Preparation method and equipment of a chlorin supermolecular assembly

Abstract

This invention discloses a method and apparatus for preparing dihydroporphyrin supramolecular assemblies, belonging to the field of functional nanomaterials technology. By designing a Y+S composite microfluidic chip, this invention utilizes Dean's vortex induced by an S-shaped flow channel to precisely control the nucleation and growth kinetics of Ce6 molecules, achieving the directional preparation of nanospherical Ce6 supramolecular assemblies. Simultaneously, it ensures that the assemblies have a narrow particle size distribution (PDI < 0.1), completely solving the problems of large morphological heterogeneity and poor controllability in traditional methods, and providing technical support for the standardized preparation of Ce6-based nanodelivery systems.

Description

Technical Field

[0001] This invention belongs to the field of functional nanomaterials technology, specifically relating to a method and equipment for preparing dihydroporphyrin supramolecular assemblies. Background Technology

[0002] Supramolecular self-assembly is the core construction method for photosensitizer nanodelivery systems in photodynamic therapy (PDT). Dihydroporphyrin (Ce6), as a high-performance photosensitizer, needs to form nanoscale assemblies through supramolecular self-assembly to adapt to transport and endocytosis processes within the body. The microstructure of the assembly is a key factor determining its physicochemical properties, biological behavior, and ultimate therapeutic effect. Among these, uniform nanospheres are considered the ideal structural form for Ce6 supramolecular assemblies due to their advantages such as controllable particle size, good dispersibility, and stable endocytosis efficiency.

[0003] In the preparation of Ce6 supramolecular assemblies, traditional bulk mixing methods (such as direct mixing in test tubes or stirring) are currently the mainstream techniques. These methods rely on passive diffusion within a macroscopic volume to achieve molecular mixing and assembly, making it impossible to precisely control the kinetic conditions of the assembly process. On the one hand, local concentration gradients inevitably form within the mixed system, leading to uneven spatial distribution of Ce6 molecule nucleation and growth rates. On the other hand, the fluid shear forces during macroscopic mixing are irregular and cannot guide the molecules to stack in an orderly manner. Therefore, the morphology of the prepared Ce6 supramolecular assemblies is entirely dominated by thermodynamic random processes, exhibiting severe morphological heterogeneity: the products often contain a mixture of nanospheres, nanofibers, and other morphologies, with extremely wide particle size distributions and significantly high polydispersity index (PDI) for particles of the same morphology. This disordered morphological characteristic directly results in poor batch-to-batch reproducibility of the assemblies, failing to meet the stringent requirements for morphological uniformity in nanomedicine delivery systems. Summary of the Invention

[0004] In view of this, the purpose of this invention is to address the problems existing in the prior art by providing a method and apparatus for preparing dihydroporphyrin supramolecular assemblies. This invention discloses a method for precisely controlling the morphology of Ce6 supramolecular assemblies based on microfluidic Dean vortex technology. By controlling the flow rate, flow ratio, Ce6 concentration, and Dean number, the morphology of single nanospheres can be controllably prepared, resulting in Ce6 supramolecular assemblies with PDI < 0.1 and particle size of 100-200 nm, exhibiting both precise morphology and particle size control. Building upon this, by introducing liposome carriers and further controlling the morphology using microfluidic dynamics, this invention successfully constructed composite assemblies with particle sizes of approximately 600 nm-1 μm. This method not only retains the precise control of the assembly's microstructure by microfluidic technology but also achieves efficient loading and structural stability in a lipid matrix.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: A method for preparing dihydroporphyrin supramolecular assemblies, using a Y+S composite microfluidic chip to achieve controllable preparation of nanospherical Ce6 supramolecular assemblies.

[0006] It is worth noting that this invention utilizes the S-shaped curved flow channel of the Y+S composite microfluidic chip to induce controllable Dean vortexes. Through the "stretching-folding" effect of this secondary flow, rapid and uniform mixing of Ce6 molecular mixtures is achieved, and the nucleation rate and growth direction of Ce6 supramolecular assembly are precisely controlled. This transforms the assembly process from thermodynamic random control to kinetic precise control, ultimately resulting in the directional formation of uniform nanosphere morphology. Thus, the optimization of cellular uptake performance is achieved through morphology regulation.

[0007] Furthermore, the preparation method of the dihydroporphyrin supramolecular assembly includes the following steps: S1. Prepare the assembly phase solution: The organic phase is a Ce6 dimethyl sulfoxide solution, and the aqueous phase is ultrapure water or an aqueous suspension of liposomes; S2. Microfluidic flow field control and assembly: The organic phase and aqueous phase are pumped into the corresponding injection channels of the Y-type chip by a syringe pump, and the flow rate of the two phases is controlled to be 1~5 μL / min. S3. Collection and Aging: The assemblies are continuously collected from the S-shaped chip outlet and allowed to stand for aging to obtain uniformly morphological nanosphere Ce6 supramolecular assemblies.

[0008] Furthermore, in step S1, the concentration of the Ce6 dimethyl sulfoxide solution is 0.05~0.1 mg / mL.

[0009] It is worth noting that the concentration of the Ce6 dimethyl sulfoxide solution is limited to 0.05~0.1 mg / mL in this invention to ensure uniform initial molecular dispersion. Using ultrapure water as the aqueous phase maintains the dispersion stability of the aqueous system and avoids non-specific aggregation during assembly.

