Anti-aging composition based on pn and natural extracts and its enhancement process

Abstract

The application belongs to the technical field of biological medicine manufacturing, and discloses an anti-aging composition based on PN and natural extract and an enhanced process thereof, which comprises: establishing a fluid mixing domain, so that a first phase fluid is injected into a continuously flowing second phase fluid according to a dynamic volume flow model, and the Reynolds number of the mixed fluid in the fluid mixing domain is maintained at 2800-4500; the mixed solvent dielectric constant at the injection interface is reduced, and the Coulomb force of the divalent metal cation on the polar group of the natural extract is amplified, so that the natural extract in a supersaturated state is induced to occur limited heterogeneous nucleation on the surface of the polynucleotide molecular chain, and a core-shell coated structure is constructed; the core-shell coated structure is introduced into an aging flow channel, and the mixed liquid in the aging flow channel is controlled to be lowered to a final locking temperature at a cooling rate of 0.5-1.5 DEG C / min; the application avoids irreversible physical rupture of the polynucleotide phosphodiester bond, maintains the complete macromolecular chain retention rate at more than 95%, and blocks the coalescence and coarsening process between microparticles.

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceutical manufacturing technology, and particularly relates to an anti-aging composition based on PN and natural extracts and its enhancement process. Background Technology

[0002] In the current context of the synergistic effects of polynucleotide and natural extract combinations in anti-aging and tissue repair, such combinations contain biomacromolecules with specific spatial conformations and natural extracts with antioxidant effects, and their activity depends on the microscopic distribution and molecular structural integrity of the components. For such sensitive systems, the industry usually adopts the method of mixing aqueous solutions of polynucleotides with organic solutions of natural extracts and using the high-intensity physical shear force of homogenizing equipment to achieve component dispersion. However, biopharmaceutical preparation processes face conflicts in physical properties: the high input energy required to maintain the homogeneous distribution of polar components will generate fluid cavitation effect, and this effect will cause physical damage to the phosphodiester bonds of polynucleotides, causing molecular chain breakage.

[0003] Besides hardware limitations, a lack of microkinetic window locking in the process control logic can lead to inaccurate formulation performance. For example, Chinese invention patent application CN107205931A discloses a method for preparing submicron-scale amorphous solid dispersions through co-precipitation, utilizing microfluidics to achieve micro-mixing of active ingredients and stabilizers within microchannels. However, this approach relies on the intense interactions under high pressure (34.5 MPa to 350 MPa). While this improves the mixing efficiency of small molecule drugs, it is problematic for flexible long-chain macromolecules like polynucleotides with ultra-high molecular weights and shear sensitivity. The high-energy shear field induces... Phosphodiester bonds break randomly. To avoid mechanical damage, existing processes attempt to use an antisolvent precipitation path, inducing extract precipitation by changing the polarity of the solvent environment. However, during fluid mixing, the solvent dielectric constant decreases rapidly, causing the supersaturated extract to exhibit a tendency for homogeneous nucleation. Due to the lack of directional traction at the microscopic level, active molecules exhibit random distribution and self-aggregation, forming a large number of free aggregates. This prevents the formation of a continuous and dense physical coating layer on the surface of long polynucleotide chains. This disorder in the microscopic phase structure weakens the pharmacological activity of the composition, leading to phase separation during storage and restricting the stability of the biological agent.

[0004] Therefore, the core technical problem to be solved by this invention is how to achieve the directional assembly of different polar components at the liquid interface while maintaining the conformational integrity of biomacromolecules, thereby resolving the inherent contradiction between the retention of polynucleotide activity and the thermodynamic stability of the formulation. Summary of the Invention

[0005] In this technical solution, an enhancement process for an anti-aging composition based on PN and natural extracts includes the following steps: Step S101: Prepare a first phase fluid and a second phase fluid; the first phase fluid includes a natural extract and an organic solvent having a first dielectric constant; the second phase fluid includes a polynucleotide, an aqueous buffer having a second dielectric constant, and a divalent metal cation, wherein the molar concentration of the divalent metal cation is from 0.8 mmol / L to 1.5 mmol / L. Step S102: Establish a fluid mixing domain with a microchannel collision structure. The first-phase fluid is injected into the continuously flowing second-phase fluid via a micro-jet flow with a collision angle of 120° to 160°, based on a dynamic volumetric flow rate model, maintaining the Reynolds number of the mixed fluid within the mixing domain at 2800 to 4500. The physical scale of the fluid mixing domain restricts the micro-vortex scale of the mixed fluid. Radius of gyration of polynucleotide molecular chains Satisfying the mathematical relation is The dynamic volumetric flow rate model constrains the injection parameters of the first-phase fluid, ensuring that the ratio of the volumetric flow rate of the first-phase fluid to that of the second-phase fluid, multiplied by the 0.5th power of the ratio of their dynamic viscosities, is between 0.05 and 0.15. Step S103: The decrease in dielectric constant of the mixed solvent at the injection interface, combined with the amplification of the Coulomb attraction of the divalent metal cations on the polar groups of the natural extract, induces the supersaturated natural extract to undergo restricted heterogeneous nucleation on the surface of the polynucleotide molecular chain, thus constructing a core-shell encapsulation structure. In step S104, the core-shell coating structure is introduced into the aging channel, and the mixture in the aging channel is controlled to decrease at a cooling rate of 0.5℃ / min to 1.5℃ / min until the final lock-in temperature.

[0006] Preferably, step S101 includes: step S1011, dissolving the natural extract in an organic solvent, controlling the mass concentration of the natural extract to be from 10 mg / mL to 30 mg / mL, wherein the organic solvent is selected from one or more of ethanol, propylene glycol or polyol; step S1012, dissolving the polynucleotide in an aqueous buffer, controlling the mass concentration of the polynucleotide to be from 1.5 mg / mL to 5.0 mg / mL, and adding an inorganic salt that provides a divalent metal cation.

