A hydrophilic drug supramolecular nanoscale drug delivery system and an assembling method thereof

Abstract

The application discloses a kind of hydrophilic drug supramolecular nanoscale drug delivery systems and its assembly method, belong to medical technical field, hydrophilic drug, such as tirapazamine (Tirapazamine, TPZ), temozolomide (Temozolomide, TMZ), gemcitabine hydrochloride (Gemcitabine hydrochloride, GEM) or 5-azacytidine (5-azacytidine, 5AC) as model drug, with dihydrophenanthrene (Ce6) is directly co-assembled under ultrasonic condition, forms nanoscale drug delivery system;The method does not need chemical synthesis and greatly simplifies the preparation of nanoscale drug delivery system. It is proved by experiment, the supramolecular hydrophilic drug (CTNPs) assembled in the application can effectively improve the intratumoral accumulation of drug in tumor-bearing mice, and can significantly inhibit the growth and metastasis of tumor through synergistic antitumor effect. Therefore, the supramolecular assembly method in the application provides a new mode for developing easy-to-manufacture and multifunctional hydrophilic nanomedicine delivery system.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to a hydrophilic drug supramolecular nanoscale drug delivery system and its assembly method. Background Technology

[0002] Nanoscale drug delivery systems (NDDS) offer numerous advantages in cancer therapy, such as high controllability, multifunctionality, and tumor selectivity (allowing for the release of effective payloads spatiotemporally). They can perform multiple functions (e.g., synergistic effects, combined diagnosis and treatment) and enhance drug accumulation in tumor tissues through passive or active targeting. Hydrophobic drugs, due to their lower bioavailability, rely more heavily on NDDS; hydrophilic drugs can be administered directly without a delivery system. However, these highly active drugs are easily biodegraded and rapidly cleared by the kidneys, resulting in short circulation times and reduced therapeutic efficacy. Liposomes are one of the most classic hydrophilic drug delivery systems, approved and commercialized by the FDA. However, the drug loading capacity of liposomes is typically no more than 20%, with over 80% being ineffective excipients, leading to low drug loading efficiency. Furthermore, the accumulation of drugs in the body after delivery by non-drug carriers can also pose potential toxicity. It has been reported that immobilizing hydrophilic drugs on polymer nanostructures can effectively increase drug loading. For example, combining hydrophilic drugs with hydrophobic molecules to form amphiphilic prodrugs and assembling them into nanomedicines can significantly increase drug loading, and to a certain extent, 100% drug-loaded NDDSs can be formed.

[0003] For example, Yan's group combined irinotecan with chloramphenicol and assembled them into nanoprodrugs with 100% drug loading. We have also previously linked hydrophilic drugs, such as doxorubicin hydrochloride and irinotecan hydrochloride, with photosensitizers and assembled them into nanomedicines with drug loading rates of 51% and 100%, respectively. However, these conjugation methods are only applicable to certain drugs with active groups in their chemical structures. Even if a drug has an active moiety, drug release remains a significant issue because the conjugation bonds are reversible and can facilitate drug dissociation. Furthermore, the synthesis of these complexes introduces organic solvent residues and additional chemicals, such as conjugating agents and catalysts. The biosafety of the corresponding solvent / chemical residues from these chemical interventions needs careful evaluation before clinical translation, significantly increasing the cost of the translation process.

[0004] Therefore, we need to rethink how to optimize and transform NDDS by avoiding the preparation of increasingly complex nanoparticles. Supramolecular assembly, consisting of molecular complexes linked together by non-covalent bonds, such as hydrogen bonds, hydrophobic forces, π-π stacking, electrostatic interactions, or metallic connections, offers a new approach for multifunctional NDDS with high drug loading that does not involve chemical synthesis. NDDS constructed by supramolecular assembly consists of only one or two therapeutic / diagnostic drugs through interaction forces, forming specific morphologies. Supramolecular assembly greatly simplifies the preparation of NDDS because it does not require chemical synthesis, and small molecule drugs are among the most diverse components in terms of structure and function, thus avoiding the challenges of chemical synthesis often faced in the construction of functional materials. Heller et al. co-assembled fluorescent dyes (Congo Red or IR783) with small molecule drugs to form stable nanoparticles with ultra-high drug loading (up to 90%), and calculated and predicted their self-assembly rules through molecular simulations. However, their method only involved hydrophobic drugs. Traverso and colleagues predicted the co-assembly of a large number of excipients and drugs through simulations and calculations, and guided similar self-assembly through molecular dynamics simulations. However, the two works mentioned above did not summarize the general rules of supramolecular assembly; they only used their computational algorithms to describe the corresponding assembly mechanisms. Therefore, a supramolecular assembly method that can guide the administration of a wide range of hydrophilic drugs is essential. Summary of the Invention