[0010] Furthermore, in step S1, the method for preparing the liposome aqueous suspension includes: mixing dimyristoylphosphatidylcholine (DMPC), (2,3-dioleoyl-propyl)-trimethylamine (DOTAP), DSPE-PEG2000 and cholesterol in a mass ratio of 16:2:1:1, hydrating at 37 °C and extruding through a membrane 11-15 times, and then dispersing in ultrapure water.

[0011] Furthermore, in step S2, the flow rate ratio of the organic phase to the aqueous phase is 7.5:5 to 5:5.

[0012] It is worth noting that after initial contact in the Y-shaped mixing zone, the fluid of this invention enters the S-shaped curved flow channel zone, where a stable Dean vortex is generated under the action of centrifugal force. The "stretching-folding" effect of the Dean vortex causes the two-phase interface to fuse rapidly, eliminating local concentration gradients and triggering explosive and uniform nucleation of Ce6 molecules. Moreover, the nucleation rate is much greater than the growth rate, forcing Ce6 molecules to grow in an isotropic direction and directionally form nanosphere structures.

[0013] This invention utilizes the S-shaped curved flow channel of a Y+S composite microfluidic chip to induce controllable Dean's vortex, achieving rapid and uniform mixing of Ce6 molecules. This allows for precise control of the nucleation rate and growth direction of Ce6 supramolecular assemblies, regulating the formation of uniform nanosphere morphology. Furthermore, in the preparation of liposome-coated dihydroporphyrin supramolecular assemblies, the centrifugal force field generated by the curved flow channel of the microfluidic chip enables highly efficient mixing of the fluid in a very short time. Combined with the electrostatic attraction of the lipid surface, this induces the formation of uniformly encapsulated assembly complexes, resulting in excellent biocompatibility and membrane affinity. Surprisingly, the Ce6 encapsulation efficiency of the liposome-coated dihydroporphyrin supramolecular assemblies disclosed in this invention is significantly higher than that obtained by traditional direct blending methods. This means that with each cellular uptake, a higher dose of drug can be delivered, thereby improving the overall intracellular drug accumulation efficiency.

[0014] A second object of the present invention is to provide an apparatus for use in the preparation method described above.

[0015] An apparatus for preparing dihydroporphyrin supramolecular assemblies, wherein the Y+S composite microfluidic chip is a Y-shaped mixing region connected to a continuous arc-shaped S-shaped curved flow channel at the rear end, the width and height of the flow channel are both 300 μm, and it has a regular square cross-section.

[0016] It is worth noting that microfluidic technology, with its precise control over fluids at the microscale, provides a key technical path for solving the problem of porphyrin assembly regulation. Its core advantage lies in its ability to construct a highly controllable flow field environment. Therefore, this invention designs a dedicated flow channel for the molecular stacking characteristics of Ce6. By controlling the flow channel configuration and fluid parameters, it precisely intervenes in the nucleation and growth kinetics of molecular self-assembly, thereby achieving directional control of the assembly morphology.

[0017] The Y+S composite microfluidic chip disclosed in this invention has a closed integrated flow channel. The sample inlet area has two independent inlets that are connected to the organic phase and the aqueous phase respectively, and the sample outlet area has a single outlet. It is suitable for the fluid control requirements of microfluidic hybrid assembly. Under low Reynolds number laminar flow conditions, stable Dean vortices can be induced through the S-shaped flow channel to achieve precise control of the Ce6 assembly morphology.

[0018] Furthermore, the flow channel intersection angle of the Y-shaped mixing zone is 75-85°.

[0019] It is worth noting that in existing technologies, when directly mixing at room temperature, Ce6 molecules tend to form irregular, large-sized assemblies through π-π stacking due to a lack of precise control over the flow field shear force. In this invention, the 75-85° angle design of the Y-shaped mixing zone predetermines the initial contact momentum of the two-phase fluids. Combined with the curvature of the inner radius, this creates a vortex center within the flow channel cross-section. This stable flow field force gradient overcomes random intermolecular aggregation, forcing the supramolecular assemblies to transform into a spherical configuration with the lowest energy during the gradual displacement of the solvent system, rather than random, disordered stacking.

[0020] Furthermore, the S-shaped curved flow channel has a total of 15 arc-shaped bends, the width of which is 300-600 μm, the inner radius is 650 μm, and the outer radius is 950 μm.

[0021] It is worth noting that this invention utilizes a Y+S composite microfluidic chip to induce a secondary flow field that stretches and folds the fluid across its cross-section, significantly increasing the contact area at the two-phase interface and shortening the diffusion distance. This hybrid dynamics enhanced by the secondary flow influences the nucleation process of Ce6. According to classical nucleation theory, the extremely high mixing efficiency of the Y+S composite microfluidic chip leads to an instantaneous high supersaturation of the system, triggering explosive nucleation. At this point, the nucleation rate is much greater than the growth rate, and monomers are rapidly consumed and undergo isotropic growth, ultimately leading to the formation of small, highly uniform spherical nanoparticles.