[0007] Preferably, in step S101: the divalent metal cation is selected from one or more of magnesium ions, zinc ions, or calcium ions; by utilizing the coordination of the divalent metal cation with the polynucleotide phosphate backbone, a local positive charge electrostatic enrichment domain is constructed along the polynucleotide molecular chain segment, providing electrostatic capture sites for the natural extract.

[0008] Preferably, step S102 includes: step S1021, adjusting the feed pressure ratio of the first phase fluid to the second phase fluid to 1:2 to 1:5; step S1022, utilizing a low-energy kinetic window with a Reynolds number of 2800 to 4500 to avoid the fluid cavitation field and reduce local shear stress in order to preserve the integrity of the phosphodiester bonds of the polynucleotide; wherein, through the coupling constraint of pressure ratio and Reynolds number, the local temperature rise in the fluid mixing domain is controlled to not exceed 3°C.

[0009] Preferably, in step S104: the aspect ratio of the aging flow channel is controlled to be 10:1 to 50:1; the cooling rate is used to guide the orderly fixation of the hydrogen bond network inside the core-shell coating structure and suppress the coarsening trend of the self-assembled system.

[0010] Preferably, the natural extract is selected from plant polyphenols or flavonoids; the weight-average molecular weight of the polynucleotide is not less than 1000 kDa; and the average particle size of the generated self-assembled microparticles is 150 nm to 350 nm.

[0011] Preferably, in step S101: the pH value of the second phase fluid is controlled to be 6.2 to 7.2; the charge state of the polynucleotide molecular chain at the pH value is used to adjust the conformation of the polynucleotide in conjunction with the divalent metal cation to produce steric contraction.

[0012] Preferably, in step S102: the micro-vortex scale of the fluid mixing domain is controlled to be 1.5 to 3.0 times the radius of rotation of the polynucleotide molecular chain based on the Reynolds number criterion; the difference between the fluid micro-mixing characteristic time and the homogeneous nucleation induction time of the natural extract is used to achieve directional pinning of the natural extract on the polynucleotide surface.

[0013] Preferably, step S103 includes: introducing a nonionic surfactant at a mass percentage of 0.1% to 0.5% into the first phase fluid to adjust the interfacial tension between the natural extract and the polynucleotide.

[0014] An anti-aging composition based on PN and natural extracts, the anti-aging composition being prepared by an enhanced process of an anti-aging composition based on PN and natural extracts.

[0015] Compared to existing technologies, the enhanced process of the anti-aging composition based on PN and natural extracts in this invention has the following beneficial effects: Firstly, this invention enables the microscopic directional assembly and core-shell topology construction of components. By pre-setting positively charged electrostatic enrichment domains in the polynucleotide (PN) phosphate backbone and utilizing the transient dielectric constant drop induced by organic phase injection, the invention induces restricted heterogeneous nucleation of polar components within a nanosecond time window. This process forcibly pins natural extracts to the surface of the polynucleotide chain, blocking homogeneous self-nucleation and disordered aggregation at the microscopic level, and constructing a defined core-shell structure. This solves the industry dilemma of different polar components being unable to form a homogeneous and stable system.

[0016] Secondly, it effectively preserves the structural integrity and activity of biomacromolecules. It adopts a low Reynolds number (2800 to 4500) fluid mixing method constrained by a dynamic volumetric flow rate model, and locks the kinetic window with the critical instability constant. This allows the process to completely avoid fluid cavitation fields and local hot spots, which avoids irreversible physical breakage of polynucleotide phosphodiester bonds and maintains the integrity retention rate of macromolecular chains at over 95%, significantly improving the bioactivity retention level of the composition.

[0017] Third, it improves the long-term thermodynamic stability of the formulation and reduces the dependence on additives. An aging flow channel with gradient cooling control (0.5℃ / min to 1.5℃ / min) is introduced to guide the orderly fixation of the hydrogen bond network inside the self-assembled particles. The kinetic energy gradient decay is used to block the aggregation and coarsening process between particles. The generated system has a thermodynamic macroscopic stability of up to 24 months at 25℃ and effectively reduces the dependence on inactive surfactants, which meets the high-purity industrial pharmaceutical standards. Attached Figure Description

[0018] Figure 1 This is a flow chart of the core-shell reinforcement preparation process of PN and natural extracts according to the present invention; Figure 2 This is a diagram showing the evolution of the microscopic topology construction and stability locking state of the composition of the present invention. Detailed Implementation

[0019] The technical solutions of the embodiments of this application will be clearly described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of this application are within the scope of protection of this application.

[0020] An enhancement process for an anti-aging composition based on PN and natural extracts includes the following steps: Step S101: Prepare a first phase fluid and a second phase fluid; the first phase fluid includes a natural extract and an organic solvent having a first dielectric constant; the second phase fluid includes a polynucleotide, an aqueous buffer having a second dielectric constant, and a divalent metal cation, wherein the molar concentration of the divalent metal cation is from 0.8 mmol / L to 1.5 mmol / L. Step S102: Establish a fluid mixing domain with a microchannel collision structure. The first-phase fluid is injected into the continuously flowing second-phase fluid via a micro-jet flow with a collision angle of 120° to 160°, based on a dynamic volumetric flow rate model, maintaining the Reynolds number of the mixed fluid within the mixing domain at 2800 to 4500. The physical scale of the fluid mixing domain restricts the micro-vortex scale of the mixed fluid. Radius of gyration of polynucleotide molecular chains Satisfying the mathematical relation is The dynamic volumetric flow rate model constrains the injection parameters of the first-phase fluid, ensuring that the ratio of the volumetric flow rate of the first-phase fluid to that of the second-phase fluid, multiplied by the 0.5th power of the ratio of their dynamic viscosities, is between 0.05 and 0.15. Step S103: The decrease in dielectric constant of the mixed solvent at the injection interface, combined with the amplification of the Coulomb attraction of the divalent metal cations on the polar groups of the natural extract, induces the supersaturated natural extract to undergo restricted heterogeneous nucleation on the surface of the polynucleotide molecular chain, thus constructing a core-shell encapsulation structure. In step S104, the core-shell coating structure is introduced into the aging channel, and the mixture in the aging channel is controlled to decrease at a cooling rate of 0.5℃ / min to 1.5℃ / min until the final lock-in temperature.

