Lipid nanodiscs for polyphenol sustained release protection and methods of making the same

Abstract

This invention discloses a lipid nanodisc for polyphenol sustained-release protection and its preparation method, relating to the field of nanodelivery materials technology. The lipid nanodisc comprises a polyphenol active ingredient, a lipid bilayer matrix, and a composite edge stabilizer. The polyphenol active ingredient and the lipid bilayer matrix form a lipid-polyphenol pre-complexed structure, and the composite edge stabilizer seals the open edges of the lipid bilayer matrix. The preparation method includes: dissolving the polyphenol active ingredient and each lipid component in an ethanol phase to form a pre-complexed solution; performing a first stage of metastable micro-mixing with a first aqueous phase, without adding the composite edge stabilizer in the first stage; performing a second stage of sequential edge-sealing micro-mixing with the first stage outlet solution and a second aqueous phase containing the composite edge stabilizer; then performing tangential flow filtration to remove the solvent using a constant-volume percolation method, and then compounding with a glassy state protectant before freeze-drying or low-temperature spray drying to obtain a polyphenol lipid nanodisc powder product that retains its disk-like structure after reconstitution.

Description

Technical Field

[0001] This invention relates to the field of nanodelivery materials technology, specifically to a lipid nanodisc for polyphenol sustained-release protection and its preparation method. Background Technology

[0002] Polyphenolic active ingredients are widely used in functional beverages, instant nutritional powders, effervescent tablets, edible coatings, active packaging films, and topical formulations. Common varieties include curcumin, resveratrol, quercetin, tea polyphenols, anthocyanins, and phenolic acids. These substances typically contain multiple phenolic hydroxyl groups and conjugated aromatic structures, exhibiting strong hydrogen bonding, hydrophobic association, and a tendency to complex with metal ions. Therefore, they are easily limited by solubility, oxidation, complexation precipitation, and interfacial migration in aqueous systems, protein-containing systems, mineral-fortified systems, and systems that have been dried and then reconstituted. Current technologies often employ liposomes, nanoemulsions, solid lipid particles, or other lipid nanocarriers to disperse and protect polyphenols. Processes such as ethanol injection, thin-film hydration, ultrasonication, high-pressure homogenization, lyophilization, or spray drying can be used to improve their processing suitability. The above technologies can improve the apparent dispersibility of polyphenols to some extent, but they are still easily affected by factors such as ethanol / water solvent replacement, local shearing, interfacial dehydration and salt ion shielding when it comes to continuous preparation, low residual solvent control, shelf-life reconstitution and adaptation to complex food matrices.

[0003] Especially during the introduction of the ethanol phase containing lipids and polyphenols into the aqueous phase, the system is not simply diluted. As the volume fraction of ethanol decreases rapidly, the local solubility of polyphenols drops abruptly before the orderly assembly of lipids. Transient supersaturation can easily occur in the feed inlet, mixing dead zones, or localized concentrated areas of the stirred tank, leading to the formation of free crystal nuclei or crystal nucleus-lipid adsorption complexes. Even if the apparent particle size is subsequently reduced through ultrasound or homogenization, it is difficult to eliminate the already formed crystal nuclei and structural mixtures. If surfactants are added prematurely or in excessive amounts to improve dispersibility, free micelles may form, altering the distribution of polyphenols between the lipid and aqueous phases.

[0004] The above problems include In systems containing proteins, polysaccharides, or high sugar content with low water activity, the effects will be further amplified, manifesting as particle size and PDI drift, unstable proportions of disc-like or layered structures, excessively rapid initial release, darkening of color, membrane surface filtration contamination, turbidity after powder reconstitution, or precipitation of crystal points. Summary of the Invention

[0005] (a) Technical problems to be solved

[0006] To address the shortcomings of existing technologies, this invention provides a lipid nanodisc for polyphenol sustained-release protection and its preparation method, solving the problems of how to suppress the generation of transient free crystal nuclei of polyphenols caused by ethanol / water solvent replacement and the resulting lipid structure mixing, free crystal residue, initial burst release and decreased stability during the continuous preparation and solidification resolution of polyphenol lipid nanocarriers.

[0007] (II) Technical Solution

[0008] To achieve the above objectives, the present invention provides the following technical solution:

[0009] A lipid nanodisc for polyphenol sustained-release protection and its preparation method are disclosed. The lipid nanodisc comprises a polyphenol active ingredient, a lipid bilayer matrix, and a composite edge stabilizer. The polyphenol active ingredient forms a lipid-polyphenol pre-complexed structure with the lipid bilayer matrix, and the composite edge stabilizer seals the open edges of the lipid bilayer matrix.

[0010] The polyphenolic active ingredients are selected from one or more of curcumin, resveratrol, quercetin, catechin, epigallocatechin gallate, tea polyphenols, anthocyanins, chlorogenic acid, ferulic acid, and gallic acid derivatives; the lipid bilayer matrix includes phosphatidylcholine lipids, hydrogenated phospholipids or saturated phospholipids, anionic phospholipids, sterol membrane rigidity regulators, and antioxidant lipids; the composite edge stabilizer includes saponin-based edge sealers and N-acyl amino acid salt-based amphiphilic edge sealers.