[0022] Therefore, this invention discloses a Y+S composite microfluidic chip and its application in the directional construction of Ce6 supramolecular assemblies with nanospheres. The core of this application utilizes Dean's vortex induced by an S-shaped flow channel to regulate assembly dynamics. Based on the microfluidic Dean's vortex-based method for precise morphology control of Ce6 supramolecular assemblies, the flow rate, flow ratio, Ce6 concentration, and Dean's number are controlled to achieve the directional preparation of single nanospheres, thus realizing a technical solution for precise morphology and particle size control of Ce6 supramolecular assemblies with a PDI < 0.1 and a particle size of 100-200 nm. Furthermore, this invention introduces liposome carriers and further regulates the morphology using microfluidic dynamics, successfully constructing composite assemblies with particle sizes of approximately 600 nm-1 μm. This method not only retains the precise control of the assembly's microstructure by microfluidic technology but also achieves efficient loading and structural stability in a lipid matrix.

[0023] Compared with the prior art, the present invention has the following advantages: (1) High uniformity: Compared with the traditional method where PDI > 1.0, the present invention can reduce PDI to below 0.1; (2) Precise morphology switching: Controlled transformation from disordered state to 100-200 nm spherical structure or 600 nm-1 μm lipid complex assembly was achieved at the nanoscale; (3) Strong environmental adaptability: The assembled structure makes it difficult to disintegrate in complex physiological environments, and its storage stability can reach more than one month; (4) Universality: This flow field control strategy can be extended to the precise assembly of various porphyrin hydrophobic molecules, and has great engineering application value. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0025] Figure 1 This is an experimental flowchart disclosed in Embodiment 1 and Comparative Example 3 of the present invention.

[0026] Figure 2 The images show 3D diagrams of the Y and YS chip channels disclosed in Embodiment 1 and Comparative Example 3 of this invention.

[0027] Figure 3 The images show SEM (top) and TEM (bottom) images of different assembly methods disclosed in Experimental Example 1 of the present invention, where (i) is Ce6 H2O disclosed in Comparative Example 1, (ii) is MF Ce6 H2O disclosed in Comparative Example 3 (Y), and (iii) is MFCe6 H2O disclosed in Example 1.

[0028] Figure 4 The particle size distributions of Ce6 supramolecular assemblies prepared by different assembly methods disclosed in Experimental Example 1 of this invention are as follows: Ce6 H2O disclosed in Comparative Example 1, MF Ce6 H2O (Y) disclosed in Comparative Example 3, and MF Ce6 H2O disclosed in Example 1.

[0029] Figure 5 The particle size distribution diagram shows the effects of bend width, concentration, and flow rate on particle size as disclosed in Experimental Example 2 of this invention. From left to right, the diagram shows the effects of bend width (300-1000 μm) on particle size, Ce6 concentration (0.02-0.5 mg / mL) on particle size, and constant flow rate (1-25 μL / min) on particle size.

[0030] Figure 6 The images show the UV absorption and fluorescence spectra of Ce6 at different liposome concentrations in Experimental Example 3 of this invention. Figure a shows the UV absorption spectrum, and Figure b shows the fluorescence spectrum.

[0031] Figure 7 The images show the SEM and TEM images of the Ce6 lip group (a) and MF CE6 lip group (b) composite nanoparticles in Experimental Example 3 of this invention, where (i) is SEM and (ii) is TEM.

[0032] Figure 8 The graph shows the Zeta potential test results of the Ce6lip and MF Ce6lip samples in Experimental Example 3 of this invention.

[0033] Figure 9 Figure 1 shows the particle size distribution stability of Ce6 supramolecular assemblies prepared by different assembly methods in Experimental Example 3 of the present invention before and after one month of storage. Figure 2a shows the Ce6 lip group and Figure 3b shows the MF Ce6 lip group.

[0034] Figure 10 This is a bar chart showing the yield of singlet oxygen in each system in Experimental Example 4 of this invention.

[0035] Figure 11 These are laser confocal microscopy images of Ce6 assemblies taken up by cells in each group in Experiment Example 4 of this invention, where (i) is the cell nucleus (DAPI), (ii) is the cytoplasm (DIO), (iii) is the Ce6 drug fluorescence, and (iv) is the merge image.

[0036] Figure 12 This is a flow cytometry statistical graph showing the uptake efficiency of cells for different assemblies in Experiment Example 4 of this invention.

[0037] Figure 13 This is a bar chart comparing the encapsulation rates of Ce6 in the Ce6 lip group and the MF CE6 lip group in Experimental Example 4 of this invention.

[0038] Figure 14 This is a particle size stability test diagram of MF Ce6 H2O and MF Ce6 lip under different physiological culture conditions in Experimental Example 4 of the present invention.

[0039] Figure 15 This is a co-localization flow cytometry statistical diagram of the cell entry efficiency of liposome carriers in the lip, Ce6 lip and MF Ce6 lip groups in Experiment Example 4 of this invention.

[0040] Figure 16 This is a bar chart showing the photodynamic killing efficiency (cell survival rate) of each group of systems in Experiment Example 4 of this invention.

[0041] Figure 17 This is a safety evaluation chart of the hemolysis experiment for each group of systems in Experiment Example 4 of this invention. Detailed Implementation

[0042] The technical solutions in the embodiments of the present invention will be clearly and completely described below. 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.

[0043] The term "embodiment" used herein, as an example, is not necessarily to be construed as superior to or better than other embodiments. Performance testing in the embodiments of this application, unless otherwise specified, employs conventional testing methods in the art. It should be understood that the terminology used in this application is merely for describing particular implementations and is not intended to limit the scope of this disclosure.

[0044] Unless otherwise stated, the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains; other experimental methods and technical means not specifically mentioned herein refer to experimental methods and technical means commonly used by one of ordinary skill in the art.