[0021] Preferably, step S101 includes: step S1011, dissolving the natural extract in an organic solvent, controlling the mass concentration of the natural extract to be from 10 mg / mL to 30 mg / mL, wherein the organic solvent is selected from one or more of ethanol, propylene glycol or polyol; step S1012, dissolving the polynucleotide in an aqueous buffer, controlling the mass concentration of the polynucleotide to be from 1.5 mg / mL to 5.0 mg / mL, and adding an inorganic salt that provides a divalent metal cation.

[0022] Preferably, in step S101: the divalent metal cation is selected from one or more of magnesium ions, zinc ions, or calcium ions; by utilizing the coordination of the divalent metal cation with the polynucleotide phosphate backbone, a local positive charge electrostatic enrichment domain is constructed along the polynucleotide molecular chain segment, providing electrostatic capture sites for the natural extract.

[0023] Preferably, step S102 includes: step S1021, adjusting the feed pressure ratio of the first phase fluid to the second phase fluid to 1:2 to 1:5; step S1022, utilizing a low-energy kinetic window with a Reynolds number of 2800 to 4500 to avoid the fluid cavitation field and reduce local shear stress in order to preserve the integrity of the phosphodiester bonds of the polynucleotide; wherein, through the coupling constraint of pressure ratio and Reynolds number, the local temperature rise in the fluid mixing domain is controlled to not exceed 3°C.

[0024] Preferably, in step S104: the aspect ratio of the aging flow channel is controlled to be 10:1 to 50:1; the cooling rate is used to guide the orderly fixation of the hydrogen bond network inside the core-shell coating structure and suppress the coarsening trend of the self-assembled system.

[0025] Preferably, the natural extract is selected from plant polyphenols or flavonoids; the weight-average molecular weight of the polynucleotide is not less than 1000 kDa; and the average particle size of the generated self-assembled microparticles is 150 nm to 350 nm.

[0026] Preferably, in step S101: the pH value of the second phase fluid is controlled to be 6.2 to 7.2; the charge state of the polynucleotide molecular chain at the pH value is used to adjust the conformation of the polynucleotide in conjunction with the divalent metal cation to produce steric contraction.

[0027] Preferably, in step S102: the micro-vortex scale of the fluid mixing domain is controlled to be 1.5 to 3.0 times the radius of rotation of the polynucleotide molecular chain based on the Reynolds number criterion; the difference between the fluid micro-mixing characteristic time and the homogeneous nucleation induction time of the natural extract is used to achieve directional pinning of the natural extract on the polynucleotide surface.

[0028] Preferably, step S103 includes: introducing a nonionic surfactant at a mass percentage of 0.1% to 0.5% into the first phase fluid to adjust the interfacial tension between the natural extract and the polynucleotide.

[0029] Preferably, an anti-aging composition based on PN and natural extracts is prepared by an enhancement process of an anti-aging composition based on PN and natural extracts.

[0030] Example 1: In the industrial-scale preparation of medical-grade high-activity polynucleotide formulations, due to the physical breakage of long polynucleotide chains under mechanical homogenization, and the random self-aggregation of natural extract components due to polarity differences, the industry generally uses high-intensity mechanical shear fields exceeding 20,000 rpm to achieve component dispersion. The resulting fluid cavitation effect causes physical damage to the phosphodiester bonds of polynucleotides. Furthermore, in the local dynamics process during the initial stage of antisolvent mixing, the lack of directional traction constraint leads to homogeneous self-nucleation of the supersaturated natural extracts, forming free aggregates and failing to build a dense physical protective layer on the surface of the polynucleotide molecular chain.

[0031] This technical solution implements an enhanced process to prepare a first-phase fluid and a second-phase fluid. The first-phase fluid includes a natural extract with a mass concentration of 10 mg / mL to 30 mg / mL and an organic solvent selected from ethanol, propylene glycol, or polyols. The second-phase fluid includes a polynucleotide with a mass concentration of 1.5 mg / mL to 5.0 mg / mL, an aqueous buffer solution with a pH of 6.2 to 7.2, and an inorganic salt providing divalent metal cations. When preparing the second-phase fluid, the molar concentration of the divalent metal cations is controlled to be 0.8 mmol / L to 1.5 mmol / L, utilizing the interaction between the divalent metal cations and polynucleotides... The coordination of the acid-phosphate backbone generates positively charged electrostatic enrichment domains along the polynucleotide molecular chain, providing electrostatic capture sites for natural extracts and inducing spatial conformational contraction of the long polynucleotide chain. This establishes a fluid mixing domain, and the feed pressure ratio of the first-phase fluid to the second-phase fluid is adjusted to 1:2 to 1:5. The first-phase fluid is then injected into the continuously flowing second-phase fluid according to a dynamic volumetric flow rate model, maintaining the Reynolds number of the mixed fluid within the mixing domain at 2800 to 4500. Simultaneously, the injection parameters of the first-phase and second-phase fluids are controlled to satisfy the following characteristic correlation: K = (Q1 / Q2) × (μ1 / μ2). 0.5 Where Q1 is the volumetric flow rate of the first phase fluid, Q2 is the volumetric flow rate of the second phase fluid, μ1 is the dynamic viscosity of the first phase fluid, μ2 is the dynamic viscosity of the second phase fluid, and parameter K is the critical instability constant of the composition system, which takes a value between 0.05 and 0.15.