[0011] This invention also provides a method for preparing the above-mentioned lipid nanodiscs, comprising: dissolving polyphenolic active ingredients and lipid components for forming a lipid bilayer matrix in an ethanol phase to obtain a lipid-polyphenol pre-complexed solution; performing a first-stage metastable micro-mixing of the pre-complexed solution with a first aqueous phase, wherein no composite edge stabilizer is added in the first stage; performing a second-stage sequential edge-sealing micro-mixing of the first-stage outlet solution with a second aqueous phase containing a composite edge stabilizer to obtain a lipid nanodisc dispersion; performing tangential flow filtration to desolventize the lipid nanodisc dispersion by constant volume percolation; optionally, compounding the desolventized nanodisc dispersion with a glassy state protectant and performing lyophilization or low-temperature nitrogen closed-loop spray drying.

[0012] (III) Beneficial Effects

[0013] This invention provides a lipid nanodisc for polyphenol sustained-release protection and its preparation method, which has the following beneficial effects:

[0014] By pre-forming lipid-polyphenol pre-complexes in the ethanol phase, polyphenols are guided to the lipid bilayer interface before entering the aqueous phase. This reduces the tendency for free polyphenols to directly nucleate during ethanol / water exchange, and avoids the subsequent formation of free crystals or crystal nucleus-lipid adsorption complexes, thereby improving the structural uniformity of lipid nanodisks from the source.

[0015] The first stage of metastable micromixing introduces only the first aqueous phase, allowing lipids to hydrate first and form a bilayer precursor without being prematurely micellized and extracted by the edge stabilizer. The second stage introduces a composite edge stabilizer to sequentially seal the open bilayer edges, shifting the system from a closed liposome or mixed sheet path to a disc-shaped bilayer sealing path, thereby improving the retention of the disc-shaped structure and reducing initial burst release. Tangential flow filtration removes ethanol, free polyphenols, and free edge stabilizers in a constant-volume percolation manner, reducing secondary recombination and membrane fouling, and improving the stability of the continuous preparation process.

[0016] When combined with a glassy protective agent for solidification, the edges of the nanodiscs can be protected during drying and resolution, reducing the risk of aggregation, turbidity, and crystal point precipitation after resolution. This makes the obtained polyphenolic lipid nanodiscs more suitable for complex applications such as functional beverages, instant powders, and active packaging films. Attached Figure Description

[0017] Figure 1 This is a schematic diagram of the lipid nanodisc structure of the present invention;

[0018] Figure 2 This is a flowchart illustrating the overall process for preparing lipid nanodisks according to the present invention.

[0019] Figure 3 This is a schematic diagram of the two-stage micro-mixing and timing edge sealing of the present invention;

[0020] Figure 4 This is a schematic diagram of the parallel interlaced herringbone micromixer structure of the present invention;

[0021] Figure 5 This is a schematic diagram of the tangential flow filtration desolventizing and glass solidification process of the present invention. Detailed Implementation

[0022] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0023] Please see Figures 1-5 This invention provides a lipid nanodisc for polyphenol sustained-release protection and its preparation method. The invention will be further described below with reference to specific embodiments.

[0024] Unless otherwise stated, all percentages, parts and proportions are based on mass or volume; the lipid nanodiscs refer to nanoscale disk-shaped dispersions with a disk surface formed by a lipid bilayer matrix and open edges sealed by a composite edge stabilizer, which are distinct from closed liposomes, nanoemulsions or solid lipid particles.

[0025] Composition and process control range

[0026] In one embodiment, the polyphenolic active ingredient is selected from one or more of curcumin, resveratrol, quercetin, catechin, epigallocatechin gallate, tea polyphenols, anthocyanins, chlorogenic acid, ferulic acid, and gallic acid derivatives; preferably, it is a polyphenol containing at least two phenolic hydroxyl groups and having a hydrophobic aromatic skeleton. The mass ratio of polyphenols to total lipids is controlled at 0.02:1-0.25:1, preferably 0.05:1-0.14:1.

[0027] The lipid bilayer matrix comprises phosphatidylcholine lipids, hydrogenated phospholipids or saturated phospholipids, anionic phospholipids, sterol membrane rigidity regulators, and antioxidant lipids. Based on the total molar amount of lipids, phosphatidylcholine lipids comprise 45-92 mol%, preferably 58-78 mol%; hydrogenated phospholipids or saturated phospholipids comprise 0.1-35 mol%, preferably 8-22 mol%; anionic phospholipids comprise 0.5-18 mol%, preferably 3-10 mol%; phytosterols or cholesterol comprise 2-28 mol%, preferably 8-18 mol%; and tocopherols or natural antioxidant lipids comprise 0.02-2.0 wt% of the total lipid mass, preferably 0.08-0.8 wt%.

