Colon-targeted pH-responsive nano-liposome and application thereof in improving oral bioavailability of saponin

Abstract

The invention relates to the technical field of pharmaceutical preparations, and particularly provides colon-targeted pH-responsive nano-liposome and application thereof in improving oral bioavailability of saponin. Aiming at the problems of gastric acid damage, small intestine metabolism inactivation and low colon absorption efficiency during oral delivery of saponin, a three-layer core-shell structure design is adopted, wherein an inner core is composed of a saponin-phospholipid crystal compound; the middle layer is a chitosan-PEG bimolecular layer, and pH response release is realized in a colon environment with pH of 7.4; and the outer layer is modified with a folic acid-PE conjugate to enhance colon cell targeted recognition. The uniform nanoparticles with the particle size of 80 + / -5 nm are prepared by combining a gradient pH drug loading method with a microfluidic focusing technology. According to the preparation, the saponin bioavailability (AUC0-12 reaches 43.31 h. Microgram / mL) is remarkably improved, and the colon targeting property (the cell uptake rate is 52.9%) is enhanced. The innovativeness is that the gastrointestinal segmented protection and accurate colon release are synergistically realized through a multi-layer functional design, and the solubilization-permeation paradox of the traditional delivery technology is broken through.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical preparations, and in particular relates to an oral delivery system based on nanocarriers, specifically nanoliposomes with colon targeting and pH-responsive dual release functions, which are used to improve the oral bioavailability of saponins derived from medicinal and edible substances. Background Art

[0002] Saponin active ingredients (such as ginsenoside Rg3 and Panax notoginsenoside R1) are the core functional substances of medicinal and edible resources. Their oral bioavailability has long been limited by three interrelated biological barriers.

[0003] The molecular size barrier is manifested by a molecular weight (784–976 Da) that significantly exceeds the 500 Da threshold for passive diffusion in the intestine; the transmembrane permeability barrier is due to strong hydrophilicity (Log P < -1), resulting in an apparent permeability coefficient (Papp) of less than 1×10 -6 cm / s; the metabolic inactivation barrier involves the hydrolysis of glycosidic bonds by intestinal β-glucuronidase (>80%) and the first-pass effect mediated by the liver CYP450 enzyme system (CYP3A4 hydroxylation metabolism rate 68%, UGT1A1 glucuronidation accounts for 30%).

[0004] These characteristics collectively result in a bioavailability generally below 5%, posing an essential challenge to the development of delivery technologies.

[0005] Existing delivery strategies have not yet been able to systematically overcome the above obstacles.

[0006] Although cyclodextrin inclusion technology can improve the solubility of saponins (for example, the saturated solubility of Rg3 increases from 0.12 mg / mL to 1.85 mg / mL), the size of the inclusion complex expands to 2.8 nm, which in turn reduces the transmembrane transport efficiency; ordinary liposomes have serious insufficient structural stability in the gastric acid environment (pH 1.2) (2-hour residual rate <50%), and suffer from bile salt depolymerization in the duodenum (24-hour leakage rate 78%); nanoemulsions partially circumvent the first-pass effect through lymphatic transport, but the liver metabolic loss still exceeds 60%.

[0007] Similar patented technologies also have significant limitations: the colon cell uptake rate of polymer nanoparticles is less than 20%, and the single-layer chitosan-coated liposomes have an operational defect of a pH response lag of more than 30 minutes.

[0008] The core contradiction of these solutions lies in the failure to synergistically achieve dynamic regulation of gastrointestinal segmental protection and precise colon release, resulting in the continued existence of the "solubilization-permeation paradox".

[0009] Therefore, the development of a new delivery system needs to simultaneously meet three functional requirements: (1) maintain gastric fluid stability through rigid coating (target residual rate > 90%); (2) construct a pH / enzyme dual-responsive mechanism to achieve colon-targeted release (target release rate > 85%); (3) enhance cellular uptake efficiency through receptor-mediated targeting (target uptake rate > 50%) and inhibit the P-gp efflux effect.

[0010] This technical path has urgent practical significance for promoting the high-value utilization of medicinal and edible resources. Summary of the Invention

[0011] In response to the triple biological barriers that exist in the oral delivery of saponin active ingredients (such as ginsenoside Rg3 and Panax notoginseng saponin R1) - gastric acid degradation barrier (the 2-hour residual rate of ordinary liposomes under pH 1.2 environment is less than 50%), small intestinal metabolic barrier (β-glucuronidase hydrolysis rate >80% and first-pass metabolic loss >60%) and colon absorption barrier (cellular uptake rate <20% accompanied by P-gp efflux effect) - the present invention constructed a core-shell structured nanoliposome with the dual functions of colon targeting and pH response.