[0032] Within the fluid mixing domain, the decrease in dielectric constant of the mixed solvent at the injection interface, combined with the amplification of the Coulomb attraction of divalent metal cations to the polar groups of the natural extract, results in a local mixing characteristic time of the first-phase fluid that is shorter than the homogeneous nucleation induction time of the natural extract. This induces the supersaturated natural extract to be captured and pinned to the surface of the polynucleotide molecular chain by the amplified electrostatic attraction before independent nucleation, forming a core-shell coating structure. This generates self-assembled microparticles with an average particle size of 150 nm to 350 nm. The generated self-assembled microparticles are introduced into an aging channel with an aspect ratio of 10:1 to 50:1 and a temperature gradient. The mixture in the aging channel is controlled to cool down to the lock-in temperature at a rate of 0.5 °C / min to 1.5 °C / min. Through the gradual decay of kinetic energy, the hydrogen bond network inside the core-shell coating structure is orderly fixed, eliminating the risk of uneven growth of microparticles. This increases the molecular weight integrity retention rate of the polynucleotide from less than 70% to more than 95%, and the composition exhibits overall thermodynamic stability for 24 months at 25 °C.

[0033] Example 2: The current experiment was conducted on a microfluidic continuous synthesis platform equipped with online pressure monitoring and a pressure acquisition resolution of 0.01 MPa to verify the effect of the divalent metal cation-induced directional pinning mechanism on the integrity of polynucleotide molecular chains and the stability of self-assembled microparticles. The experimental platform integrated a dynamic flow meter with a sampling frequency of 100 Hz to monitor material fluctuations in the fluid mixing domain in real time. The core parameter Reynolds number Re was set to balance the update rate of the mixing interface with the mechanical damage of fluid shear stress to biomacromolecules. When Re is in the range of 2800 to 4500, the system generates microturbulence sufficient to cover the thickness of the diffusion layer to improve mass transfer efficiency, while avoiding the breakage of polynucleotide phosphodiester bonds caused by excessively high local shear stress. 3512 in the mixing condition was selected as the baseline parameter point.

[0034] For the injection logic of the first-phase and second-phase fluids, a critical instability constant K of 0.11 was selected for testing. This parameter value considers the interfacial instability kinetics and droplet breakup equilibrium during solvent displacement. When the K value is between 0.05 and 0.15, the first-phase fluid generates a jet interface with a specific curvature upon entering the second-phase fluid. The molar concentration of divalent metal cations in the second-phase fluid was controlled at 1.2 mmol / L to serve as nucleation anchoring centers. The potential difference was used to induce spatial conformational contraction of the long polynucleotide chain and construct a positive charge enrichment domain along the molecular chain. The control sample did not contain divalent metal cations. The cations generate disordered aggregates with an average particle size of 1052.4 nm at the mixing interface. The present invention utilizes the coordination of divalent metal cations with the polynucleotide phosphate backbone to shorten the micro-mixing characteristic time to 8.2 ms. This timescale is less than the homogeneous nucleation induction time of the natural extract at the current supersaturation level (25.6 ms). This guides the supersaturated natural extract to be captured and pinned to the polynucleotide molecular chain surface by amplified Coulomb attraction before independent nucleation. In this physical process, the 8.2 ms micro-mixing characteristic time refers to the time it takes for the first-phase fluid solvent to diffuse into the second-phase fluid. The timescale required for critical supersaturation, while the nanosecond time window refers to the instantaneous induction period during which a supersaturated solute overcomes the nucleation energy barrier and undergoes a phase transition. This invention utilizes a high charge density region pre-constructed on a polynucleotide chain by divalent metal cations to significantly reduce the chemical potential energy required for heterogeneous nucleation. This allows natural extract molecules to trigger nanosecond-level confined nucleation within a micro-region the instantaneous moment of reaching supersaturation, ensuring that the nucleation event is confined to the diffusion layer thickness in time series. This solves the spatiotemporal coupling problem between macroscopic mixing lag and microscopic rapid precipitation. A parallel competitive reaction model is used to determine the microscopic mixing characteristic time, thus promoting mixing... The system is injected with reference solutions containing potassium iodide and potassium iodate, as well as a test solution containing hydrochloric acid. The absorbance of the effluent is measured using a spectrophotometer, and the extraction is calculated based on the reaction kinetic constant. The homogeneous nucleation induction time is calibrated using the stop-flow spectroscopy method. A dynamic light scattering probe records the abrupt change in the scattered light intensity of the supersaturated natural extract solution, and the time corresponding to this abrupt change is extracted as the homogeneous nucleation induction time. The microchannel inlet diameter in the fluid mixing domain is set to be 0.3 mm to 0.8 mm, and the first-phase fluid and the second-phase fluid collide and merge at an angle of 120 degrees to 160 degrees. The above geometric dimensions, combined with the fluid dynamic viscosity, form the basis for calculating the critical instability constant.

