Preparation method of nanomedicine carrier and application thereof

Abstract

The application belongs to the technical field of biological pharmacy, and particularly relates to a preparation method of a nano drug carrier and application thereof. The application proposes a preparation process based on a pre-aggregation-coordination-shaping sequence: first, mild combination of chitosan and polyphenol is caused under near neutral conditions to form small particles with uniform size and good dispersibility, thereby effectively reducing the adhesion between particles; then, slowly introducing trivalent iron ions under the regulation of a weak chelating agent enables the trivalent iron ions to be uniformly coordinated on the particle surface, thereby constructing an inner layer with dense structure and responsiveness to acidic environment or competitive ligands; finally, introducing sodium alginate and cross-linking through slow release of calcium ions to form a flexible outer layer, and the outer layer and the inner layer synergistically act to significantly enhance the anti-dilution capacity and anti-protein adsorption performance of the carrier.

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceutical technology, specifically relating to a method for preparing a nanomedicine carrier and its application. Background Technology

[0002] Nanoparticles have been a significant research area in drug delivery over the past two decades. Typical systems include polymer nanoparticles (such as biodegradable polyester nanoparticles, polymer micelles, and dendritic macromolecules), lipid carriers (liposomes, solid lipid nanoparticles, and lipid nanoparticles), and inorganic and hybrid carriers (mesoporous silica, metal oxides, metal-organic / phenolic coordination networks, etc.). These carriers achieve encapsulation, protection, targeting, and controlled release at the nanoscale, improving the in vivo exposure of poorly soluble or unstable active ingredients, reducing distribution to non-target tissues and adverse reactions, and endowing them with responsiveness to microenvironmental signals such as acids, bases, enzymes, and redox reactions. However, current technologies still face a difficult trade-off between structural uniformity, environmental responsiveness, process scalability, and regulatory friendliness of raw materials and excipients.

[0003] From a materials perspective, polymer self-assembled supports prepared under organic solvent / antisolvent conditions are prone to problems such as wide particle size distribution and difficulty in suppressing first-dose burst release. Lipid systems undergo protein crown remodeling in the presence of plasma proteins, often masking previously reported active targeting groups, leading to inconsistencies between in vivo distribution and in vitro evaluation. While multilayer layer-by-layer (LBL) self-assembly strategies can improve interfaces, they involve numerous cyclic deposition steps, high time and equipment costs, and batch-to-batch consistency is significantly affected by operator error. Metal-polyphenol networks have attracted attention due to their ability to form dense shells under mild conditions and their sensitivity to acidity and competing ligands; however, common one-step rapid mixing easily induces cross-particle bridging and uncontrolled precipitation, making it difficult to stably reproduce shell thickness and pore structure. Simultaneously, hydrolysis of metal ions, secondary phase deposition, and competitive coordination of anions in buffer systems can all cause unpredictable structural differences. From a process perspective, many high-performance nanocarriers rely on microfluidics, precision jetting, or high-end homogenization equipment to achieve instantaneous mixing and scale control, which poses a high barrier to equipment investment and process transfer. When ion-crosslinked polysaccharides (such as alginate-calcium) are used for outer layer stabilization, instantaneous calcium delivery often results in uneven shell thickness and surface roughness. The lack of process-based control over the rate of crosslinking advancement makes it difficult to achieve a reproducible balance between dilution resistance and environmental response. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the present invention aims to provide a method for preparing nanomedicine carriers and their applications. Addressing the problems of existing metal-polyphenol carriers, such as flocculation, poor stability in bodily fluids, and poor release behavior, this invention proposes a preparation process based on a prepolymerization-coordination-shaping sequence: First, chitosan and polyphenols are lightly bound under near-neutral conditions to form uniformly sized, well-dispersed small particles, effectively reducing particle adhesion; then, ferric ions are slowly introduced under the control of a weak chelating agent, achieving uniform coordination on the particle surface and constructing a dense inner layer that is responsive to acidic environments or competing ligands; finally, sodium alginate is introduced and cross-linked through the slow release of calcium ions to form a flexible outer layer. This outer layer and the inner layer work synergistically to significantly enhance the carrier's anti-dilution ability and anti-protein adsorption performance. Combined with appropriate ripening and desalination treatment, a metal-polyphenol carrier with high stability, narrow particle size distribution, and good release behavior is finally obtained.

[0005] To achieve the above objectives, the present invention provides the following technical solution: a method for preparing a nanomedicine carrier, comprising the following steps:

[0006] S1: Add chitosan to glacial acetic acid, adjust the pH to 6-6.5 with NaOH solution to obtain a 0.01-0.05 wt% chitosan solution; add tannic acid to deionized water, adjust the pH to 6-6.5 with acetic acid solution and NaOH solution to obtain a 0.08-0.15 wt% tannic acid solution;

[0007] S2: Mix equal volumes of chitosan solution and tannic acid solution from step S1, add NaCl, perform high-speed shearing treatment, and let stand to obtain a nano-co-deposited core system.