[0028] The composite edge stabilizer is used to seal the open edges of the lipid bilayer, and is preferably formed by combining a saponin-based edge sealer and an N-acyl amino acid salt-based amphiphilic edge sealer. The mass ratio of the edge stabilizer to the total lipids is controlled at 0.015:1-0.12:1, preferably 0.030:1-0.080:1. The saponin-based edge sealer accounts for 20-95 wt% of the edge stabilizer, preferably 45-80 wt%; the N-acyl amino acid salt-based amphiphilic edge sealer accounts for 5-80 wt% of the edge stabilizer, preferably 20-55 wt%.

[0029] In one embodiment, the saponin-based edge-sealing agent includes one or more of saponins, tea saponins, and glycyrrhizic acid salts; the N-acyl amino acid salt amphiphilic edge-sealing agent includes one or more of N-lauroyl-L-glutamate sodium salt, N-stearoyl glutamate salt, and N-acyl glycinate salt.

[0030] The ethanol volume fraction in the ethanol-phase pre-complexing solution is controlled at 82-96 vol%, preferably 88-94 vol%; the total lipid concentration is 10-180 mg / mL, preferably 35-95 mg / mL; the polyphenol concentration is 0.2-35 mg / mL, preferably 2-10 mg / mL. The pre-complexing temperature is controlled at 30-52℃, preferably 38-46℃; the pre-complexing time is 5-30 min, preferably 8-20 min; and the dissolved oxygen is preferably not higher than 1.5 mg / L.

[0031] The first stage of micromixing introduces only the first aqueous phase without adding edge stabilizers, allowing the ethanol phase to enter the metastable region where lipids are hydrated but polyphenols have not yet crystallized significantly under millisecond-level mixing conditions. The volumetric flow rate ratio of the first aqueous phase to the ethanol phase is 1.2:1-5.5:1, preferably 2.0:1-3.5:1; the ethanol volume fraction at the first stage outlet is 18-42 vol%, preferably 24-34 vol%; the first stage mixing time is 10-180 ms, preferably 20-80 ms; the online turbidity variation coefficient in the stable stage is preferably no greater than 20%, more preferably no greater than 8%.

[0032] The second stage of micromixing brings the first stage outlet feed liquid into contact with a second aqueous phase containing a composite edge stabilizer, thereby sequentially closing the open double-layer edge. The volume ratio of the second aqueous phase to the first stage outlet feed liquid is 0.5:1-6.0:1, preferably 1.0:1-3.2:1; the volume fraction of ethanol at the second stage outlet is 5-20 vol%, preferably 8-14 vol%; the mixing time in the second stage is 10-500 ms, preferably 25-150 ms; and the pH in the second stage is controlled at 5.5-8.2, preferably 6.3-7.3.

[0033] The second-stage effluent preferably enters the tangential flow filtration system within 5 minutes to remove ethanol, free polyphenols, and free edge stabilizers via constant-volume percolation. The TFF membrane has a molecular weight cutoff of 30-500 kDa, preferably 50-150 kDa; a transmembrane pressure of 0.03-0.16 MPa, preferably 0.05-0.10 MPa; and a cross-flow shear rate of... Preferred The percolation volume is 3-15 DV, preferably 5-9 DV.

[0034] The purified nanodisc dispersion is compounded with a glassy protective agent and then subjected to lyophilization or low-temperature nitrogen closed-loop spray drying. The mass ratio of the protective agent to the unprotected nanodisc solids is 1.5:1-8.0:1, preferably 2.5:1-5.0:1; the protective agent includes one or more of trehalose, inulin, low-DE maltodextrin, gum arabic, whey protein, or modified starch. Before spray drying, the residual ethanol in the feed solution is not higher than 1000 ppm, and the mass fraction of the feed solution solids is adjusted to 16.0-22.0 wt%; the inlet air temperature for spray drying is 80-130℃, preferably 90-110℃; the outlet air temperature is 45-65℃, preferably 52-58℃; and the oxygen content in the column is controlled to be not higher than 3.0 vol%, preferably not higher than 2.0 vol%.

[0035] In one embodiment, the obtained lipid nanodiscs have a D50 of 15-120 nm, preferably 25-65 nm; a PDI of no more than 0.30, preferably no more than 0.18; an AFM height of 3.5-8.0 nm, preferably 4.0-6.5 nm; a disk-like morphology ratio of no less than 50%, preferably no less than 75%; and a free crystalline polyphenol ratio of no more than 10 wt%, preferably no more than 3 wt%.

[0036] Example 1: Preparation of Continuously Sealed Curcumin Lipid Nanodisk Powder

[0037] Raw materials and proportions

[0038] Continuous-sealed lipid nanodisc powder was prepared using curcumin as a representative hydrophobic polyphenol. Starting with 500.0 mL of ethanol-phase pre-complexing solution, 2.550 g of curcumin and 30.000 g of total lipids were weighed, with a polyphenol to total lipid mass ratio of 0.085:1. The total lipids consisted of 19.800 g of soybean phosphatidylcholine, 4.800 g of hydrogenated soybean phospholipids, 1.800 g of sodium phosphatidylglycerol, 3.450 g of phytosterols, and 0.150 g of mixed tocopherols.