[0035] The particle size distribution and stability indicators under different operating conditions were monitored using a dynamic light scattering instrument. Data showed that when the concentration of divalent metal cations was 0.6 mmol / L, insufficient charge density to support the construction of a continuous coating layer led to uneven growth of particles within the aging channel, increasing the average particle size from the initial 210.5 nm to 582.4 nm. When the concentration was set at 1.8 mmol / L, excessive ionic strength triggered a salting-out effect of polynucleotides, resulting in flocculent precipitation and a rapid expansion of the particle size distribution, with the polydispersity index (PDI) increasing to 0.42. Within the 0.8 mmol / L to 1.5 mmol / L window defined in this invention, by controlling the cooling rate of the aging channel at 1.0 °C / min, the average particle size of the core-shell coating structure remained stable at 245.8 nm, and the PDI was [not specified]. 0.12; The measured intact retention rate of the polynucleotide molecular weight was 96.5%, which is different from the 68.2% obtained by the traditional 20,000 rpm high-intensity mechanical shearing process; To verify the contribution of the directional pinning mechanism of this invention to the long-term stability of the formulation, the system initiated a stability simulation evaluation based on the Arrhenius kinetic model, which involved placing the prepared composition in a thermally accelerated aging environment at 40°C and a relative humidity of 75% ± 5% for 90 days; According to chemical kinetic calculations, this accelerated condition is equivalent to storing the composition at room temperature of 25°C for 24 months. The test results showed that the decrease rate of the effective ingredient content after equivalent storage of 24 months was only 3.1%, and the weight-average molecular weight retention level of the polynucleotide remained stable; This data not only confirms the physical protective effect of the core-shell coating structure on the active component, but also reflects the excellent thermodynamic locking state of this system.

[0036] The method for determining key dynamic time parameters in this invention is limited as follows: micro-mixing characteristic time ( The parallel competitive reaction assay procedure employs a modified iodate-iodide redox competitive reaction system. A reference solution containing potassium iodide, potassium iodate, and borate buffer is used as the second phase, while sulfuric acid is injected as the test solution into the first phase. The physical process occurs within a fluid mixing domain. If the micro-mixing rate is fast, the acid is rapidly diluted, resulting only in a neutralization reaction; if mixing is delayed, localized excess acid will trigger side reactions producing iodine molecules. ), which then combines with excess iodide ions to form triiodide anions ( Quantitative extraction is performed by continuously monitoring the mixture at the outlet using a UV-Vis spectrophotometer. exist Absorbance at wavelength, calculate the segregation index By combining the flow model of this specific mixer, the micro-mixing characteristic time at the current Reynolds number is calculated. The measurements obtained in the embodiments of the present invention The fluctuation range should be between 5ms and 15ms; the homogeneous nucleation induction time of natural extracts ( The stop-flow spectral calibration procedure is as follows: using a stop-flow analysis device equipped with a high-sensitivity dynamic light scattering (DLS) probe, the operation steps are to introduce an organic solution containing natural extracts (first phase) and an aqueous buffer solution without polynucleotides (second phase) into the stop-flow mixing chamber at a preset ratio, and lock the fluid instantly after mixing is completed, the DLS probe... The system's scattered light intensity is monitored in real time at a sampling rate of times per second. The time interval from the start of fluid mixing to the appearance of the first characteristic jump point on the scattered light intensity curve is recorded. This time interval is defined as the homogeneous nucleation induction time at that specific supersaturation. The technical objective is to ensure, by adjusting the Reynolds number and pressure ratio, that... This allows natural extract molecules to undergo restricted heterogeneous nucleation on the surface of polynucleotides via charge capture before they can undergo disordered self-aggregation.

[0037] Example 3: This example combines Figures 1 to 2 The description of anti-aging compositions based on PN and natural extracts and their enhancement processes, such as... Figure 1 As shown, the enhanced process includes step S101, which involves preparing a first-phase fluid comprising a natural extract and an organic solvent having a first dielectric constant, and a second-phase fluid comprising polynucleotides, an aqueous buffer having a second dielectric constant, and divalent metal cations with a molar concentration of 0.8 mmol / L to 1.5 mmol / L. Step S102 involves establishing a fluid mixing domain to inject the first-phase fluid into the continuously flowing second-phase fluid according to a dynamic volumetric flow rate model, and maintaining the Reynolds number of the mixed fluid within the fluid mixing domain at 2800 to 4500. Then, in step S103, the decrease in the dielectric constant of the mixed solvent at the injection interface, combined with the amplification of the Coulomb attraction of the divalent metal cations on the polar groups of the natural extract, induces confined heterogeneous nucleation of the supersaturated natural extract on the surface of the polynucleotide molecular chain to construct a core-shell coating structure. Finally, in step S104, the core-shell coating structure is introduced into the aging channel, and the mixture in the aging channel is controlled to decrease at a cooling rate of 0.5 °C / min to 1.5 °C / min to the final lock-in temperature.

[0038] like Figure 2As shown, the microscopic logical evolution process from raw materials to stable formulation is as follows: the system initially exists in a dispersed state, where polynucleotides and natural extracts are freely distributed in their respective solvent systems. Through coordination with divalent metal cations, it is transformed into an electrostatic pre-enriched state, thereby constructing local positive-charge electrostatic enrichment domains along the polynucleotide molecular chain segments. The pH value is used to adjust the strength of the coordinating cations, causing the molecular chains to undergo controlled spatial contraction to form a spatial contraction conformation. After establishing a mixing domain with a Reynolds number of 2800 to 4500, it enters a directional pinning nucleation state. At this time, the microscopic mixing characteristic time is less than the nucleation induction time. Under the constraint of the microscopic eddy current scale, a restricted heterogeneous nucleation kinetic path occurs. Through Coulomb attraction amplification and synergistic interface charge neutralization, a core-shell topological steady state is achieved, constructing a defined core-shell coated topological structure and realizing the directional assembly of components at the molecular interface. Finally, through gradient cooling, the hydrogen bond network is guided to fix and enter a thermodynamically locked state, ensuring the orderly fixation of the hydrogen bond network and ensuring that the system exhibits a long-term stable particle size distribution.