[0008] S3: The nano-co-deposited core system from step S2 was replaced with phosphate buffer solution by dialysis. Then, 1 mM sodium citrate was added to adjust the pH to 6.9–7.1. The mixture was stirred continuously while 1–2 mM FeCl3 solution was slowly added dropwise. During the addition, NaOH solution was continuously used to maintain the pH at 6.9–7.1. After the addition was completed, stirring was continued to complete the rearrangement.

[0009] S4: After completing step S3, add 0.03-0.1 wt% sodium alginate solution evenly and let it stand to complete surface adsorption; add Ca-EDTA to make the final concentration 1-3 mM, and then slowly reduce the pH to 6.6-6.8 within 10-15 min, and let it stand for aging treatment.

[0010] S5: After completing the ripening treatment in step S4, the salt is removed by dialysis, the pH is adjusted to 6, then 0.01wt% chitosan solution is added, the mixture is allowed to stand, trehalose is added, the mixture is pre-frozen, dried, and desorbed to obtain the nano-drug carrier.

[0011] In step S2, the amount of NaCl added is 5–15 mmol / L;

[0012] In step S2, the high-speed shearing parameters are: rotation speed 3000-6000 rpm, time 1-2 min;

[0013] In step S3, the specific operation of solvent replacement is as follows: 8-14kDa dialysis bag, the volume of the external liquid is 20 times the volume of the sample, the external liquid is 10mM phosphate buffer, the external liquid is replaced 3-4 times, each time for 30-60 minutes, and the temperature is maintained at 20-25℃ throughout the process;

[0014] In step S3, the volume ratio of the FeCl3 solution to the tannic acid solution in step S1 is 10-24:100.

[0015] In step S4, the amount of sodium alginate solution added is 100 mL / L;

[0016] In step S5, the dialysis desalting parameters are as follows: 8-14 kDa dialysis bag, external liquid volume is 20 times the sample volume, first use 10 mM phosphate buffer as external liquid for 1-2 replacements, then use deionized water containing 5% trehalose as external liquid for 2-3 replacements, each for 30-60 minutes, and maintain 20-25℃ throughout the process;

[0017] In step S5, the amount of chitosan solution added is 20-40 mL / L.

[0018] The technical effects described in this invention are achieved through the following technical solution: a method for preparing a nanomedicine carrier, comprising the following steps:

[0019] S1: Add chitosan to 0.1M glacial acetic acid, adjust the pH to 6-6.5 with NaOH solution to obtain a 0.01-0.05wt% chitosan solution; add tannic acid to deionized water, adjust the pH to 6-6.5 with acetic acid solution and NaOH solution to obtain a 0.08-0.15wt% tannic acid solution;

[0020] S2: Mix equal volumes of chitosan solution and tannic acid solution from step S1, add a trace amount of NaCl, maintain the temperature at 20-25℃ for high-speed shearing treatment, let stand for 5 minutes, and obtain a nano-co-deposited core system.

[0021] S3: The nano-co-deposited core system from step S2 was replaced by dialysis to replace the solvent with 10 mM phosphate buffer. Then, 1 mM sodium citrate was added to adjust the pH to 6.9–7.1. The mixture was stirred continuously at 600–800 rpm, while 1–2 mM FeCl3 solution was slowly added dropwise at a rate of 0.5–1.5 mL / min. During the addition, 0.1 M NaOH solution was continuously used to maintain the pH at 6.9–7.1. After the addition was completed, the mixture was stirred for 10 min to complete the rearrangement.

[0022] S4: After completing step S3, uniformly add 0.03-0.1 wt% sodium alginate solution over 10-20 minutes, and let it stand for 5-10 minutes to complete surface adsorption; add Ca-EDTA to make its final concentration 1-3 mM; then slowly lower the pH to 6.6-6.8 over 10-15 minutes, and let it stand for 10-30 minutes for aging treatment;

[0023] S5: After completing the ripening treatment in step S4, dialyze to remove salt, adjust the pH to 6, then add 0.01wt% chitosan solution, let stand for 5-10 min, add 3-5% trehalose, pre-freeze at -40℃ for 2 h, dry at -20℃ for 6-12 h, and decompose at 20℃ for 2-4 h to obtain nano-drug carriers.

[0024] Preferably, in step S2, the amount of NaCl added is 5–15 mmol / L;

[0025] Preferably, in step S2, the high-speed shearing parameters are: rotation speed 3000-6000 rpm, time 1-2 min;

[0026] Preferably, in step S3, the specific operation of solvent replacement is as follows: 8-14kDa dialysis bag, the volume of the external liquid is 20 times the volume of the sample, the external liquid is 10mM phosphate buffer, the external liquid is replaced 3-4 times, each time for 30-60 minutes, and the temperature is maintained at 20-25°C throughout the process;

[0027] Preferably, in step S3, the volume ratio of the FeCl3 solution to the tannic acid solution in step S1 is 10-24:100.