[0045] To better illustrate the content of this application, numerous specific details are provided in the following detailed embodiments. Those skilled in the art should understand that this application can be implemented even without certain specific details. In the embodiments, some methods, means, instruments, and devices well-known to those skilled in the art are not described in detail in order to highlight the main points of this application.

[0046] In the description of this invention, it should be understood that the terms "inner," "outer," "first," "last," etc., indicating orientation or positional relationships are based on the orientation or positional relationships shown in the accompanying drawings and are only for the convenience of describing this invention, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation, and therefore should not be construed as a limitation of this invention. Similarly, the terms "first," "second," etc., indicating differences in components are only for the convenience of describing this invention, and do not indicate or imply that the device or element referred to must have a specific order or sequence, and therefore should not be construed as a limitation of this invention.

[0047] Without conflict, the technical features disclosed in the embodiments of this application can be combined arbitrarily, and the resulting technical solution belongs to the content disclosed in the embodiments of this application.

[0048] This invention discloses a method and apparatus for preparing dihydroporphyrin supramolecular assemblies, belonging to the field of functional nanomaterials technology. By designing a Y+S composite microfluidic chip, this invention utilizes Dean's vortex induced by an S-shaped flow channel to precisely control the nucleation and growth kinetics of Ce6 molecules, achieving controllable preparation of nanospherical Ce6 supramolecular assemblies and lipid composite assemblies. Simultaneously, it ensures that the assemblies have a narrow particle size distribution (PDI < 0.1), completely solving the problems of large morphological heterogeneity and poor controllability in traditional methods, and providing technical support for the standardized preparation of Ce6-based nanodelivery systems.

[0049] To better understand the present invention, the following embodiments are provided for further detailed description of the present invention, but they should not be construed as limiting the present invention. Any non-essential improvements and adjustments made by those skilled in the art based on the above-described invention are also considered to fall within the protection scope of the present invention. Example 1

[0050] A method for preparing a dihydroporphyrin supramolecular assembly includes the following steps: Raw material preparation: Dissolve the photosensitizer dihydroporphyrin (Ce6) in dimethyl sulfoxide (DMSO) to prepare an organic phase solution with a concentration of 0.1 mg / mL; use ultrapure water as the aqueous phase solvent.

[0051] Equipment: A composite microfluidic chip with a Y-shaped inlet and a continuous S-shaped curved flow channel is used. The Y-shaped mixing zone has an 80° channel intersection angle, and both the width and height of the channel are 300 μm. The width of the S-shaped curved flow channel is 300 μm.

[0052] Parameter settings: Set the flow rate ratio of organic phase to aqueous phase to 5:5, and the constant flow rate to 5 μL / min.

[0053] Assembly process: After the two-phase fluids come into contact at the Y-shaped intersection, they enter the S-shaped flow channel. The centrifugal force generated by the bend induces the Dean vortex, which achieves intense stretching and folding of the two-phase interface.

[0054] Product collection and aging: Assembly samples were continuously collected from the S-shaped chip outlet and allowed to stand for aging for 24 h to obtain uniformly morphological nanosphere Ce6 supramolecular assemblies MF Ce6 H2O. Example 2

[0055] A method for preparing a dihydroporphyrin supramolecular assembly includes the following steps: Raw material preparation: DMPC, DOTAP, DSPE-PEG2000 and cholesterol were dissolved in methanol / chloroform at a mass ratio of 16:2:1:1. After being formed into a film by rotary evaporation, the film was hydrated at 37 °C and extruded to prepare a monolayer liposome suspension as the aqueous phase; a 0.1 mg / mL Ce6 / DMSO solution was prepared as the organic phase.

[0056] Equipment: A composite microfluidic chip with a Y-shaped inlet and a continuous S-shaped curved flow channel is used.

[0057] Parameter settings: Set the flow rate ratio of organic phase to water phase to 5:5, and the constant flow rate to 5 μL / min.

[0058] Assembly process: After the two-phase fluids come into contact at the Y-shaped junction, they enter the S-shaped flow channel, where the centrifugal force generated by the bend induces a high-frequency Dean vortex. Driven by this interfacial dynamics, Ce6 molecules in the organic phase undergo explosive and uniform nucleation, and are directionally migrated to the liposome surface by the electrostatic attraction generated by DOTAP.

[0059] Product collection and aging: Assemblies were continuously collected from the S-shaped chip outlet and allowed to stand for aging for 24 hours to obtain Ce6 supramolecular assemblies MF Ce6 lip with uniform morphology.

[0060] Compared to traditional bulk mixing, which can lead to lipid bilayer rupture due to localized high concentrations of DMSO, this invention greatly eliminates the concentration gradient through flow field convection, protects the integrity of the phospholipid membrane structure, and promotes controlled recombination and dense coating of liposomes on the Ce6 template, ultimately forming a uniform "cotton-like" dense composite structure. This results in the construction of a Ce6 supramolecular composite assembly MF Ce6 lip with a particle size of approximately 600 nm. Example 3

[0061] A method for preparing a dihydroporphyrin supramolecular assembly includes the following steps: Raw material preparation: Dissolve the photosensitizer dihydroporphyrin (Ce6) in dimethyl sulfoxide (DMSO) to prepare an organic phase solution with a concentration of 0.05 mg / mL; use ultrapure water as the aqueous phase solvent.