[0039] Example 4: In the biopharmaceutical manufacturing scenario facing batch viscosity fluctuations of natural extracts, the dynamic viscosity of plant-derived components can deviate by ±15% due to the influence of extraction purity and ambient temperature. The traditional constant flow injection mode causes the critical instability constant K in the fluid mixing domain to deviate from the preset range, resulting in uncontrolled particle size of self-assembled microparticles and incomplete polynucleotide coating. The system deployment has an enhanced process architecture with online viscosity monitoring and closed-loop flow regulation to collect the dynamic viscosity μ1 of the first phase fluid and the dynamic viscosity μ2 of the second phase fluid, wherein the viscosity acquisition resolution is better than 0.05 mPa·s.

[0040] The regulation system uses a calculation module to lock the critical instability constant K value at 0.12 in real time. In the hardware-level closed loop, the regulation system is connected to a high-precision dual-channel constant flow pump and an online pressure sensor with a sampling frequency of 100Hz. The calculation module acquires the real-time viscosity values ​​of the first and second phase fluids and calculates the current flow deviation value with a control cycle of 10ms. When viscosity fluctuations cause K value drift, the system adjusts the pump motor speed in real time via a frequency converter to ensure that the ratio of Q1 to Q2 always meets the dynamic balance of the characteristic correlation. This millisecond-level response feedback mechanism ensures that even when the rheological properties of the fluid fluctuate, the curvature of the jet interface at the injection interface remains stable within the preset kinetic window. When the dynamic viscosity μ1 of the first phase fluid increases, the calculation module synchronously increases the volumetric flow rate Q1 of the first phase fluid according to the characteristic correlation to offset the attenuation of interfacial shear stress caused by the increase in viscosity, maintaining the Reynolds number Re in the quasi-steady-state range of 3200 to 3800 in the fluid mixing domain. At this time, the molar concentration of divalent metal cations in the second phase fluid is determined according to the mass concentration C of the polynucleotide. PN Implement dynamic compensation, setting C PNThe molar concentration M of the divalent metal cation is 3.5 mg / mL, and it satisfies the following linear fit rule: M = k × C PN +δ, where M is the molar concentration of divalent metal cations, k is the charge neutralization coefficient (0.35 L / g), and δ is the background electrolyte correction constant (0.05 mmol / L). The constant δ reflects the contribution of residual sodium ions and buffer salts carried by the polynucleotide raw materials in the aqueous buffer system to the charge shielding effect. Before actual production, the background electric field of this batch of systems was obtained by calibrating the conductivity of the pure buffer solution and converting it into equivalent ionic strength. This value was set to 0.05 mmol / L to correct the effective coordination concentration of divalent metal cations and ensure that the calculated M value can truly reflect the net cation flux used to construct the positive charge enrichment domain. Through the coupling of the above parameters, a local electrostatic attraction higher than the homogeneous nucleation energy barrier is generated at the injection interface.

[0041] During the kinetic energy decay stage of the aging flow channel, the system monitors the instantaneous temperature T of the mixture at different nodes in the flow channel. i The cooling rate R is set to satisfy R = ΔT / Δt, where ΔT is the temperature change and Δt is the cooling time interval. When the temperature of the mixture is in the hydrogen bond network recombination sensitive region between 45℃ and 30℃, the cooling rate is adjusted to 0.8℃ / min. This rate makes the microscopic Brownian motion frequency of the polynucleotide chain segment lower than the collision frequency of hydrogen bond formation, inducing the polyphenol groups in the natural extract to form a highly oriented hydrogen bond locking structure with the nitrogen-containing bases of the polynucleotide backbone, blocking the cross-interface diffusion of active components inside the particles. The generated self-assembled particles have an average particle size of 242.5nm, and the polynucleotide degradation rate after 48 hours of simulated sunlight irradiation is less than 1.5%. The process parameters have the ability to directionally regulate the molecular-level microstructure.

[0042] To ensure the ratio of the key parameter dynamic viscosity in the dynamic volumetric flow rate model of this invention ( To ensure accuracy, the following viscosity calibration and calculation procedures were performed during the preparation process of each embodiment of this invention. The measuring equipment and conditions were the dynamic viscosity of the first-phase fluid and the second-phase fluid. , All measurements were performed online or offline using a high-precision oscillating viscometer equipped with a micro-temperature control unit. The measured temperature was strictly locked at the instantaneous temperature when the material entered the fluid mixing zone, and the reference temperature was set to [temperature value missing]. To eliminate viscosity fluctuations caused by temperature drift; fluid characteristics were identified for second-phase fluids containing high molecular weight polynucleotides (PN), which exhibit non-Newtonian fluid characteristics. During the calculation, a shear rate within a certain range was selected. to Apparent viscosity within the linear range is used as dynamic viscosity. The effective value; for low-viscosity organic first-phase fluids, the measured value of their dynamic viscosity at the same temperature is directly extracted. The dynamic calculation logic of this invention's online viscosity monitoring system involves using a pre-established component concentration-temperature-viscosity characteristic mapping matrix, combined with the Arrhenius equation, to perform real-time fitting of the fluid viscosity-temperature curve. The system synchronously acquires the instantaneous temperature of the two-phase fluid at a 10ms period, automatically calculating the dynamic viscosity ratio under the current operating conditions. The viscosity acquisition resolution is maintained at a level better than [previous value missing]. This ensures that the fluctuation range of the critical instability constant K converges to within 0.002, thereby achieving microscopic directional control of the core-shell coating structure.