[0028] Preferably, in step S4, the amount of sodium alginate solution added is 100 mL / L;

[0029] Preferably, in step S5, the dialysis desalination parameters are: 8-14 kDa dialysis bag, external liquid volume is 20 times the sample volume, first use 10 mM phosphate buffer as external liquid for 1-2 replacements, then use deionized water containing 5% trehalose as external liquid for 2-3 replacements, each for 30-60 minutes, and maintain 20-25℃ throughout the process;

[0030] Preferably, in step S5, the amount of chitosan solution added is 20-40 mL / L.

[0031] Preferably, another aspect of the present invention provides an application of a nanomedicine carrier, specifically, this drug carrier is preferably suitable for oral administration; as one application implementation, the desired loaded drug can be directly added in step S2, and co-deposited with chitosan solution and tannic acid solution; the outer alginate-calcium ion network maintains colloidal integrity and inhibits early leakage in gastric acid, and after entering the intestinal segment, with the increase of pH and the competitive ligand action of bile salts / citrate, the inner metal-polyphenol coordination bonds gradually loosen, realizing on-demand release; the non-covalent interaction between the thin layer of chitosan and the mucus chain enhances intestinal mucosal adhesion and retention, promotes permeability and smooths the initial release.

[0032] The beneficial effects of this invention are as follows:

[0033] Compared to existing technologies, the structure and function of the nanomedicine carrier prepared in this invention are based on multi-level interactions between materials and multi-level synergistic construction in terms of process technology. First, under weakly acidic to near-neutral conditions, this invention forms a size-constrained co-deposition core through hydrogen bonding, hydrophobic interactions, and π-π stacking between chitosan and polyphenol molecules. This finite-size core spatially localizes the subsequent metal coordination reaction, significantly reducing interparticle bridging and amorphous aggregation tendencies. This ensures that the coordination growth process is under the joint control of geometric constraints and diffusion boundaries from the outset, laying the foundation for obtaining a narrowly distributed, highly reproducible carrier structure. Subsequently, ferric ions with activity regulated by a weak chelating agent are introduced into the buffer system to trigger directional coordination crosslinking of polyphenol sites. The weak chelating agent effectively reduces the activity of free metal ions, prolongs the effective reaction time, and promotes the preferential orderly deposition of metal ions at the polyphenol-rich core-shell interface, rather than rapid hydrolysis or cross-particle crosslinking in the bulk phase. Simultaneously, a constant acid-base environment inhibits side reactions such as hydroxyl precipitation, promoting the continuous densification of the coordination network from the inside out. The resulting metal-polyphenol layer possesses dynamic reversibility, and its bonding energy is sensitive to pH and competing ligands, thereby achieving threshold response regulation of carrier permeability and the construction of programmable release channels at the molecular level. Beyond the metal-polyphenol layer, a second polysaccharide network is constructed through the physical adsorption of alginate and subsequent slow-release crosslinking with calcium ions. A segmented calcium ion addition strategy is employed to control the rate of crosslinking advancement, avoiding uneven shell thickness and pore distribution caused by instantaneous crosslinking. This ion-crosslinked outer and inner coordination networks are complementary in mechanical and permeability behavior. The inner layer imparts structural strength and chemical reversibility to the carrier, while the outer layer provides resistance to dilution, protein adsorption, and interfacial stability. An energy dissipation interface is formed between the two layers through hydrogen bonding and electrostatic adsorption, significantly enhancing the morphological stability and colloidal dispersibility of the carrier in complex physiological media. During the later maturation stage, the metal-polyphenol complex undergoes site exchange and pore reconstruction under near-steady-state conditions, effectively reducing local stress and structural defects, and further converging interparticle differences. At the same time, the outer polysaccharide segments rearrange and homogenize under the drive of ion migration, thereby alleviating the overall structural heterogeneity of the carrier across scales. Attached Figure Description

[0034] Figure 1 The results of cytotoxicity tests on drug carriers at a concentration of 50 μg / mL in Examples 1-3 and Comparative Examples 1-4 of this invention are as follows;

[0035] Figure 2 The results of cytotoxicity tests on drug carriers at a concentration of 200 μg / mL in Examples 1-3 and Comparative Examples 1-4 of this invention are as follows;

[0036] Figure 3 The results are the routine stability test results of the drug carriers in Examples 1-3 and Comparative Examples 1-4 of this invention;

[0037] Figure 4 The results of accelerated stability tests on drug carriers in Examples 1-3 and Comparative Examples 1-4 of this invention;

[0038] Figure 5 These are the cell uptake results of drug carriers at a concentration of 50 μg / mL in Examples 1-3 and Comparative Examples 1-4 of the present invention;

[0039] Figure 6 These are the cell uptake results of drug carriers at a concentration of 200 μg / mL in Examples 1-3 and Comparative Examples 1-4 of the present invention;

[0040] Figure 7 The UV-Vis absorption spectra are shown for each stage of the drug carrier preparation process in Example 1 of this invention. Detailed Implementation

[0041] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Unless otherwise specified, the raw materials involved in the present invention are all purchased through conventional commercial channels. Experimental methods without specific conditions are conventional methods and conditions well known in the art, or according to the conditions recommended by the instrument manufacturer.

