are loaded with curcumin.

2. The application of PLGA-chitosan nanoparticles for delivering curcumin in PC12 cell differentiation according to claim 1, characterized in that, The encapsulation efficiency of curcumin in the nanoparticles ranged from 14.33±0.08% to 19.85±0.04%, and the drug loading ranged from 4.09±0.02% to 5.67±0.01%.

3. The application of PLGA-chitosan nanoparticles for delivering curcumin in PC12 cell differentiation according to claim 1, characterized in that, The release of curcumin from the nanoparticles follows a non-Fick diffusion process (diffusion index n=0.53), achieving slow and continuous release. The nanoparticles are non-toxic to PC12 cells, with cell survival rate ≥80% in the concentration range of 5-80 μg / mL, and antioxidant activity ≥70% (detected by DPPH free radical scavenging method).

4. The application of PLGA-chitosan nanoparticles for delivering curcumin in PC12 cell differentiation according to claim 1, characterized in that, The nanoparticles promote neurite elongation and neuronal branching in PC12 cells by enhancing cellular uptake of curcumin, protecting curcumin bioactivity, and neutralizing acidic byproducts of PLGA degradation.

5. A method for preparing PLGA-chitosan nanoparticles loaded with curcumin, characterized in that, Includes the following steps: S1. Dissolve PLGA and curcumin in dichloromethane to form an organic phase, and dissolve PVA in water to form an aqueous phase; S2. The organic phase is added dropwise to the aqueous phase, and the emulsion is formed by ultrasonic treatment. After stirring to evaporate the solvent, the emulsion is centrifuged, washed, and freeze-dried to obtain PLGA nanoparticles. S3. Disperse PLGA nanoparticles in chitosan solution, stir and centrifuge to collect the solution, and obtain PLGA-chitosan nanoparticles loaded with curcumin.

6. The preparation method according to claim 5, characterized in that, In step S1, the amount of dichloromethane used is 2-20 ml, the concentration of the mixed solution of PLGA and curcumin is 1-10 mg / ml, the amount of PLGA added is 0-100 mg, the amount of curcumin added is 5-50 mg, the concentration of PVA solution is 1-10 mg / ml, the dissolution temperature is 60-100℃, the concentration of chitosan solution is 1-10 mg / ml, and it is dissolved in 1%-5% acetic acid solution at a dissolution temperature of 25-30℃.

7. The preparation method according to claim 5, characterized in that, In step S2, the ultrasonic treatment time is 2-10 min with a cycle of 5 seconds on and 3 seconds off; the stirring temperature is 25-30℃ for 2-10 hours; the centrifugation speed is 9000-12000 rpm for 10-30 min; and 1% w / v of polyoxyethylene-polyoxypropylene copolymer 188 or F127 is added to the aqueous phase as a stabilizer in 10 ml.

8. The application of PLGA-chitosan nanoparticles for delivering curcumin in PC12 cell differentiation according to any one of claims 1-4, characterized in that, The curcumin-loaded PLGA-chitosan nanoparticles are used to prepare a neuroprotective agent suitable for intervention in neurodegenerative diseases associated with PC12 cell differentiation.

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