[0043] Example 5: When the system encounters situations where the physicochemical properties of different batches of natural extract raw materials vary, a pre-calibration procedure is initiated to determine the baseline value of the charge neutralization coefficient k. Test solutions with mass concentration gradients of 10, 20, and 30 mg / mL are prepared using the natural extract from that batch. The surface zeta potential of the polynucleotide solution at different concentrations is measured using a potentiometer under a constant temperature environment of 25°C. The critical mass ratio at which the potential transitions from negative to positive is extracted, and the calculation module calculates the value using the formula k=M. limit / C PN Fitting parameters, where M limit C represents the molar concentration of divalent metal cations in a charge-neutralized state. PN The k value represents the mass concentration of the polynucleotide. This value is used to modify the dynamic compensation model of the production control system to adapt to the electrochemical characteristics of this batch of raw materials.

[0044] In the scenario of starting a production cycle on a microfluidic continuous synthesis platform, a system resistance characteristic calibration procedure is implemented using organic solvents and aqueous buffer solutions. The metering pump is controlled to perform step-by-step frequency conversion within a flow rate range of 10 mL / min to 50 mL / min. The sensor array collects the pressure loss ΔP at the inlet of the fluid mixing domain in real time. The system is adjusted to calculate the system's flow resistance factor λ based on the correlation curve between the collected flow rate Q and the pressure loss ΔP. ​​The feed pressure prediction algorithm in the dynamic volumetric flow rate model is then corrected based on λ. When the feed pressure ratio of the first phase fluid to the second phase fluid stabilizes at the calibration point of 1:3.5, the fluctuation amplitude of the critical instability constant K converges to within 0.002, enabling physical consistency in the preparation of enhanced compositions across different equipment terminals.

[0045] Example 6: The final lock-in temperature T of the aging channel was set during the preparation of polynucleotide formulations with molecular weights from 500 kDa to 2000 kDa. lock The offline parameter-finding procedure uses a differential scanning calorimetry monitoring unit to acquire the phase transition enthalpy change curve of the composition system and extracts the characteristic exothermic peak T during the hydrogen bond network fixation process. pThe final lockout temperature T is determined by optimizing the energy-sensitive point. lock Satisfying relation T lock =T p -8, where T lock To ultimately lock in the temperature, T p To determine the characteristic exothermic peak temperature, the procedure controls the metering pump to adjust the volumetric flow rate Q2 of the second phase fluid at a gradient of 5 mL / min, and simultaneously collects the pressure pulsation frequency f at the inlet of the fluid mixing zone. When the coefficient of variation of the pressure pulsation frequency f reaches 0.05, the Reynolds number Re under the current state is recorded as the critical stable value of the batch of material, and a parameter matrix is ​​constructed based on the mass concentration of the natural extract as a reference standard for production operation.

[0046] In the pre-deployment calibration scenario of a microfluidic system employing a collision-type chip, the channel parameter adaptation procedure for the fluid mixing domain selects a structure with an inlet channel diameter of 0.5 mm and a jet collision angle of 150°. Pressure sensors collect the instantaneous pressure gradient between the first and second phase fluids at their confluence in real time. A laser scattering unit measures the sphericity of the self-assembled particles. When the sphericity measurement value is below 0.92, the feed pressure of the second phase fluid is increased to accelerate the droplet fragmentation rate of the first phase fluid. When the online viscometer detects a viscosity fluctuation exceeding 20% ​​in the mixture, the system... The system automatically initiates a calibrated fault-tolerant procedure, maintaining a constant feed pressure ratio of 1:5 between the first and second phase fluids. It also activates a proportional control valve at the end of the aging channel to maintain a static pressure of 0.85 MPa within the system. The average particle size deviation of the generated self-assembled microparticles is less than 5.0 nm over 72 hours of continuous operation. In the end-filling scenario for large-scale biopharmaceutical production, the system initiates a procedure to verify the integrity of the composition's microparticle structure. It controls the aseptic filtration of the mixture through a 0.22 μm polyethersulfone membrane, simultaneously monitoring the pressure difference ΔP across the membrane. filter The system adjusts the zeta potential of self-assembled particles in the filtrate in real time. When ΔP filter When the pressure is stable within the range of 0.02 MPa to 0.05 MPa and the polydispersity index (PDI) offset of the filtered particles is less than 0.05, the core-shell coating structure is deemed to have resistance to deformation when passing through narrow physical pores. This procedure feeds back the physical test indicators to the feed pressure regulation algorithm, realizing full-chain process control from liquid intermediates to sterile finished products, ensuring that the composition maintains its preset particle size distribution characteristics in the filling process of biopharmaceutical manufacturing.

[0047] In the experimental scenario evaluating the retention of enhanced bioactivity of the composition, the system initiated a molecular weight distribution index (MWDI) monitoring program. High-performance liquid chromatography (HPLC) was used to collect the polynucleotide elution signal after aging process, and the ratio of the weight-average molecular weight to the number-average molecular weight of the polynucleotide chain was calculated to obtain the MWDI value. Under parameter coupling conditions of a cooling rate of 1.0 °C / min and a divalent metal cation molar concentration of 1.2 mmol / L, the MWDI change rate of the composition after 90 days of thermally accelerated aging at 40 °C was found to be less than 2.5%. This result confirms that the hydrogen-bonded locking structure constructed by the directional pinning mechanism can effectively restrict the thermal motion of long polynucleotide chains, solving the problem of conformational unfolding and degradation of bioactive macromolecules in solvent systems, and establishing a closed-loop logical mapping between process parameters, microscopic locking state, and formulation stability.