[0042] Example 1: A method for preparing a nanomedicine carrier, comprising the following steps:

[0043] S1: Add 0.15g of chitosan to 500mL of 0.1M glacial acetic acid, and adjust the pH to 6.3 with NaOH solution to obtain a 0.03wt% chitosan solution; add 0.6g of tannic acid to 500mL of deionized water, and adjust the pH to 6.3 with acetic acid solution and NaOH solution to obtain a 0.12wt% tannic acid solution;

[0044] S2: Mix equal volumes of the chitosan solution and tannic acid solution from step S1, add 10 mmol NaCl; maintain the temperature at 22℃ and perform high-speed shearing treatment at 4500 rpm for 1.5 min, then let stand for 5 min to obtain the nano-co-deposited core system.

[0045] S3: The nano-co-deposited core system from step S2 was replaced by dialysis. The solvent was replaced with 10 mM phosphate buffer. A 12 kDa dialysis bag was used, and the external liquid volume was 20 times the sample volume. The external liquid was 10 mM phosphate buffer. The external liquid was replaced 3 times, 45 min each time, and the temperature was maintained at 22°C throughout. Then, 1 mM sodium citrate was added to adjust the pH to 7. The mixture was stirred continuously at 700 rpm. At the same time, 120 mL of 1 mM FeCl3 solution was slowly added dropwise at a rate of 0.8 mL / min. During the dropwise addition, 0.1 M NaOH solution was continuously used to maintain the pH at 7. After the dropwise addition was completed, the mixture was stirred for 10 min to complete the rearrangement.

[0046] S4: After completing the treatment in step S3, add 100 mL of 0.06 wt% sodium alginate solution evenly over 15 min, let stand for 8 min to complete surface adsorption; add 2 mmol Ca-EDTA, then slowly lower the pH to 6.7 over 12 min, and let stand for 20 min for aging treatment.

[0047] S5: After completing the ripening treatment in step S4, the sample was desalted by dialysis using a 12kDa dialysis bag with an external liquid volume 20 times that of the sample. First, a 10mM phosphate buffer solution was used as the external liquid for one replacement, followed by two replacements using deionized water containing 5% trehalose, each lasting 45 minutes, while maintaining the temperature at 22°C throughout. The pH was adjusted to 6, and then 30mL of 0.01wt% chitosan solution was added. The sample was allowed to stand for 8 minutes, then 4% trehalose was added. The sample was pre-frozen at -40°C for 2 hours, dried at -20°C for 10 hours, and desorbed at 20°C for 3 hours to obtain the nanomedicine carrier.

[0048] Example 2: A method for preparing a nanomedicine carrier, comprising the following steps:

[0049] S1: Add chitosan to 0.1M glacial acetic acid, adjust the pH to 6.5 with NaOH solution to obtain a 0.05wt% chitosan solution; add tannic acid to deionized water, adjust the pH to 6.5 with acetic acid solution and NaOH solution to obtain a 0.15wt% tannic acid solution;

[0050] S2: Mix equal volumes of the chitosan solution and tannic acid solution from step S1, add a trace amount of NaCl to make the final concentration 15 mmol / L; maintain the temperature at 20℃ and perform high-speed shearing treatment at 6000 rpm for 2 min, then let stand for 5 min to obtain the nano-co-deposited core system.

[0051] S3: The nano-co-deposited core system from step S2 was replaced by dialysis. The solvent was replaced with 10 mM phosphate buffer. A 14 kDa dialysis bag was used, and the external liquid volume was 20 times the sample volume. The external liquid was 10 mM phosphate buffer. The external liquid was replaced 4 times, 60 min each time, and the temperature was maintained at 20°C throughout. Then, 1 mM sodium citrate was added to adjust the pH to 7.1. The mixture was stirred continuously at 800 rpm. At the same time, 50 mL of 2 mM FeCl3 solution was slowly added dropwise at a rate of 1.5 mL / min. During the addition, 0.1 M NaOH solution was continuously used to maintain the pH at 7.1. After the addition was completed, the mixture was stirred for 10 min to complete the rearrangement.

[0052] S4: After completing the treatment in step S3, add 100 mL of 0.1 wt% sodium alginate solution evenly over 20 min, let stand for 10 min to complete surface adsorption; add 3 mmol Ca-EDTA, then slowly lower the pH to 6.8 over 15 min, and let stand for 30 min for aging treatment.