[0039] The total amount of edge stabilizer was 1.560 g, composed of 1.014 g of saponins and 0.546 g of sodium N-lauroyl-L-glutamate, with a mass ratio of edge stabilizer to total lipids of 0.052:1. The ethanol phase consisted of 460.0 mL of anhydrous ethanol and 40.0 mL of deoxygenated purified water, with an ethanol volume fraction of 92.0 vol%. The volume of the first aqueous phase was 1086.2 mL, and the volume of the second aqueous phase was 2794.8 mL. The solidification protectant consisted of 53.730 g of trehalose, 29.850 g of inulin, and 35.820 g of low-DE maltodextrin, with a mass ratio of protectant to non-protective solids in the nanodiscs of 3.50:1.

[0040] Equipment preparation and aqueous phase preparation

[0041] A jacketed 316L stainless steel light-proof mixing tank with an effective volume of 1.0L is used as the ethanol phase tank, featuring an internal anchor-type agitator and a bottom reflux port. The first and second aqueous phases are handled in 2.0L and 5.0L glass-lined or 316L mixing tanks, respectively. The ethanol phase tank, metering pump, and micromixer area are equipped with explosion-proof electrical systems and are grounded, with a grounding resistance not exceeding 10Ω. The oxygen content at the top of the ethanol phase tank is controlled to be no higher than 8.0 vol%, with a process operating target of no higher than 5.0 vol.

[0042] The first micromixer employs a sixteen-channel parallel staggered herringbone micromixing plate. Each channel has an effective width of 280.0 μm, an effective depth of 180.0 μm, a hydraulic diameter of approximately 219.1 μm, and an effective mixing length of 55.0 mm. Each channel contains a continuous staggered herringbone ridge with a height of 22.0% of the channel depth and an angle of 45.0° with the mainstream direction. The period between adjacent ridges is 600.0 μm. The second micromixer employs a forty-eight-channel parallel staggered herringbone micromixing plate. Each channel has an effective width of 320.0 μm, an effective depth of 220.0 μm, a hydraulic diameter of approximately 260.7 μm, and an effective mixing length of 80.0 mm. The pre-complexed ethanol phase, the first aqueous phase, and the second aqueous phase are all distributed via equal-impedance manifolds to ensure that the flow deviation between channels does not exceed ±3.0%. Before use, all feed lines should be flushed with deoxygenated purified water at 38.0℃ for 20.0 min and then purged with nitrogen for 10.0 min.

[0043] The first aqueous phase was a phosphate-citrate buffer system with pH 6.80 and an ionic strength of 35.0 mmol / L. After bubbling with nitrogen for 30.0 min, the dissolved oxygen was reduced to below 0.50 mg / L. The second aqueous phase was a similar buffer system with pH 6.80 and an ionic strength of 35.0 mmol / L. During preparation, 80.0% of the target volume of deoxygenation buffer was added to the second aqueous phase container, the temperature was adjusted to 38.0℃, and low-shear stirring at 150 rpm was used to form a smooth liquid surface. Then, saponins were slowly added along the container wall at a rate of 0.20 g / min below the liquid surface, and stirring continued for 20.0 min until the solution changed from a slightly turbid state to a light yellow and transparent state, with the foam layer thickness not exceeding 5.0 mm.

[0044] Sodium N-lauroyl-L-glutamate was pre-wetted into a slurry using a small amount of 38.0℃ buffer solution, and then pumped into the saponin solution at a rate of 1.0 mL / min. The pH was then adjusted to 6.80 ± 0.05 using 0.10 mol / L citrate buffer. After the second aqueous phase was prepared, it was degassed under vacuum at −0.06 MPa for 8.0 min, then refilled with nitrogen, filtered through a 0.45 μm low-adsorption hydrophilic membrane, and a bubble trap was installed before it entered the second-stage micromixer.

[0045] Ethanol phase pre-complexation

[0046] Add 460.0 mL of anhydrous ethanol and 40.0 mL of deoxygenated purified water to a light-protected ethanol phase container, raise the jacket temperature to 42.0 °C, and set the stirring speed to 350 rpm. After the temperature inside the container stabilizes, add 2.550 g of curcumin. Initially, curcumin forms an orange-yellow powder island on the liquid surface. After about 2.0 min, it is wetted by ethanol and sinks, and the liquid in the container gradually turns into a transparent amber color. After stirring for another 5.0 min, there is no floating fine powder on the liquid surface, and no needle-like crystal flashes are observed when viewed under transmitted light.

[0047] Subsequently, the lipid components were added in the following order: soybean phosphatidylcholine, hydrogenated soybean phospholipids, sodium phosphatidylglycerol, phytosterols, and mixed tocopherols. Upon addition of soybean phosphatidylcholine, the liquid viscosity increased slightly, and the vortex changed from a sharp funnel shape to a blunt, rounded funnel shape. After addition of hydrogenated soybean phospholipids, a brief filamentous tail appeared on the liquid surface, disappearing after approximately 3.0 minutes. Upon addition of phytosterols, the system changed from a transparent amber color to a clear liquid with a slightly golden-yellow refractive index. After all lipids were added, pre-complexation was continued for 12.0 minutes at 42.0℃ and 350 rpm. At the end of the pre-complexation, 1.0 mL of the ethanol phase was placed in a quartz cuvette; it was observed to be a homogeneous, transparent amber liquid. The absorption peak was measured at 420 nm, with a peak position deviation of no more than 1.0 nm for three consecutive measurements.