[0005] To address the aforementioned problems, this invention aims to provide a hydrophilic drug supramolecular nanoscale drug delivery system and its assembly method, which can broadly guide the assembly of hydrophilic drug supramolecular nanoscale drug delivery systems.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] A method for assembling a hydrophilic drug supramolecular nanoscale delivery system, characterized by comprising the following steps:

[0008] S1: Premix the hydrophilic drug with the photosensitizer to obtain a premix;

[0009] S2: Add the premix from step S1 to deionized water under ultrasonic conditions;

[0010] S3: The system obtained in step S2 is subjected to sonication, followed by vortexing, to finally obtain supramolecular self-assembled nanomedicine.

[0011] Furthermore, the porphyrin photosensitizer is Ce6.

[0012] Furthermore, the hydrophilic drug is telatazamine, temozolomide, gemcitabine hydrochloride, or 5-azocytidine.

[0013] Furthermore, in step S1, the molar ratio of photosensitizer to hydrophilic drug is 1:0.5 to 3.

[0014] Furthermore, when the hydrophilic drug is telatazamine, the molar ratio of photosensitizer to hydrophilic drug is 1:2.

[0015] Furthermore, when the hydrophilic drug is temozolomide, gemcitabine hydrochloride, or 5-azocytidine, the molar ratio of the photosensitizer to the hydrophilic drug is 1:1.

[0016] Furthermore, in step S3, the system obtained in step S2 is subjected to ultrasonic treatment for 10 seconds, and the system is applied to a vortex for 5 seconds.

[0017] Furthermore, a hydrophilic drug supramolecular nanoscale drug delivery system was obtained by assembling using the assembly method described above.

[0018] The beneficial effects of this invention are:

[0019] 1. This invention proposes an assembly method for a hydrophilic drug supramolecular nanoscale drug delivery system. Hydrophilic drugs (such as TPZ, TMZ, GEM, or 5AC) are selected as model drugs and directly co-assembled with Ce6 at different molar ratios, greatly simplifying the preparation of NDDS without the need for chemical synthesis. Based on the assembly results between Ce6 and TPZ, this invention summarizes the rational rules for the supramolecular assembly of hydrophilic drugs and selects more hydrophilic drugs, such as TMZ, GEM, and 5AC, to successfully assemble them into nanostructures using similar assembly methods. This indicates that the assembly method proposed in this invention can broadly guide the improvement of drug delivery through supramolecular assembly of hydrophilic drugs.

[0020] 2. In this invention, Ce6 and TPZ-assembled nanoparticles were further selected as representatives to verify the antitumor activity of the assembled NDDS. The results showed that CT NPs could effectively accumulate in tumor tissues of tumor-bearing mice and significantly inhibit tumor growth and metastasis through synergistic antitumor effects. Therefore, it can be predicted that the assembly method in this invention will provide a new model for developing easily manufactured and powerful nanomedicines and guide a wider range of hydrophilic drug delivery. Attached Figure Description

[0021] Figure 1 This is the supramolecular assembly result of Ce6 and TPZ in this invention.

[0022] Figure 2 This refers to the fluorescence quenching after co-assembly of Ce6 and TPZ in this invention.

[0023] Figure 3 The results for hydrogen bonds / molecules in different simulation systems are shown in Table 1 of this invention.

[0024] Figure 4This is a simulation of the self-assembly molecular process of CT NP in this invention.

[0025] Figure 5 This is the supramolecular assembly result of Ce6 with temozolomide, gemcitabine hydrochloride and 5-azocytidine in this invention.

[0026] Figure 6 This invention represents the result of pyrophyllate a co-assembly with TPZ, TMZ, GEM, and 5AC following a supramolecular assembly mechanism.

[0027] Figure 7 This is the result of the self-assembly between doxorubicin and MSA-2 in this invention.

[0028] Figure 8 These are the characteristic results of CT NPs for the supramolecular assembly of hydrophilic drugs in this invention.

[0029] Figure 9 This presents the in vitro antitumor efficacy results of CT NPs against 4T1 cells in this invention.

[0030] Figure 10 This is the in vivo tissue distribution result of CT NPs in 4T1 tumor-bearing mice in this invention.

[0031] Figure 11 This presents the in vivo antitumor efficacy results of CT NPs on 4T1 tumor-bearing mice in this invention.

[0032] Figure 12 The results of H&E staining of tumor tissue at the experimental endpoint in different treatment groups in Example 5 of the present invention are shown.

[0033] Figure 13 This is the biocompatibility result of CT NPs in this invention.