[0053] S5: After completing the ripening treatment in step S4, the sample was desalted by dialysis using a 14kDa dialysis bag with an external liquid volume 20 times that of the sample. Two replacements were performed using 10mM phosphate buffer as the external liquid, followed by three replacements using deionized water containing 5% trehalose, each lasting 60 minutes, maintained at 20°C throughout. The pH was adjusted to 6, and then 40mL of 0.01wt% chitosan solution was added. The sample was allowed to stand for 10 minutes, then 5% trehalose was added. The sample was pre-frozen at -40°C for 2 hours, dried at -20°C for 12 hours, and desorbed at 20°C for 4 hours to obtain the nanomedicine carrier.

[0054] Example 3: A method for preparing a nanomedicine carrier, comprising the following steps:

[0055] S1: Add chitosan to 0.1M glacial acetic acid, adjust the pH to 6 with NaOH solution to obtain a 0.01wt% chitosan solution; add tannic acid to deionized water, adjust the pH to 6 with acetic acid solution and NaOH solution to obtain a 0.08wt% tannic acid solution;

[0056] S2: Mix equal volumes of the chitosan solution and tannic acid solution from step S1, add a trace amount of NaCl to make the final concentration 5 mmol / L; maintain the temperature at 25℃ and perform high-speed shearing treatment at 3000 rpm for 1 min, then let stand for 5 min to obtain the nano-co-deposited core system.

[0057] S3: The nano-co-deposited core system from step S2 was replaced by dialysis. The solvent was replaced with 10 mM phosphate buffer. An 8 kDa dialysis bag was used, and the external liquid volume was 20 times the sample volume. The external liquid was 10 mM phosphate buffer. The external liquid was replaced 3 times, 30 min each time, and the temperature was maintained at 25°C throughout. Then, 1 mM sodium citrate was added to adjust the pH to 6.9. The mixture was stirred continuously at 600 rpm. At the same time, 80 mL of 1.5 mM FeCl3 solution was slowly added dropwise at a rate of 0.5 mL / min. During the addition, 0.1 M NaOH solution was continuously used to maintain the pH at 6.9. After the addition was completed, the mixture was stirred for 10 min to complete the rearrangement.

[0058] S4: After completing the treatment in step S3, add 100 mL of 0.03 wt% sodium alginate solution evenly over 10 min, let stand for 5 min to complete surface adsorption; add 1 mmol Ca-EDTA, then slowly lower the pH to 6.6 over 10 min, and let stand for 10 min for aging treatment.

[0059] S5: After completing the ripening treatment in step S4, the sample was desalted by dialysis using an 8kDa dialysis bag with an external liquid volume 20 times the sample volume. First, a 10mM phosphate buffer solution was used as the external liquid for one replacement, followed by two replacements using deionized water containing 5% trehalose, each lasting 30 minutes, while maintaining the temperature at 25°C throughout. The pH was adjusted to 6, and then 20mL of 0.01wt% chitosan solution was added. The sample was allowed to stand for 5 minutes, then 3% trehalose was added. The sample was pre-frozen at -40°C for 2 hours, dried at -20°C for 6 hours, and desorbed at 20°C for 2 hours to obtain the nanomedicine carrier.

[0060] Comparative Example 1: The operation process of Comparative Example 1 is basically the same as that of Example 1. The main difference is that the high shear co-deposition treatment in step S2 is removed in Comparative Example 1, and only equal volume mixing and magnetic stirring are used to form co-deposition nuclei; the other operating parameters remain the same.

[0061] Comparative Example 2: The operation process of Comparative Example 2 is basically the same as that of Example 1. The main difference is that Comparative Example 2 does not perform system buffering in step S3, that is, it does not replace the system obtained in step S2 with 10mM phosphate buffer, but directly carries out the subsequent addition of sodium citrate and slow drop of FeCl3 in the original system; the other operating parameters remain the same.

[0062] Comparative Example 3: The operation process of Comparative Example 3 is basically the same as that of Example 1. The main difference is that in step S3, sodium citrate is not added as a weak chelating rate regulator, and FeCl3 is added slowly to complete the coordination densification directly; the other operating parameters remain the same.

[0063] Comparative Example 4: The operation process of Comparative Example 4 is basically the same as that of Example 1. The main difference is that in step S4, only Ca-EDTA is removed and no calcium ion crosslinking is performed. After the alginic acid adsorption is retained, it directly enters the subsequent steps such as desalination, surface fine-tuning and freeze-drying. The other operating parameters remain the same.