[0048] First stage of metastable micromixing

[0049] Three high-precision metering pumps were started. The ethanol phase flow rate was set to 12.00 mL / min, the first aqueous phase flow rate was set to 26.07 mL / min, and the total flow rate of the first stage was 38.07 mL / min, which translates to a single-channel flow rate of approximately 2.38 mL / min. The back pressure valve at the micromixer inlet was set to 0.35 MPa, and the jacket temperature of the first-stage micromixer was maintained at 38.0 °C. After the ethanol phase entered the first-stage micromixer, the transparent amber ethanol phase was cut into continuous thin bands by the colorless first aqueous phase, and lateral folds were formed under the induction of staggered herringbone-shaped turbulence ridges. The ethanol volume fraction decreased from 92.0 vol% to 29.0 vol% at the first-stage outlet within 35.0 ms.

[0050] In the first stage of the outlet observation window, the feed liquid changes from a transparent amber color at the inlet to a clear orange-yellow dispersion with a very weak blue Tyndall effect. Short-term fluctuations in online turbidity are permissible within the initial 3.0 minutes of startup; after stabilization, the coefficient of variation of turbidity should not exceed 3.0%. If milky white clumps, sparkling crystals, tailing deposits along the pipe wall, or back pressure pulses exceeding 0.05 MPa appear in the observation window, it is considered localized crystallization or lipid sheet deposition; feed should be stopped and the line switched to the waste liquid branch. The feed liquid obtained in this stage is a lipid-curcumin bilayer precursor, and the edges are not yet completely sealed.

[0051] Second segment of time-series edge sealing

[0052] The first-stage effluent bypasses the storage tank and directly enters the second-stage micro-mixer. The second aqueous phase flow rate is set to 67.08 mL / min, and the total effluent flow rate at the second-stage outlet is approximately 105.15 mL / min. This translates to a single-channel flow rate of approximately 2.19 mL / min for the second stage. The entire batch is processed in approximately 41.7 minutes of continuous operation.

[0053] After the second aqueous phase enters the second-stage micromixer, the outlet liquid's orange-yellow color slightly lightens, and the Tyndall effect changes from the weak blue of the first stage to a stable, delicate pale blue opalescence; the liquid remains transparent. Within 20-40 seconds after the second aqueous phase starts up, the online back pressure rises slightly by 0.015-0.030 MPa, then falls back to a steady state within 60-120 seconds. This back pressure peak serves as a fingerprint of the process by which the edge stabilizer transitions from a free state to an anchored state at the edge of the nanodisc. The pH of the outlet liquid in the second stage is 6.80 ± 0.05. After a 20-fold dilution, a rapid DLS analysis was performed, yielding a D50 of 32-48 nm and a PDI below 0.18.

[0054] Online dilution and TFF desolvation

[0055] The second-stage effluent enters the tangential flow filtration system within 5.0 min. The TFF uses a 100 kDa polyethersulfone flat sheet membrane, with an experimental batch membrane area of ​​0.05 m². Before use, it is pre-washed sequentially with purified water, pH 6.80 buffer, and buffer containing 10.5 vol% ethanol until the permeate conductivity, pH, and UV background are stable. The effluent passes through a 1.2 μm low-adsorption protective filter before entering the TFF, and the effluent temperature is reduced to 25.0 °C.

[0056] When starting the TFF, first circulate at a transmembrane pressure of 0.040 MPa for 5.0 minutes for wettation and venting. After confirming that there are no foam vortices in the circulation tank, increase the transmembrane pressure to 0.080 MPa, and control the cross-flow shear rate as follows: First, dilute online 3.0 times with the same pH buffer solution, then perform 7.0 DV perfiltration using a constant volume perfiltration method until the residual ethanol is below 800 ppm and the free curcumin ratio is below 1.0 wt%. In the initial stage of TFF startup, the permeate is very pale yellow; after 3.0 DV, the color of the permeate becomes significantly lighter; at 7.0 DV, the permeate is basically colorless.

[0057] After TFF (Transient Fluorescence), the dispersion was concentrated to approximately 6.0 wt% of the non-protectant solids. The resulting dispersion was a uniform, orange-yellow, low-viscosity nano-dispersion with a bluish microemulsion. After standing for 30.0 min, no yellow sediment ring was found at the bottom of the container, and no oily floating matter was observed on the surface.

[0058] Addition of glassy preservative and low-temperature spray drying

[0059] Trehalose, inulin, and low-DE maltodextrin were dissolved in 260.0 g of deoxygenated purified water to obtain a clear to slightly milky white protective agent aqueous solution. This protective agent aqueous solution was added to the nanodisc concentrate at a rate of 8.0 mL / min, with stirring at 220 rpm and a temperature of 25.0 °C. Initially, the viscosity of the solution increased slightly after addition, and after approximately 15.0 min, a homogeneous orange-yellow milky liquid was formed.