[0062] Equipment: A composite microfluidic chip with a Y-shaped inlet and a continuous S-shaped curved flow channel is used, with a curve width of 300 μm.

[0063] Parameter settings: Set the flow rate ratio of organic phase to aqueous phase to 5:5, and the constant flow rate to 5 μL / min.

[0064] Assembly process: After the two-phase fluids come into contact at the Y-shaped intersection, they enter the S-shaped flow channel. The Dean vortex generated by the bend is used to achieve intense stretching and folding of the two-phase interface.

[0065] Product collection and aging: Assembly samples were continuously collected from the S-type chip outlet and allowed to stand for 24 h to obtain uniformly morphological nanosphere Ce6 supramolecular assemblies MF Ce6 H2O (0.05 mg / mL). Example 4

[0066] The dihydroporphyrin supramolecular assembly was prepared using the same steps as in Example 1, except for the equipment: a composite microfluidic chip with a Y-shaped inlet and a continuous S-shaped curved flow channel was used, with a curve width of 600 μm. Uniformly morphological nanosphere Ce6 supramolecular assemblies MFCe6H2O (600 μm) were obtained. Example 5

[0067] Dihydroporphyrin supramolecular assemblies were prepared using the same steps as in Example 1, except for the parameter settings: the flow rate ratio of the organic phase to the aqueous phase was set to 5:5, and the constant flow rates were 1 μL / min and 2.5 μL / min, respectively. Uniformly morphological nanosphere Ce6 supramolecular assemblies MF Ce6 H2O (1 μL / min) and MF Ce6 H2O (2.5 μL / min) were obtained.

[0068] To further demonstrate the beneficial effects of the present invention and to better understand the present invention, the following comparative examples and experimental examples further illustrate the characteristics and application performance of the preparation method and equipment for dihydroporphyrin supramolecular assemblies disclosed in the present invention. However, these should not be construed as limiting the present invention. Other methods and applications obtained by those skilled in the art based on the above-described invention and their results are also considered to fall within the protection scope of the present invention. Comparative Example 1

[0069] Ce6 was dissolved in DMSO to prepare an organic phase stock solution of 0.1 mg / mL. At room temperature, the stock solution was rapidly mixed with an equal volume of ultrapure water (1:1, v / v), subjected to pipetting / shaking, and aged at room temperature for 24 h to obtain Ce6 supramolecular assemblies Ce6H2O. Comparative Example 2

[0070] The photosensitizer dihydroporphyrin (Ce6) was dissolved in anhydrous dimethyl sulfoxide (DMSO) to prepare a stock solution with a concentration of 0.1 mg / mL. A hydrated liposome suspension with a concentration of 1 mg / mL was prepared in advance using a thin-film hydration method. The Ce6 / DMSO stock solution and the hydrated liposome suspension were mixed at a volume ratio of 1:1 (v / v). The two phases were brought into full contact by conventional physical stirring or shaking. The mixed system was aged at room temperature for 24 h to obtain Ce6 supramolecular assemblies Ce6lip. Comparative Example 3

[0071] 0.1 mg / mL Ce6 / DMSO solution and ultrapure water were separately loaded into syringes and injected into the Y-shaped microfluidic chip through the injection port. The total flow rate was controlled at 5 μL / min, and the two fluid phases contacted and mixed at the Y-shaped junction. The sample was collected from the outlet and aged at room temperature for 24 h to obtain the Ce6 supramolecular assembly MF Ce6 H2O (Y chip). The Y-shaped microfluidic chip consists of two symmetrical inlet channels and one confluence channel, with the confluence end forming an 80° angled mixing zone. The channel width and height are both 300 μm. Figure 2 ). Comparative Example 4

[0072] The dihydroporphyrin supramolecular assembly was prepared using the same steps as in Example 1, except for the equipment: a composite microfluidic chip with a Y-shaped inlet and a continuous S-shaped curved flow channel was used, with a curve width of 1000 μm. The resulting Ce6 supramolecular assembly MF Ce6 H2O (1000 μm) was obtained. Comparative Example 5

[0073] Dihydroporphyrin supramolecular assemblies were prepared using the same steps as in Example 1, except for the preparation of the raw materials: the photosensitizer dihydroporphyrin (Ce6) was dissolved in dimethyl sulfoxide (DMSO) to prepare organic phase solutions with a concentration of 0.2 mg / mL or 0.5 mg / mL; ultrapure water was used as the aqueous phase solvent. Ce6 supramolecular assemblies with the following morphologies were obtained: MF Ce6 H2O (0.2 mg / mL) and MF Ce6 H2O (0.5 mg / mL). Comparative Example 6

[0074] Dihydroporphyrin supramolecular assemblies were prepared using the same steps as in Example 1, except for the parameter settings: the flow rate ratio of the organic phase to the aqueous phase was set to 5:5, and the constant flow rates were 10 μL / min and 25 μL / min, respectively. Ce6 supramolecular assemblies MF Ce6 H2O (10 μL / min) and MF Ce6 H2O (25 μL / min) were obtained. Experimental Example 1

[0075] The Ce6H2O, MF Ce6H2O (Y), and MFCe6H2O groups prepared in Comparative Examples 1, 3, and 1, respectively, were characterized, and the results are as follows: Figure 3 As shown, compared to the Ce6H2O group ( Figure 3 i) disordered fibrous aggregates and MF Ce6 H2O (Y) group ( Figure 3 ii) 600 nm two-dimensional nanosheets, the present invention MF Ce6 H2O group ( Figure 3 iii) The dynamic transformation to uniform spherical nanoparticles was achieved through Dean's vortex.