[0064] Performance testing:

[0065] Cytotoxicity assay: The drug carrier samples prepared in Examples 1-3 and Comparative Examples 1-4, along with the culture medium negative control and blank wells containing "nanocarrier + detection system but no cells" (to eliminate background absorption / scattering interference from particles), were evenly distributed in 96-well plates with 6 wells per group of samples; HT-29 epithelial cells were selected and cultured in DMEM containing 10% fetal bovine serum to the logarithmic growth phase, and the cells were diluted to 1×10⁻⁶. 5 After inoculating with 50 μL of the sample at a concentration of 100 cells / mL, the cells were incubated at 37°C and 5% CO2 for 24 h. Then, 50 μL of different concentrations of carrier (50 μg / mL carrier solution; 200 μg / mL carrier solution) were added, and incubation continued for 24 h and 48 h. Cell viability was detected using the CCK-8 assay, with a microplate reader at 570 nm and 630 nm as a reference to subtract the background. Cell viability (%) was calculated as [sample well (subtracted background) OD / control group (subtracted background) OD] × 100%. Each sample was tested at least three times independently, and the average results were taken. Figure 1 and Figure 2 As shown.

[0066] based on Figure 1 and Figure 2The results showed that the drug carrier prepared in the embodiments of the present invention exhibited high cell viability at both 50 μg / mL and 200 μg / mL concentrations, demonstrating excellent biocompatibility. Based on the results of Comparative Example 1 and Example 1, it can be seen from the results of Comparative Example 1 that after canceling the high-shear co-deposition in step S2, Comparative Example 1 showed poor cell viability, and the difference was greater with time and high concentration conditions. This may be because the nuclei formed by relying solely on equal volume mixing and magnetic stirring lack sufficient geometric constraints and energy input, resulting in larger primary nuclei and wider distribution, which leads to a higher probability of cross-particle bridging and local shell inhomogeneity in the subsequent coordination stage. Based on the analysis of the results of Comparative Example 2 and Example 1, Comparative Example 2 exhibited extremely poor cell viability. This may be due to the direct addition of an iron source and a weak chelating agent to the original system without adjusting the buffer system, leading to drastic changes in ionic strength and pH. This triggered partial metal hydrolysis and non-directional cross-linking in the bulk phase, weakening the selective coordination at the polyphenol enrichment interface. In a serum environment, this type of network is more easily disturbed by protein substitution and ion fluctuations, resulting in a more significant decrease in cell compatibility over time and dosage, and exacerbating the curve fluctuations. Based on the analysis of the results of Comparative Example 3 and Example 1, the cell viability of Comparative Example 3 showed a greater degree of deterioration and dispersion at high concentrations and during the 48-hour period. This may be due to the excessively high iron ion activity and short effective reaction time after omitting weak chelating rate modulators such as citric acid, resulting in a significantly increased coordination rate. This triggered the formation of an excessively cross-linked layer with cross-particle bridging and rapid densification, lacking uniform channels and site rearrangement time, and leaving many uncoordinated metal sites and stress concentration areas inside. Based on the analysis of the results of Comparative Example 4 and Example 1, there was no significant difference between Comparative Example 4 and Example 1 under low and medium doses and 24h conditions. However, the cell viability fluctuated more when the concentration was high and the incubation was extended to 48h. This may be because only Ca-EDTA was removed while alginate adsorption was retained. The outer soft shell lacked a uniform and controllable ionic cross-linking network. The surface polysaccharides were more easily replaced or locally loosened under salt dilution and protein competition, making it difficult to form a stable energy dissipation interface with the inner dynamic coordination layer.

[0067] Stability testing: Examples 1-3 and Comparative Examples 1-4 were aliquoted and sealed according to the final drug delivery medium and stored at 4°C and 65% RH. Samples were taken at 0, 1, 2, 3, and 6 months. An accelerated testing condition of 40°C and 75% RH was also set up, with samples taken at 0, 2, 4, and 8 weeks. The drug encapsulation efficiency was measured at each sampling. The results are as follows: Figure 3 and Figure 4 As shown.

[0068] based on Figure 3 and Figure 4The results analysis showed that the encapsulation efficiency of the drug carriers prepared in the embodiments of the present invention exhibited a slow and stable decreasing trend under both conventional and accelerated conditions, demonstrating excellent overall stability. Based on the results analysis of Comparative Example 1 and Example 1, Comparative Example 1 showed a more significant decrease in encapsulation efficiency over the entire time span. This may be because, after the elimination of high-shear co-deposition, the primary nuclei lacked sufficient geometric constraints and energy input, resulting in larger nucleation sizes and wider distributions. Subsequently, the coordination stage was more prone to cross-particle bridging and uneven splicing of shell thickness / porosity, forming more early permeation defect sites. With the increase in storage time and temperature, the dynamic coordination network underwent site exchange and local rearrangement along these defect paths, accelerating the leakage flux. Based on the analysis of the results of Comparative Example 2 and Example 1, Comparative Example 2 showed the most significant deterioration in encapsulation efficiency under both conventional and accelerated conditions. This may be due to the lack of buffer system adjustment; the direct addition of an iron source and a weak chelating agent to the original system caused drastic changes in ionic strength and pH, triggering partial metal hydrolysis and non-directional cross-linking in the bulk phase, weakening the selective coordination of the polyphenol enrichment interface and entraining impurities. With time and the accumulation of thermal and humid stress, site exchange and pore expansion became more likely to occur, leading to a rapid decrease in encapsulation efficiency. Based on the analysis of the results of Comparative Example 3 and Example 1, Comparative Example 3 exhibited nonlinear and accelerated encapsulation efficiency decay in the middle and later stages and under accelerated conditions. This may be due to the excessively high iron ion activity and excessively fast coordination rate after omitting weak chelating rate regulators such as citric acid, resulting in an overly dense and poorly pore-forming coordination layer. Internal stress and insufficiently coordinated metal sites became the initiation centers of microcracks and micropores during storage, with dynamic rearrangement preferentially occurring along defects. Temperature rise and humidity promoted segment migration and coordination exchange, significantly increasing the permeation flux in the later stages and increasing the degree of encapsulation efficiency deterioration. Based on the analysis of the results of Comparative Example 4 and Example 1, Comparative Example 4 showed no significant difference from the Example under early and low stress conditions, but the encapsulation efficiency decreased gradually and faster under long-term and accelerated conditions. This may be because only Ca-EDTA was removed while alginate adsorption was retained, resulting in a lack of uniform and controllable ionic cross-linking network in the outer layer, making it difficult to form a stable energy dissipation interface with the inner dynamic coordination layer; the surface polysaccharide is easily replaced by environmental ions or becomes locally loose, and the shell permeation resistance decreases over time.