[0060] Before spray drying, ensure the residual ethanol in the feed solution is no higher than 1000 ppm and adjust the solids content to 16.0-22.0 wt%. Stir the feed solution at 25.0℃ with low shear for 15.0 min, ensuring no persistent foam appears on the surface and no orange-yellow oil rings on the cup walls. The spray drying uses a closed-loop nitrogen system, controlling the oxygen content in the tower below 2.0 vol%, and implementing online interlocking between oxygen content and ethanol vapor. If the oxygen content in the tower exceeds 4.0 vol% or the ethanol concentration in the tail gas abnormally increases, the feed will be automatically cut off and the system will enter nitrogen purging mode.

[0061] Spray drying employs two-fluid atomization, with a fixed inlet air temperature of 98.0℃ and an outlet air temperature controlled at 54.0℃, allowing a control window of 52.0-56.0℃. The atomization pressure is fixed at 0.45MPa, and the feed rate is fixed at 5.50mL / min. During normal atomization, a uniform light orange mist cone forms below the nozzle, with only a very thin layer of dry powder adhering to the tower wall; no wet, shiny streaks should appear. The collected powder is a light orange-yellow, loose, fine powder without oil spots or charring points, with a moisture content of 3.2wt%.

[0062] Reconstitution observation

[0063] Take 1.000g of the obtained powder and add it to 99.000g of purified water. Gently stir magnetically for 5.0min to form a uniform orange-yellow transparent to semi-transparent dispersion. No yellow crystals adhere to the cup wall, and uniform blue Tyndall effect can be seen when irradiated with a laser. The test results of three batches after reconstitution are shown in the experimental example.

[0064] II. Experimental Examples

[0065] Experimental Example 1: Preparation and Performance Testing of Control Samples

[0066] The following uses curcumin as a representative polyphenol to test the performance of the lipid-curcumin pre-complexed driven continuous edge-sealing lipid nanodisc powder prepared in Example 1, and five comparative groups are set up for control. Unless otherwise stated, the total amount of curcumin, lipid matrix and edge stabilizer used in each group is the same as in Example 1.

[0067] Sample preparation

[0068] Example 1 was prepared according to the preparation steps of Example 1 above.

[0069] Comparative Example 1 employed a traditional one-step ethanol injection-ultrasound-rotary evaporation route, omitting lipid-curcumin pre-complexation, two-stage microchannel sealing, and TFF deethanolization. Curcumin, phospholipids, and sterols were dissolved in the ethanol phase and injected into a stirred aqueous reactor at 38.0℃ at a rate of 20.0 mL / min, with a stirring speed of 1200 rpm. This was followed by probe sonication for 6.0 min, batch rotary evaporation for ethanol removal, and lyophilization. Upon entering the aqueous phase, an orange-yellow plume appeared around the inlet, with the plume edges rapidly turning white. After approximately 30 seconds, the clear yellow liquid in the reactor transformed into a milky-yellow coarse dispersion. During the sonication stage, fine foam and localized dark yellow ring-shaped deposits appeared on the surface, and the system temperature rose from 38.0℃ to approximately 46.5℃. During rotary evaporation, a thin yellow film formed on the flask wall, and after lyophilization and reconstitution, small yellow crystals were visible on the flask wall.

[0070] Comparative Example 2 retained the two-stage microchannel, second-stage edge sealing, TFF, and solidification steps, but omitted the lipid-curcumin pre-complexation stage at 42.0℃ for 12.0 min. After curcumin and lipids reached visual dissolution in the ethanol phase, they directly entered the first-stage micromixer. After entering the first-stage micromixer, intermittent fine bright spots appeared in the outlet observation window, and the online turbidity curve showed a sharp peak with slight upward fluctuations every 20-40 seconds. After adding the edge stabilizer in the second stage, the liquid showed a blue opalescence, but the opalescence contained a slight grayish-white haze.

[0071] Comparative Example 3 retained the pre-complexed formulation, edge stabilizer composition, and total feed amount, but eliminated the two-stage microchannel. Instead, the first aqueous phase was added dropwise in a jacketed stirred tank, followed by the second aqueous phase. The subsequent TFF desolventizing and glassy protective agent solidification steps were the same as in Example 1. The first aqueous phase was added dropwise at 26.0 mL / min, and the stirring speed was increased to 1500 rpm. When the first aqueous phase was added, a milky white hydration cloud formed below the feed point on the liquid surface. The cloud was pulled into a ribbon shape by the impeller and diffused throughout the tank after about 60 seconds. Inside the tank, localized areas of concentrated orange-yellow and milky white turbidity alternated and rolled. In the subsequent TFF stage, the membrane pressure increased, and the permeate became distinctly yellow.

[0072] Comparative Example 4 retained the microchannel device and overall formulation, but all saponins and N-lauroyl-L-glutamate were pre-dissolved in the first aqueous phase, allowing the first-stage hydration and edge-sealing agent contact to occur simultaneously, thus eliminating the process sequence of the first-stage hydration without edge-sealing agent and the second-stage sequential edge-sealing. During the preparation process, the foam on the surface of the first aqueous phase tank increased significantly, and the first-stage outlet exhibited a strong but not fine opalescence, displaying a bluish-white light. After dilution in the second stage, no clear edge-sealing optical transition was observed, and SEC-DLS analysis revealed the coexistence of small-diameter micelle peaks and nanodisc peaks.