[0076] Specifically, in the Ce6 H2O group ( Figure 3 i) Both SEM and TEM images show a highly irregular and disordered hybrid structure, composed of numerous fibrous branches and spherical aggregates, exhibiting an amorphous morphology with uneven density and demonstrating extremely high polydispersity. In contrast, the MF Ce6 H2O (Y) group ( Figure 3 ii) After being processed by the Y-type chip, the morphology of Ce6 undergoes a significant transformation, evolving from a disordered state into a highly ordered two-dimensional nanosheet structure with a smooth surface, clear edges, and a lateral dimension of approximately 600 nm. Figure 4 Under transmission electron microscopy, it exhibits a translucent, flake-like texture with significantly improved monodispersity. When using a Y+S composite microfluidic chip, the MF Ce6 H2O group ( Figure 3 The Ce6 supramolecular assembly in iii) underwent a second morphological mutation, evolving into highly uniform, dense spherical nanoparticles. Electron microscopy revealed that the particles were spherical, with smooth surfaces and no adhesion, and their size was precisely reduced to the range of 100-200 nm. Figure 4 Furthermore, the internal structure exhibits a dark solid projection, demonstrating that Dean's vortex-induced explosive nucleation achieves extreme control over the morphology and particle size distribution of the assembly.

[0077] Therefore, the dihydroporphyrin supramolecular assemblies prepared in this invention exhibit precise morphological characteristics, as observed by transmission electron microscopy (TEM), showing a transition from irregular sheet-like structures to highly uniform nanosphere structures; regarding particle size distribution, dynamic light scattering (DLS) testing reveals… Figure 4 The resulting spherical particles have a particle size distribution between 100 and 200 nm, and the polydispersity index (PDI) is optimized to below 0.1, exhibiting excellent monodispersity. Experiment Example 2

[0078] The particle size of the dihydroporphyrin supramolecular assemblies prepared in Examples 1, 3-5, and Comparative Examples 4-6 was characterized, and the results are as follows: Figure 5 As shown.

[0079] As can be seen, when the S-bend width is 300 μm (Example 1) and 600 μm (Example 4), the particle size of the assemblies is highly consistent (175±2 nm and 176±3 nm, respectively), and the PDI is extremely low (0.09±0.1 for both), demonstrating excellent uniformity. Once the S-bend width increases to 1000 μm (Comparative Example 4), the particle size surges to 2114±465 nm, and the PDI soars to 0.7±0.2. This confirms that when the channel width is too high, the abnormal increase in the Dean number (De) disrupts the flow field stability, leading to a sharp decrease in mixing efficiency, and the assembly process changes from controlled laminar mixing to disordered aggregation.

[0080] Furthermore, the Ce6 concentration and particle size exhibit a significant non-linear relationship: within the range of 0.05–0.1 mg / mL, the particle size stabilizes at around 172–175 nm, and the PDI remains below 0.1. When the concentration is as low as 0.02–0.05 mg / mL, although the PDI remains low (0.04), the particle size is slightly larger (249 ± 3 nm), which may be due to excessive growth of single particles caused by insufficient nucleation sites. As the concentration increases to 0.2–0.5 mg / mL, the particle size significantly increases to 383 ± 3 nm, and the PDI also increases accordingly. This verifies that the sharp increase in nucleation rate at high concentrations induces frequent molecular collisions, and the local high concentration effect leads to the transformation of the assembly from a densely packed state to a disordered aggregate state.

[0081] Furthermore, the effect of flow rate on particle size exhibits a wide adaptation window, but stability is compromised at high flow rates: within the range of 1 μL / min to 5 μL / min, the particle size remains within a narrow range of 169-175 nm, and the PDI is extremely stable. When the flow rate is further increased to 10-25 μL / min, the particle size begins to increase (up to a maximum of 261±3 nm), and the PDI increases significantly to 0.25±0.02. The data indicate that medium to low flow rates (1-5 μL / min) are the optimal process selection for this invention. Excessively high flow rates generate strong local turbulence and uneven shear forces, thereby disrupting the stable mixing environment of the nanoparticles.

[0082] Therefore, the present invention preferably uses an S-shaped curved channel with a width of 300-600 μm, a Ce6 concentration of 0.05-0.1 mg / mL, and a flow rate of 1-5 μL / min. Within this process range, the Dean flow intensity can accurately cover the channel cross-section, in conjunction with optimal nucleation kinetics. Experimental Example 3

[0083] The MF Ce6 lip composite assembly prepared in Example 2 and the Ce6 lip composite assembly prepared in Comparative Example 2 were characterized, and the experimental results are as follows: First, spectroscopic analysis confirmed the regulatory role of liposomes on the arrangement of Ce6 molecules. Figure 6 As the liposome concentration increased from 0.1 mg / mL to 1 mg / mL, the Q-band absorption peak of Ce6 in the MF Ce6 lip group showed a continuous red shift. Simultaneously, the aggregation-related characteristic peak at 650 nm in the fluorescence spectrum gradually disappeared, becoming more similar to monomer emission characteristics. This indicates that liposomes can block the disordered aggregation of Ce6 molecules. The core role of microfluidics is to regulate assembly through "directional movement + uniform mixing." Positive charge attraction fixes the orientation of Ce6, leading to the appearance of positive and negative Cotton effects. This demonstrates that microfluidics alters the aggregation state of Ce6 during the mixing process.