[0034] Figure 14 Images of the spleen at the experimental endpoint of different treatment groups in Embodiment 5 of the present invention. Detailed Implementation

[0035] To enable those skilled in the art to better understand the technical solutions of the present invention, the technical solutions of the present invention will be further described below in conjunction with the accompanying drawings and embodiments.

[0036] Example 1:

[0037] A method for assembling a hydrophilic drug supramolecular nanoscale delivery system includes the following steps:

[0038] S1: Premix the working solution of the hydrophilic drug and the photosensitizer to obtain a premix;

[0039] Specifically, the hydrophilic drug is telazamine (TPZ), the photosensitizer is Ce6, and the molar ratio of Ce6 to TPZ is 1:2.

[0040] S2: Add the premix from step S1 to deionized water under ultrasonic conditions;

[0041] S3: The system obtained in step S2 is subjected to ultrasonic treatment for 10s and then vortexed for 5s to obtain hydrophilic drug supramolecular assembly nanoparticles CT NPs, which is a hydrophilic drug supramolecular nanoscale drug delivery system.

[0042] Furthermore, to investigate the dynamic properties of Ce6 and TPZ molecules and the self-assembly process at different molar ratios, the molar ratios of Ce6 to TPZ were adjusted to 1:0.5, 1:1, 1:1.5, 1:2.5, and 1:3. Molecular dynamics simulations and comparative studies of the self-assembly process of Ce6 and TPZ at different molar ratios were then conducted using Amber 22.0 software.

[0043] The chemical molecular structures of Ce6 and TPZ were obtained from PubChem, and the force field parameters for Ce6 / TPZ were the CHARMM universal force field (CGenFF). Each system was immersed in an arbitrary solute atom extension. A cubic TIP3P water box was constructed, and appropriate amounts of Na+ or Cl- were added using VMD 1.9.4 software to neutralize the negative charge of Ce6. Furthermore, the ionization factors of Ce6 and TPZ were considered in the simulation, and different molecular ionization states were constructed using Chem3D and Chimera 1.16 software. The system was energy optimized using the conjugate gradient method and the steepest descent method, and the time step for all simulations was set to 2 fs. After heating the system and isothermating it at 250 K, a 600 ns molecular dynamics simulation was performed. Considering both the number of molecules in the simulation system and the simulation time, two simulation systems of different sizes were constructed to more comprehensively analyze the objectivity of the simulation results. In the smaller system, the molar ratio of Ce6 to TPZ was 1:0.5, 1:1, 1:1.5, 1:2, 1:2.5, and 1:3, with the number of Ce6 molecules set to 32, and the number of TPZ molecules adjusted accordingly. The small system consists of approximately 5000 atoms, and the simulation time is approximately 600 ns. In the large system, the number of Ce6 molecules is set to 100, and the TPZ number varies according to the molar ratio. The total number of atoms in the large system ranges from 80,000 to 125,045, and the simulation time is approximately 1000 ns.

[0044] To elucidate binding affinity and self-assembly processes, the number of hydrogen bonds was calculated using Chimera 1.16 software to assess the dynamic binding processes in different systems. The ratio of hydrogen bonds to molecules was used for calculation and trend plotting. Hydrogen bonds are considered stable if the number of hydrogen bonds is within the confidence interval [ρ-3σ, ρ+3σ].

[0045] The particle size distribution and polydispersity index (PDI) of CTNPs were measured using a Zetasizer (ZS90, Brookhaven, USA). Their morphology was observed using a transmission electron microscope (TEM, Philips CM120) at an accelerating voltage of 120 kV. To prepare the TEM samples, an aqueous suspension of CT NPs was dropped onto a copper grid and dried at room temperature. The UV / Vis absorption and fluorescence spectra of the CTNPs were measured using a UV / Vis spectrometer (UV-1800) and a fluorescence spectrometer (RF-6000), respectively.

[0046] The results are attached. Figure 1 As shown, in the appendix Figure 1 In the diagram, a represents the chemical structures of Ce6 and TPZ; b represents the particle size distribution, PDI, morphology, and MD simulation of CTNPs assembled with Ce6 and TPZ at a 1:0.5 ratio (scale bar: 100 nm); c represents the particle size distribution, PDI, morphology, and MD simulation of CTNPs assembled with Ce6 and TPZ at a 1:1 ratio; d represents the particle size distribution, PDI, morphology, and MD simulation of CTNPs assembled with Ce6 and TPZ at a 1:1.5 ratio; e represents the particle size distribution, PDI, morphology, and MD simulation of CTNPs assembled with Ce6 and TPZ at a 1:2.0 ratio; f represents the particle size distribution, PDI, morphology, and MD simulation of CTNPs assembled with Ce6 and TPZ at a 1:2.5 ratio; and g represents the particle size distribution, PDI, morphology, and MD simulation of CTNPs assembled with Ce6 and TPZ at a 1:3.0 ratio. Particle size distribution, PDI, morphology, and MD simulation plots of NPs; h represents the NN-C32T64 system, with Ce6-TPZ in a non-ionized state; i represents the IN-C32T64 system, with Ce6 ionized and TPZ non-ionized; j represents the NI-C32T64 system, with Ce6 non-ionized and TPZ ionized; k represents the II-C32T64 system, with Ce6 and TPZ in an ionized state.