[0073] The material composition, pre-complexation, microchannel, time-sequential sealing, TFF, and solidification steps of Comparative Example 5 were the same as those of Example 1, except that the volume fraction of ethanol at the first outlet was reduced from 29.0 vol% to 16.0 vol%, and the aqueous phase flow rate was adjusted to make the volume fraction of ethanol at the second outlet close to 10.5 vol%. In the observation window at the first outlet, the feed liquid did not show a delicate pale blue milky sheen, but instead exhibited a brief orange smoky turbidity, with localized whitening at the hydration front accompanied by tiny bright spots flashing; the online turbidity could not be stabilized after startup, with the maximum peak value being approximately 2.8 times that of Example 1.

[0074] (II) Testing Methods

[0075] Table 1 Test Methods

[0076]

[0077] (III) Test Results

[0078] The test results for each group are shown in the table below. The data are expressed as mean ± standard deviation of the three batches. Comparative Example 1 did not use TFF desolventizing, so the TFF flux retention rate and permeate water consumption per unit of non-protective agent solids are expressed as "not using TFF". Although the two-stage microchannel was cancelled in Comparative Example 3, it was still processed according to the subsequent TFF and glass solidification steps of Example 1.

[0079] Table 2. Technological Process and Industrial Operation Indicators

[0080]

[0081] Table 3. Indicators of Industrialization Costs

[0082]

[0083] Table 4 Terminal Structure Indicators (I)

[0084]

[0085] Table 5 Terminal Structure Indicators (II)

[0086]

[0087] Table 6 Release, Challenge Stability, and Shelf Stability Indicators

[0088]

[0089] Results Analysis

[0090] As can be seen from Tables 2 to 6, Example 1 is not superior to the comparative example in all industrial indicators. However, it has advantages in core indicators such as low crystal residue, preservation of disc-shaped morphology, low initial burst release, resistance to Fe³⁺ challenge and resolution stability. At the same time, it pays the industrialization cost of higher percolation water consumption, lower measured content of curcumin in powder and certain solid loss.

[0091] Compared with the conventional one-step ethanol injection-ultrasound-rotary evaporation route of Comparative Example 1, the D50 of the sample obtained in Example 1 decreased from 164.9 nm to 41.7 nm, the PDI decreased from 0.397 to 0.128, the proportion of disk-shaped morphology in TEM increased from 9.6% to 82.6%, and the proportion of residual curcumin crystals in PXRD decreased from 33.9% to 3.2%. These results indicate that Example 1 can reduce the residual free curcumin crystals and form a higher proportion of disk-shaped nanostructures.

[0092] Regarding release performance, the 2-hour release rate of Example 1 was 18.9%, lower than the 49.6% of Comparative Example 1; the 24-hour release rate of Example 1 was 58.7%, indicating that it has low initial burst release and sustained release characteristics. After the Fe³⁺ challenge, the curcumin retention rate of Example 1 was 84.3%, higher than the 44.9% of Comparative Example 1; after storage at 40℃ / 75%RH for 30 days, the D50 growth rate of reconstituted Example 1 was 14.8%, lower than the 188.5% of Comparative Example 1.

[0093] In Comparative Example 2, after removing the lipid-curcumin pre-complexation, the residual peak of PXRD crystals increased to 16.7%, and the 2-hour release rate increased to 35.8%. In Comparative Example 3, after removing the two-stage microchannels, the D50 increased to 203.5 nm, the PDI increased to 0.461, and the TFF flux retention decreased to 41.8%. In Comparative Example 4, after adding the edge stabilizer to the first aqueous phase in advance, although the D50 was smaller, the 2-hour release rate increased to 42.1%, and the proportion of TEM disk-shaped morphology was only 48.2%. In Comparative Example 5, after reducing the volume fraction of ethanol at the first-stage outlet to 16.0 vol%, the residual peak of PXRD crystals increased to 42.8%, and the 2-hour release rate increased to 53.7%.

[0094] The above comparative results indicate that lipid-polyphenol pre-complexation, the first stage of metastable micromixing, the second stage of time-sequential edge sealing, and the first stage of ethanol volume fraction control collectively affect the structure retention, free crystal residue, and sustained-release performance of lipid nanodisks.

[0095] The above description is merely a specific embodiment of this application, but the scope of protection of this application is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in this application should be included within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of the claims.

Claims

1. A lipid nanodisc for polyphenol sustained-release protection, characterized in that, The formulation includes a polyphenolic active ingredient, a lipid bilayer matrix, and a composite edge stabilizer. The polyphenolic active ingredient forms a lipid-polyphenol pre-complexed structure with the lipid bilayer matrix, and the composite edge stabilizer seals the open edges of the lipid bilayer matrix. The lipid bilayer matrix includes phosphatidylcholine lipids, hydrogenated phospholipids or saturated phospholipids, anionic phospholipids, and sterol membrane rigidity modifiers. The composite edge stabilizer includes saponin-based edge sealing agents and N-acyl amino acid salt amphiphilic edge sealing agents. The lipid nanodisks have a D50 of 15-120 nm, a PDI of no more than 0.30, and an AFM height of 3.5-8.0 nm.