[0069] Cell uptake rate: After Caco-2 cells were resuscitated, cultured, differentiated, and matured, they were seeded into 96-well plates (1×10⁻⁶ cells / wells). 5Cells were cultured for 24 hours. The working solutions for the drug carriers prepared in Examples 1-3 and Comparative Examples 1-4 (containing 10% serum, 50 μg / mL carrier solution; 200 μg / mL carrier solution), negative controls (blank), and positive controls (cationic liposomes) were added to each well (100 μL per well, 6 wells per group). Cells were incubated at 37℃ and 5% CO2 for 0.5 h, 2 h, 6 h, and 24 h, respectively. After the endpoint, the cells were washed with PBS to remove surface adhesion. Fluorescence intensity was quantified by flow cytometry, and internalization localization was observed on parallel plates using a confocal microscope. Cell uptake efficiency was measured and analyzed by flow cytometry. The experimental results are as follows: Figure 5 and Figure 6 As shown.

[0070] based on Figure 5 and Figure 6Analysis of the results showed that the carrier prepared in Example 1 exhibited a smooth, monotonically increasing cell uptake curve with time and dosage under serum-containing conditions, with minimal fluctuations. Based on the results of Comparative Example 1 and Example 1, the cell uptake rate of Comparative Example 1 showed a significantly worse increase, with larger fluctuations. This may be because the removal of high-shear co-deposition resulted in insufficient geometric constraints and instantaneous energy input for primary nucleus formation, leading to larger nucleus size, wider distribution, and heterogeneous density and composition of the chitosan-polyphenol phase. This heterogeneity was further transferred to the subsequent coordination stage, causing cross-particle bridging and uneven shell thickness distribution. Based on the analysis of the results of Comparative Example 2 and Example 1, Comparative Example 2 showed the smallest increase in cell uptake rate. This may be because the iron source and weak chelating agent were added directly to the original system without buffering, introducing a sudden and drastic change in ionic strength and pH. Some iron ions hydrolyzed and non-directionally bound to polyphenols, producing heterogeneous phases or non-uniform coordination domains that weakened the selectivity of the polyphenol enrichment interface and the densification trend from the inside out. This resulted in poor shell structure continuity, decreased protein crown stability, and enhanced reversible competition, making it difficult for cell surface receptors to form effective clusters and maintain contact. Based on the analysis of the results of Comparative Example 3 and Example 1, the cell uptake rate of Comparative Example 3 showed a slow increase in the early and mid-stages, with a significant deterioration in the overall increase. This may be because the omission of weak chelating rate modulators such as citric acid resulted in higher iron ion activity and a faster coordination rate, forming a dense but non-uniform microporous rapid coordination layer in a short time. During the culture process, this structure underwent slow internal stress release and site exchange, resulting in increased surface rigidity, decreased permeability, and membrane bending and encapsulation becoming more dependent on high-energy events, thereby inhibiting endocytosis efficiency. Based on the analysis of the results of Comparative Example 4 and Example 1, the cell uptake rate of Comparative Example 4 was not significantly different from that of Example 1 at short time points and low doses, but the difference in the curves was more obvious after the time was extended and the dose was increased. This may be because only Ca-EDTA was removed while alginate adsorption was retained, and the outer layer lacked a uniform and controllable ionic cross-linking network. The soft shell was more prone to ion replacement and protein crown remodeling under serum-containing conditions, resulting in a loose outer layer structure and unstable surface charge. Although the inner layer still had reversible coordination characteristics, the outer layer was not sufficiently buffered against dilution and shearing, which shortened the residence and effective contact time of particles in the microenvironment around the cells, and interfered with the formation of receptor-mediated endocytic pits.