[0084] This invention further explores the influence of hybrid dynamics on the spatial construction of composite assemblies. Figure 7 SEM results showed that Ce6 lip exhibited significant morphological heterogeneity, with MF Ce6 lip forming dense composite nanoparticles. The particle surface was coated with a soft film structure, exhibiting a typical fried egg-shaped structure under TEM, which corroborated the morphological characteristics observed by SEM. SEM and TEM characterization results showed that in the Ce6 lip group, Ce6 was randomly adsorbed, aggregated, or dispersed on the surface of liposomes in the form of small particles, accompanied by some structurally disordered liposomes, resulting in a relatively disordered overall assembly morphology. The main reason for this is that in the Ce6 lip group, due to the uneven concentration distribution, Ce6 molecules first self-aggregated in the chaotic environment. Subsequently, these aggregates were adsorbed onto the outside of the liposomes under the attraction of their charge. For the MF Ce6 lip group, the composite surface was positively charged due to the presence of DOTAP; at the microfluidic interface, through the synergistic effect of electrostatic attraction and centrifugal force, the system continuously captured and induced Ce6 to assemble in a controlled manner on the composite surface, forming a uniform coating morphology. Combined with the increase of Zeta potential ( Figure 8 This fully demonstrates that microfluidic interface dynamics enable lipid complexes to densely coat the outer surface of Ce6 in a "quilt-like" morphology.

[0085] To test its stability, the MF Ce6 lip group maintained a relatively uniform particle size distribution even after one month of storage. Figure 9This is because microfluidics facilitates the structural integration of Ce6 with the lipid bilayer. Simple mixing, primarily characterized by random convection and non-uniform diffusion, lacks laminar flow constraints, leading to instantaneous imbalances in the local concentrations of Ce6 and liposomes, unstable contact interfaces, and disordered assembly, ultimately forming chaotic aggregates with uncontrollable morphology and non-uniform particle size. Therefore, the Ce6 supramolecular assemblies prepared by the microfluidic chip in this invention exhibit a more stable structure within the lipid matrix compared to traditional mixing assembly methods. Experiment Example 4

[0086] To evaluate the effect of microfluidically regulated assembly morphology on Ce6 photodynamic therapy (PDT) activity, this invention used 9,10-anthracene dimethyl diamaronic acid (ABDA) as a ROS probe, and used the Ce6 supramolecular assemblies prepared in Examples 1-2 and Comparative Examples 1-2 as experimental subjects. The detection results showed that... Figure 10 The introduction of liposomes significantly improved the ¹O2 yield of the system, mainly due to the inhibition of Ce6 aggregation and the maintenance of molecular state distribution by the liposomes. Notably, MF Ce6 lip and Ce6 lip did not show a statistically significant difference in ¹O2 yield. This phenomenon profoundly reveals the core value of microfluidics: it does not work by altering the inherent photophysical properties of molecules (such as ¹O2 quantum yield), but rather by constructing specific spatial morphologies and stability to regulate their biodistribution and utilization efficiency.

[0087] Because cells are sensitive to dimethyl sulfoxide (DMSO) content, samples aged for 24 h were subjected to 24 h of dialysis treatment, with the dialysis solution being changed every 8 h. After dialysis, the samples were quantified by an enzyme-linked immunosorbent assay (ELISA) reader, and samples of equal concentration and volume were added to the cell system at a volume ratio of 5%. After incubation for 5 h, imaging observation and CCK8 assay were performed.

[0088] Cellular experiments further elucidated the structure-activity relationship between assembly morphology and biological behavior. Flow cytometry and confocal microscopy revealed that all assemblies exhibited cellular entry capabilities, but the uptake efficiency showed a clear gradient: MFCe6lip > Ce6lip > MF Ce6H2O > Ce6H2O. Figure 11(12). MF Ce6lip showed better uptake than Ce6lip, mainly because Ce6 is stably coated by liposomes, exhibiting strong anti-interference ability and easy cellular uptake; while Ce6lip is only adsorbed on the surface and has poor stability. MF Ce6lip uptake was higher than MF Ce6 H2O, thanks to the excellent intracellular delivery capability of liposomes, which significantly promoted drug entry into cells. Surprisingly, although the MF Ce6lip group ultimately formed a large-sized composite structure with a particle size close to 1 μm, far larger than the ideal particle size of around 200 nm usually considered most favorable for cellular uptake, this structure, significantly exceeding the conventional size range, did not weaken its cellular uptake ability; on the contrary, it maintained a high level of internalization. Compared to a single size effect, the ordered assembly structure formed by microfluidics, the continuous and flexible lipid outer layer, the uniform interfacial properties, and the stable charge characteristics can more effectively trigger multiple uptake pathways such as cell adhesion, membrane fusion, and macropinocytosis, thereby breaking through traditional particle size limitations. This unique hierarchical structure is made possible primarily by the precise fluid dynamics control provided by the microfluidic chip, which enables the tight encapsulation of Ce6 on the outer layer.