2. The lipid nanodisc for polyphenol sustained-release protection according to claim 1, characterized in that, The polyphenolic active ingredient is selected from one or more of curcumin, resveratrol, quercetin, catechin, epigallocatechin gallate, tea polyphenols, anthocyanins, chlorogenic acid, ferulic acid, and gallic acid derivatives; the mass ratio of the polyphenolic active ingredient to total lipids is 0.02:1-0.25:

1.

3. The lipid nanodisc for polyphenol sustained-release protection according to claim 1, characterized in that, Based on the total molar amount of lipid components in the lipid bilayer matrix, the phosphatidylcholine lipids are 45-92 mol%, the hydrogenated phospholipids or saturated phospholipids are 0.1-35 mol%, the anionic phospholipids are 0.5-18 mol%, and the sterol membrane rigidity regulators are 2-28 mol%; the lipid bilayer matrix also includes antioxidant lipids, which are 0.02-2.0 wt% of the total lipid mass.

4. The lipid nanodisc for polyphenol sustained-release protection according to claim 1, characterized in that, The mass ratio of the composite edge stabilizer to total lipids is 0.015:1-0.12:1; the saponin-based edge sealer accounts for 20-95 wt% of the mass of the composite edge stabilizer, and the N-acyl amino acid salt amphiphilic edge sealer accounts for 5-80 wt% of the mass of the composite edge stabilizer.

5. The lipid nanodisc for polyphenol sustained-release protection according to claim 1, characterized in that, The proportion of free crystalline polyphenols in the lipid nanodiscs is no more than 10 wt%, and the proportion of disk-shaped morphology is no less than 50%. In the powder containing the lipid nanodiscs and the glassy protective agent, the glassy protective agent includes one or more of trehalose, inulin, low DE maltodextrin, gum arabic, whey protein or modified starch, and the mass ratio of the glassy protective agent to the solids of the non-protective agent of the nanodiscs is 1.5:1-8.0:

1.

6. A method for preparing lipid nanodiscs for polyphenol sustained-release protection, characterized in that, include: The polyphenol active ingredient and the lipid components used to form the lipid bilayer matrix were dissolved in the ethanol phase to obtain a lipid-polyphenol pre-complexed solution. The lipid-polyphenol pre-complexed solution is subjected to a first stage of metastable micro-mixing with a first aqueous phase, without the addition of a composite edge stabilizer, so that the ethanol volume fraction at the first stage outlet is 18-42 vol%, and the first stage mixing time is 10-180 ms; the first stage outlet solution is subjected to a second stage of sequential edge-sealing micro-mixing with a second aqueous phase containing a composite edge stabilizer to obtain a lipid nanodisc dispersion; the lipid nanodisc dispersion is subjected to tangential flow filtration to remove solvent by constant volume percolation.

7. The method for preparing lipid nanodisks for polyphenol sustained-release protection according to claim 6, characterized in that, The lipid-polyphenol pre-complexing solution contains 82-96% ethanol, 10-180 mg / mL total lipids, and 0.2-35 mg / mL polyphenols. The pre-complexing temperature is 30-52℃, the pre-complexing time is 5-30 min, and the dissolved oxygen is not higher than 1.5 mg / L.

8. The method for preparing lipid nanodisks for polyphenol sustained-release protection according to claim 6, characterized in that, The volumetric flow rate ratio of the first aqueous phase to the lipid-polyphenol pre-complexed solution is 1.2:1-5.5:1; the volume ratio of the second aqueous phase to the first stage outlet liquid is 0.5:1-6.0:1; the volume fraction of ethanol at the second stage outlet is 5-20 vol%; the mixing time of the second stage is 10-500 ms; and the pH of the second stage is 5.5-8.

2.

9. The method for preparing lipid nanodisks for polyphenol sustained-release protection according to claim 6, characterized in that, The first stage of metastable micromixing uses a 16-channel parallel staggered herringbone micromixing plate, with each channel having an effective width of 280.0 μm, an effective depth of 180.0 μm, and a hydraulic diameter of 219.1 μm. The second stage of time-sequential sealing micromixing uses a 48-channel parallel staggered herringbone micromixing plate, with each channel having an effective width of 320.0 μm, an effective depth of 220.0 μm, and a hydraulic diameter of 260.7 μm. The pre-complexed ethanol phase, the first aqueous phase, and the second aqueous phase are distributed via an equal-impedance shunt manifold, with the flow deviation between channels not exceeding ±3.0%.

10. The method for preparing lipid nanodisks for polyphenol sustained-release protection according to claim 6, characterized in that, The tangential flow filtration desolvation uses a membrane with a molecular weight cutoff of 30-500 kDa, a transmembrane pressure of 0.03-0.16 MPa, and a cross-flow shear rate of [missing information]. The percolation volume was 3-15 DV; after tangential flow filtration and desolventization, the obtained nanodisc dispersion was compounded with a glassy protective agent and then freeze-dried or subjected to low-temperature nitrogen closed-circuit spray drying.

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