[0012] Its core innovation lies in achieving dynamic delivery control through a three-layer collaborative architecture: 1) The core crystalline complex is self-assembled from saponin (1-5 wt%) and hydrogenated soybean lecithin (60-70 wt%) via intermolecular hydrogen bonding. The incorporation of cholesterol (8-12 wt%) reduces the interlayer spacing of the lecithin membrane from 4.3 nm to 3.8 nm, significantly improving drug loading stability (encapsulation efficiency 87.5±1.2%). 2) The pH-responsive middle bilayer is constructed using chitosan-PEG2000 (15-20 wt%), which forms a dense protective structure (Zeta potential +32.1 mV) through electrostatic contraction in the gastric acid environment (pH ≤ 3.0). However, at pH 7.4 in the colon, PEG chain dissociation triggers disintegration, achieving targeted release (release rate > 90% in 2 hours). 3) Targeted modification of the outer layer mediated folate receptor-specific endocytosis through folic acid-phosphatidylethanolamine conjugates (0.5-1 wt%), resulting in a colon cell uptake rate of 52.9±3.8% (a 237% increase compared to unmodified liposomes) and effectively inhibiting the P-gp efflux effect.

[0013] The system's innovative preparation process uses a dual-stage control system consisting of gradient pH drug loading and microfluidic focusing: 1) In the gradient drug loading stage, saponin was dissolved in a pH 12.0 ethanol solution, mixed with the phospholipid membrane at 45°C, and then cooled to pH 7.0 at a rate of 0.5 pH / min to promote the directional embedding of the molecules into the crystallization sites. 2) During the microfluidic molding stage, laminar shear force was applied in a Y-shaped channel with a sheath flow ratio of 10:1 (the sheath liquid was PBS, and the total flow rate was 5 mL / min). The liposome particle size was precisely controlled to 80 ± 5 nm (PDI < 0.1), overcoming the uneven particle size defect of traditional ultrasonic preparation (PDI > 0.3).

[0014] Validated in vitro and in animal models, this system exhibits triple delivery synergy: 1) Breakthrough in stability: Encapsulation efficiency decreased by less than 5% after 6 months of storage at 4°C (DSC showed no phase change); 2) Improved targeting: In vivo imaging showed that the Cy5 fluorescence intensity in the colon was 3.2 times that in the small intestine; 3) Bioavailability increased significantly: AUC0-12 in vivo reached 34.98 h·μg / mL, which is 9.5 times that of ordinary tablets. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The accompanying drawings are used to provide further understanding of the present invention and constitute a part of the specification. They are used to explain the present invention together with the embodiments of the present invention and do not constitute a limitation of the present invention.

[0016] Figure 1 The release rate of the core-shell structure nanoliposomes of the present invention in an in vitro simulated colon environment and gastric fluid environment.

[0017] Figure 2 Pharmacokinetic plasma concentration-time curves in beagle dogs.

[0018] Comparative preparations (n=6): (●) present invention (AUC0-12 43.31 h·μg / mL); (■) conventional liposomes (AUC0-12 16.34 h·μg / mL); (▲) commercial tablets (AUC0-12 3.68 h·μg / mL).

[0019] Specific implementation method.

[0020] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0021] The present invention discloses a targeted delivery system for improving the oral bioavailability of herbal saponins. Embodiments of the present invention are described in detail below with reference to the following examples. However, those skilled in the art should understand that the following examples are intended to illustrate the present invention only and should not be construed as limiting the scope of the present invention. Where specific conditions are not specified in the examples, conventional conditions or those recommended by the manufacturer were used.

[0022] Example 1: Basic preparation and targeting property verification of core-shell structured nanoliposomes.

[0023] Core-shell nanoliposomes were constructed using gradient pH drug delivery combined with microfluidic focusing technology. Ginsenoside Rg3 (3.0 wt%), hydrogenated soybean lecithin (65.0 wt%), cholesterol (10.0 wt%), chitosan-PEG2000 (18.0 wt%), and folic acid-phosphatidylethanolamine (0.8 wt%) were accurately weighed.

[0024] Drug loading stage: saponin was dissolved in a pH 12.0 ethanol solution (45°C), injected into a chloroform mixture of phospholipids and cholesterol, and cooled to pH 7.0 at a rate of 0.5°C / min to allow the molecules to assemble in a directed manner through hydrogen bonds (crystallization and aging for 12 h).

[0025] Microfluidic molding: A Dolomite Mitos system (Y-shaped chip, channel width 200 μm) was used. The flow rate ratio of sheath fluid (PBS pH 7.4) to lipid precursor was set at 10:1 (total flow rate 5 mL / min). Laminar shear force was used to control the particle size to 80 ± 3 nm (PDI 0.07), and the encapsulation efficiency reached 87.5 ± 1.2%.

[0026] Verification of colon targeting performance showed that the 2-hour release rate in simulated gastric fluid (pH 1.2) was only 8.3 ± 1.9%, and the 6-hour cumulative release was less than 16%, indicating structural integrity in the gastric acid environment. In the simulated colon environment (pH 7.4), the 2-hour release rate increased sharply to 92.3 ± 7.5% (*** p < 0.001 vs. Gastric juice group), confirming the disintegration specificity of the pH-responsive layer.

[0027] In the Caco-2 cell uptake experiment, the uptake rate of the folate receptor-mediated group was 52.9 ± 3.8%, which was reduced to 18.7 ± 2.1% after competitive inhibition by free folic acid ( t = 9.24, p < 0.001), demonstrating the biological effectiveness of targeted modification.