[0048] The embodiments of this application have been described above with reference to the accompanying drawings. Unless otherwise specified, the embodiments and features in the embodiments of this application can be combined with each other. This application is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of this application without departing from the spirit of this application and the scope of protection of this invention, and all of these forms are within the protection scope of this application.

Claims

1. An enhancement process for an anti-aging composition based on PN and natural extracts, comprising the following steps: Step S101: Prepare a first phase fluid and a second phase fluid; the first phase fluid includes a natural extract and an organic solvent having a first dielectric constant; the second phase fluid includes a polynucleotide, an aqueous buffer having a second dielectric constant, and a divalent metal cation, wherein the molar concentration of the divalent metal cation is from 0.8 mmol / L to 1.5 mmol / L. Step S102: Establish a fluid mixing domain with a microchannel collision structure. The first-phase fluid is injected into the continuously flowing second-phase fluid via a micro-jet flow with a collision angle of 120° to 160°, based on a dynamic volumetric flow rate model, maintaining the Reynolds number of the mixed fluid within the mixing domain at 2800 to 4500. The physical scale of the fluid mixing domain restricts the micro-vortex scale of the mixed fluid. Radius of gyration of polynucleotide molecular chains Satisfying the mathematical relation is The dynamic volumetric flow rate model constrains the injection parameters of the first-phase fluid, ensuring that the ratio of the volumetric flow rate of the first-phase fluid to that of the second-phase fluid, multiplied by the 0.5th power of the ratio of their dynamic viscosities, is between 0.05 and 0.

15. Step S103: The decrease in dielectric constant of the mixed solvent at the injection interface, combined with the amplification of the Coulomb attraction of the divalent metal cations on the polar groups of the natural extract, induces the supersaturated natural extract to undergo restricted heterogeneous nucleation on the surface of the polynucleotide molecular chain, thus constructing a core-shell encapsulation structure. In step S104, the core-shell coating structure is introduced into the aging channel, and the mixture in the aging channel is controlled to decrease at a cooling rate of 0.5℃ / min to 1.5℃ / min until the final lock-in temperature.

2. The enhancement process of the anti-aging composition based on PN and natural extracts according to claim 1, characterized in that, Step S101 includes: Step S1011, dissolving the natural extract in an organic solvent, controlling the mass concentration of the natural extract to be from 10 mg / mL to 30 mg / mL, wherein the organic solvent is selected from one or more of ethanol, propylene glycol or polyol; Step S1012, dissolving the polynucleotide in an aqueous buffer, controlling the mass concentration of the polynucleotide to be from 1.5 mg / mL to 5.0 mg / mL, and adding an inorganic salt that provides a divalent metal cation.

3. The enhancement process of the anti-aging composition based on PN and natural extracts according to claim 1, characterized in that, In step S101: the divalent metal cation is selected from one or more of magnesium ions, zinc ions or calcium ions; by utilizing the coordination of the divalent metal cation with the polynucleotide phosphate backbone, a local positive charge electrostatic enrichment domain is constructed along the polynucleotide molecular chain segment, providing electrostatic capture sites for the natural extract.

4. The enhancement process of the anti-aging composition based on PN and natural extracts according to claim 1, characterized in that, Step S102 includes: Step S1021, adjusting the feed pressure ratio of the first phase fluid to the second phase fluid to 1:2 to 1:5; Step S1022, utilizing a low-energy kinetic window with a Reynolds number of 2800 to 4500 to avoid the fluid cavitation field and reduce local shear stress in order to preserve the integrity of the phosphodiester bonds of the polynucleotide; wherein, through the coupling constraint of pressure ratio and Reynolds number, the local temperature rise in the fluid mixing domain is controlled to not exceed 3°C.

5. The enhancement process of the anti-aging composition based on PN and natural extracts according to claim 1, characterized in that, In step S104: the aspect ratio of the aging flow channel is controlled to be 10:1 to 50:1; the cooling rate is used to guide the orderly fixation of the hydrogen bond network inside the core-shell coating structure and suppress the coarsening trend of the self-assembled system.

6. The enhancement process of the anti-aging composition based on PN and natural extracts according to claim 1, characterized in that, The natural extracts are selected from plant polyphenols or flavonoids; the weight-average molecular weight of the polynucleotides is not less than 1000 kDa; and the average particle size of the generated self-assembled microparticles is 150 nm to 350 nm.

7. The enhancement process of the anti-aging composition based on PN and natural extracts according to claim 1, characterized in that, In step S101: the pH of the second phase fluid is controlled to be between 6.2 and 7.2; the charge state of the polynucleotide molecular chain at the pH value is used to adjust the conformation of the polynucleotide in conjunction with divalent metal cations to produce steric contraction.

8. The enhancement process of the anti-aging composition based on PN and natural extracts according to claim 1, characterized in that, In step S102: the micro-eddy scale of the fluid mixing domain is controlled to be 1.5 to 3.0 times the radius of rotation of the polynucleotide molecular chain based on the Reynolds number criterion; the difference between the fluid micro-mixing characteristic time and the homogeneous nucleation induction time of the natural extract is used to realize the directional pinning of the natural extract on the polynucleotide surface.

9. The enhancement process of an anti-aging composition based on PN and natural extracts according to claim 1, characterized in that, Step S103 includes introducing a nonionic surfactant at a mass percentage of 0.1% to 0.5% into the first phase fluid to adjust the interfacial tension between the natural extract and the polynucleotide.

10. An anti-aging composition based on PN and natural extracts, characterized in that, The anti-aging composition is prepared by an enhanced process of the anti-aging composition based on PN and natural extracts as described in claim 1.

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