[0071] Spectral Testing: UV-Vis absorption curves of the drug carrier described in Example 1 were determined and plotted for each preparation step using UV-Vis spectroscopy. It should be noted that for acidity-triggered verification, an equal portion of the sample after step S4 was taken, transferred to a 10mM citric acid / sodium citrate buffer, the pH was adjusted to 5.3, and the sample was allowed to stand at 25°C for 10 minutes before immediately measuring UV-Vis. The test results are as follows: Figure 7 As shown.

[0072] based on Figure 7Results analysis showed that the S2 co-deposition core curve exhibited strong, slightly broadened polyphenol absorption in the UV region (300-360 nm), while the visible region remained close to the baseline with only a slight baseline rise. At this stage, the iron-polyphenol coordination network had not yet formed, and colloidal scattering was dominant. The FeCl3 background curve corresponded to a weak and broad absorption band in the near-UV region, with almost no characteristic peaks in the visible region, serving as a background reference for metal salts. With the slow addition of Fe in step S3... 3+ Maintaining a near-neutral pH, a broad-shouldered peak appeared in the 540-580 nm region after approximately 5 minutes of dropwise addition, indicating the beginning of charge transfer band formation from ligand to metal and the initial growth stage of the coordination network. Further dropwise addition for 15 minutes resulted in a significant enhancement of the main band in the visible region, shifting towards longer wavelengths to approximately 580-620 nm, while the peak shape became fuller, reflecting the advancement of polyphenol deprotonation and coordination number enhancement, and the densification process from the intraparticle to the outer layer. Upon acidic triggering, the curve showed a significant attenuation and slight decline of the main band in the visible region, while the polyphenol band in the ultraviolet region relatively rebounded, indicating reversible loosening of the metal-polyphenol complex, consistent with the mechanism of permeability and release of this support under acidic conditions. Overall, the curves at each stage maintained relatively stable intersections in the ultraviolet region, and no significant scattering increase was observed over time, indicating that weak chelation deceleration and pH control prevented cross-particle bridging and secondary phase precipitation.

[0073] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a nanomedicine carrier, characterized in that, Includes the following steps: S1: Add chitosan to glacial acetic acid, adjust the pH to 6-6.5 with NaOH solution to obtain a 0.01-0.05 wt% chitosan solution; Tannic acid was added to deionized water, and the pH was adjusted to 6-6.5 with acetic acid solution and NaOH solution to obtain a 0.08-0.15 wt% tannic acid solution. S2: Mix equal volumes of chitosan solution and tannic acid solution from step S1, add NaCl, perform high-speed shearing treatment, and let stand to obtain a nano-co-deposited core system. S3: The nano-co-deposited core system from step S2 was replaced with phosphate buffer solution by dialysis. Then, 1 mM sodium citrate was added to adjust the pH to 6.9–7.

1. The mixture was stirred continuously while 1–2 mM FeCl3 solution was slowly added dropwise. During the addition, NaOH solution was continuously used to maintain the pH at 6.9–7.

1. After the addition was completed, stirring was continued to complete the rearrangement. S4: After completing step S3, add 0.03-0.1 wt% sodium alginate solution evenly and let it stand to complete surface adsorption; add Ca-EDTA to make the final concentration 1-3 mM, and then slowly reduce the pH to 6.6-6.8 within 10-15 min, and let it stand for aging treatment. S5: After completing the ripening treatment in step S4, the salt is removed by dialysis, the pH is adjusted to 6, then 0.01wt% chitosan solution is added, the mixture is allowed to stand, trehalose is added, the mixture is pre-frozen, dried, and desorbed to obtain the nano-drug carrier. In step S2, the amount of NaCl added is 5–15 mmol / L; In step S2, the high-speed shearing parameters are: rotation speed 3000-6000 rpm, time 1-2 min; In step S3, the specific operation of solvent replacement is as follows: 8-14kDa dialysis bag, the volume of the external liquid is 20 times the volume of the sample, the external liquid is 10mM phosphate buffer, the external liquid is replaced 3-4 times, each time for 30-60 minutes, and the temperature is maintained at 20-25℃ throughout the process; In step S3, the volume ratio of the FeCl3 solution to the tannic acid solution in step S1 is 10-24:

100. In step S4, the amount of sodium alginate solution added is 100 mL / L; In step S5, the dialysis desalting parameters are as follows: 8-14 kDa dialysis bag, external liquid volume is 20 times the sample volume, first use 10 mM phosphate buffer as external liquid for 1-2 replacements, then use deionized water containing 5% trehalose as external liquid for 2-3 replacements, each for 30-60 minutes, and maintain 20-25℃ throughout the process; In step S5, the amount of chitosan solution added is 20-40 mL / L.

2. The application of the nanomedicine carrier according to claim 1 in the preparation of oral drug carriers.

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