[0089] This "tight encapsulation" mode not only gives the system an extremely high encapsulation rate ( Figure 13 This significantly enhances its structural robustness in complex physiological environments (such as extracellular matrix charge interference). In vitro environmental simulation tests ( Figure 14 The results showed that MFCe6 lip and MF Ce6 H2O had uniform particle size in water, PBS, FBS, and DMEM++ for 24 h and could be stably present in the cell culture environment.

[0090] To eliminate differences in the cell entry ability of liposomes themselves, this invention used DSPE-FITC labeled liposomes for colocalization analysis. Figure 15 The results showed no significant difference in fluorescence intensity, indicating that the cell entry efficiency of the liposome carriers in each group was basically the same. This strongly suggests that the increase in Ce6 uptake depends on the physical tightness of its binding with liposomes. Interfacial regulation under microfluidic conditions endowed the assemblies with coating complex characteristics, enabling Ce6 to form a stable physicochemical integration with liposomes. This tightly bound state played a crucial "carrier effect," breaking the limitation of low Ce6 monomer cell entry efficiency and achieving carrier-mediated high-throughput cell uptake. In contrast, the simple mixed group showed lower encapsulation efficiency and unstable surface adsorption, making Ce6 easily detached during cell transport, resulting in a lower final effective intracellular concentration. The final photodynamic killing efficiency and cell uptake kinetics showed a high degree of consistency. Figure 16 Under conditions of extremely low dark toxicity (survival rate > 95%), the MFCe6 lip group exhibited the most significant phototoxicity, and its efficacy was far superior to that of the traditional assembly system.

[0091] Furthermore, hemolysis experiments confirmed the excellent biocompatibility of this composite system. Figure 17 In summary, this invention, through the engineered construction of a highly stable, high-encapsulation-rate hierarchical composite structure, enhances the efficacy of photodynamic therapy (PDT) by optimizing cellular endocytosis efficiency and environmental adaptability while maintaining intrinsic photophysical activity, providing a novel technical pathway for precisely regulating the biomedical effects of photosensitizers.

[0092] In summary, the dihydroporphyrin supramolecular assemblies prepared in this invention exhibit the following morphological characteristics: transmission electron microscopy (TEM) shows a precise transition from irregular lamellar structures to highly uniform nanosphere structures; regarding particle size distribution, dynamic light scattering (DLS) testing reveals that the obtained spherical particles have a size distribution between 100-200 nm, with a polydispersity index (PDI) optimized to below 0.1, demonstrating excellent monodispersity; and in terms of cell-killing ability (PDT activity), the spherical assemblies exhibit the most significant phototoxicity, with a killing efficiency far superior to traditional assembly systems. After introducing liposomes, the assemblies successfully transitioned from molecular adsorption to a highly uniform encapsulated structure; in terms of encapsulation performance, the introduction of liposomes significantly increased the encapsulation efficiency (EE) from 28.2% to 79.7%; and in terms of cellular uptake, the assemblies exhibit uptake efficiency far superior to traditional physical mixing systems. Thanks to the above advantages, this composite assembly exhibited the most significant cytotoxicity under light conditions, with a killing efficiency superior to other experimental groups.

[0093] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for producing a chlorin supramolecular assembly, characterized by, The Y+S composite microfluidic chip is used to realize controllable preparation of nanospherical Ce6 supermolecular assembly.

2. The method for preparing a chlorin supermolecule assembly according to claim 1, wherein The method comprises the following steps: S1, configuring an assembly phase solution: the organic phase is a dimethyl sulfoxide solution of Ce6, and the aqueous phase is ultrapure water or a liposome aqueous phase suspension; S2, microfluidic flow field regulation and assembly: the organic phase and the aqueous phase are respectively pumped into the corresponding sample inlet channels of the Y-type chip through a syringe pump, and the flow rates of the two phases are controlled to be 1-5 μL / min; S3, collection and aging: the assembly is continuously collected from the sample outlet of the S-type chip, and is aged to obtain nanospherical Ce6 supermolecular assembly with uniform morphology.

3. The method for preparing a chlorin supermolecular assembly according to claim 2, characterized by, In step S1, the concentration of the dimethyl sulfoxide solution of Ce6 is 0.05-0.1 mg / mL.

4. The method of preparing a chlorin supermolecule assembly according to claim 2, wherein In step S1, the preparation method of the liposome aqueous phase suspension comprises the following steps: mixing dimyristoyl phosphatidylcholine, (2,3-dioleoyl-propyl)-trimethylamine, DSPE-PEG2000 and cholesterol according to a mass ratio of 16:2:1:1, hydrating at 37 °C, and then performing membrane extrusion for 11-15 times, and then dispersing in ultrapure water to obtain.

5. The method of preparing a chlorin supermolecule assembly according to claim 2, wherein In step S2, the flow rate ratio of the organic phase to the aqueous phase is 7.5:5-5:

5.

6. The apparatus for use in the production method according to any one of claims 1 to 5, characterized in that, The Y+S composite microfluidic chip is a Y-type mixing zone connected with a continuous arc-shaped S-type curved flow channel at the rear end, the width and height of the flow channel are both 300 μm, and the flow channel has a regular square cross section.

7. The apparatus of claim 6, wherein, The flow channel intersection angle of the Y-type mixing zone is 75-85°.

8. The apparatus of claim 6, wherein, The S-type curved flow channel is provided with 10-15 arc-shaped bends, the width of the bends is 300-600 μm, the inner radius is 650 μm, and the outer radius is 950 μm.

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