[0028] Example 2: Pilot scale process scale-up and stability optimization.

[0029] To address the pain points of industrialization, three key improvements were implemented: ① Crystallization uniformity control: 0.05% polysorbate 80 was added as a crystallization directing agent to reduce the liquid-liquid interfacial tension, thereby reducing the encapsulation efficiency fluctuation from ±5.2% to ±1.3% (DLS confirmed a 62% reduction in the standard deviation of the particle size distribution); ② Microfluidic anti-clogging design: A 21 μm in-line filter (made of 316L stainless steel) was placed in front, and dual channels were operated alternately in parallel to achieve continuous production for 24 hours with a failure rate of less than 1%; ③ Lyoprotectant optimization: A combination of 5% trehalose and 2% mannitol was used. After reconstitution, the particle size only increased by 5.3% (the group without trehalose increased by 22.7%), and differential scanning calorimetry (DSC) showed that the glass transition temperature increased to 78°C.

[0030] Long-term stability studies (n=3) showed that after 6 months of storage at 4°C, the encapsulation efficiency decreased from 87.5±1.2% to 83.1±1.8%, and the particle size increased from 80 nm to 85 nm (PDI < 0.12), which is in line with the guidelines for microparticle preparations in the Chinese Pharmacopoeia.

[0031] Accelerated testing (40°C / 75% RH, 3 months) showed no phase change or precipitation.

[0032] In terms of cost control: hydrogenated soybean lecithin is replaced with egg yolk lecithin (cost reduction by 35%), and cholesterol is simultaneously adjusted to 12 wt%, and crystallization stability is maintained through membrane fluidity compensation.

[0033] Example 3: Expansion of the polysaponin system and verification of inflammatory bowel disease treatment.

[0034] Adaptability study of Panax notoginsenoside R1: The active ingredient was replaced with Panax notoginsenoside R1 with a smaller molecular weight (loading 4.5wt%), and the chitosan-PEG2000 ratio was increased to 20 wt% to enhance the acidic barrier.

[0035] In vitro release showed that the 2 h release rate was 89.7 ± 9.1% at pH 7.4, which was 8% delayed compared with the Rg3 system, attributed to the increased SB cmn resistance caused by the polarity difference of the R1 molecule.

[0036] Uhu7 pharmacokinetics in beagle dogs (n=6): The AUC0-12 of the present invention (Rg3 system) reached 43.31 ± 4.3 h·μg / mL, Cmax was 9.2 ± 0.7 μg / mL, and the relative bioavailability reached 1177% of that of commercial tablets (common liposomes were only 265%).

[0037] In vivo imaging showed that the fluorescence intensity of the colon region was 3.2 times that of the small intestine 6 h after oral administration (ROI analysis, p <0.001), supporting the colon targeting effect.

[0038] DSS-induced colitis mouse model: The drug concentration in the colon tissue of the R1 liposome group reached 28.7 μg / g, an increase of 293% compared with the conventional preparation (7.3 μg / g).

[0039] Western blot analysis showed that TNF-α expression was downregulated by 62.3% ( p < 0.001), and the NF-κB signaling pathway inhibition rate reached 58.9%, confirming that folate receptor-mediated targeted delivery can significantly enhance the anti-inflammatory effect.

[0040] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art will be able to modify the technical solutions described in the aforementioned embodiments or substitute equivalents for some of the technical features. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A colon-targeted pH-responsive nanoliposome, characterized in that Include: 1) Core: a crystalline complex formed by saponin and phospholipids through intermolecular hydrogen bonds; 2) Shell: A chitosan-PEG bilayer encapsulating the core, which triggers disintegration and release at pH ≥ 7.4; 3) Surface modification layer: folate-phosphatidylethanolamine conjugate for folate receptor-mediated colon cell targeting.

2. The nanoliposome according to claim 1, wherein: The saponin is selected from ginsenoside, notoginsenoside R1 or gypenosapside XLIX.

3. The nanoliposome according to claim 1, wherein: The particle size is 70-90 nm and the polydispersity index (PDI) is less than 0.

15.

4. The nanoliposome according to claim 1, wherein: The 2-hour residual rate in simulated gastric fluid (pH 1.2) is >90%, and the 2-hour release rate in simulated colon fluid (pH 7.4) is >85%.

5. A method for preparing the nanoliposome according to any one of claims 1 to 4, comprising the following steps: 1) Gradient pH drug loading: saponin was dissolved in an ethanol solution at pH 12, mixed with the phospholipid membrane, and then cooled to pH 7 at a rate of 0.5 pH / min to form a crystalline complex; 2) Microfluidic focusing: The product from step 1) was injected into a Y-shaped microchannel and focused at a sheath fluid flow rate ratio of 10:

1. The total flow rate was controlled at 5 mL / min, and the liposome dispersion was collected.

6. Use of the nanoliposomes according to any one of claims 1 to 4 in the preparation of a medicament for improving the oral bioavailability of saponins, wherein the oral bioavailability of the saponins is improved to more than 11 times that of conventional preparations.

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