[0047] from Figure 1 As can be seen, Ce6 has a large planar structure, which can provide hydrophobicity and π-π stacking interactions to drive supramolecular assembly. Figure 1(a) It also contains three ionizable carboxylic acids that interact with drugs via hydrogen bonding or electrostatic interactions. These properties make Ce6 an ideal assembly core for forming nanostructures through supramolecular assembly. Ce6 molecules themselves can form submicron-scale nanoparticles, indicating that hydrophobic and π-π stacking interactions can drive the assembly of Ce6 molecules into specific structures.

[0048] From the appendix Figure 1 As shown in figures b to g, Ce6 and TPZ co-assembled into spherical nanoparticles at ratios of 1:0.5, 1:1, 1:1.5, and 1:2. In these combinations, all CTNPs exhibited reasonable particle size distribution and good polydispersity index (PDI). When the ratio increased from 1:2 to 1:2.5, the PDI significantly increased from 0.2 to 0.4, indicating that the CT NPs formed at 1:2.5 were not uniform. At a ratio of 1:3, the PDI was higher, and the DLS results were poorer, suggesting that a higher proportion of TPZ could disrupt the co-assembly of Ce6-TPZ. Since the CT NPs were directly assembled from pure photosensitizer (Ce6) and chemotherapeutic agent (TPZ), they essentially possessed 100% drug loading. MD simulations showed similar results compared to transmission electron microscopy (TEM) results.

[0049] Since π-π stacking leads to fluorescence quenching of Ce6, the fluorescence behavior of CTNPs at different ratios was also investigated in this invention. When Ce6 and TPZ were co-assembled at a ratio of 1:0.5, fluorescence quenching was significant, as shown in the attached figure. Figure 2 As shown, in the appendix Figure 2 In the figures, a represents the fluorescence spectra of Ce6 and TPZ mixed in different ratios, with a ratio of 1:0 indicating pure Ce6; b represents the fluorescence intensity of a at 660 nm. The concentration of Ce6 was set to 100 μM, and the amount of TPZ was adjusted accordingly. With increasing TPZ content, the fluorescence quenching became stronger. The quenching was strongest at a ratio of 1:2.5, while fluorescence recovered somewhat when the TPZ ratio reached three times that of Ce6. The optical properties of the synthesized CT NPs reflected that the excessive TPZ molecules disrupted co-assembly, which showed good consistency with the TEM and MD results. Considering particle size distribution, PDI, morphology, and TPZ loading, a Ce6 to TPZ molar ratio of 1:2 (C32T64) was selected in this invention to further investigate the assembly mechanism of CT NPs.

[0050] In summary, Ce6 can co-assemble with TPZ into nanostructures, indicating that certain intermolecular interactions drive the supramolecular assembly between Ce6 and TPZ. To better understand the assembly mechanism, the aforementioned 600 ns MD simulations were performed on different Ce6 and TPZ self-assembly systems. Using hydrogen bonding as a characterization of the self-assembly process, we calculated the hydrogen bond / molecule ratio and visually plotted the trend. The results are shown in Table 1 and Appendix. Figure 3As shown, in the appendix Figure 3 In the diagram, (a) compares the number of hydrogen bonds in different ratios between Ce6 and TPZ. (b) compares the number of hydrogen bonds in different ionization states of Ce6 and TPZ. C represents Ce6; T represents TPZ; I - ionized, N - non-ionized; II - both Ce6 and TPZ are ionized; IN - Ce6 is ionized, TPZ is non-ionized; NN - both Ce6 and TPZ are non-ionized. It is evident that C32T64 has the highest hydrogen bond / molecule ratio among the six combinations, indicating that the 1:2 ratio of Ce6 and TPZ forms a significantly more compact structure than other simulated systems (high consistency with characterization data). Based on the relative radial particle density and hydrogen bond network of the drug molecules, we chose a 1:2 ratio to systematically elucidate the assembly mechanism. Considering different ionization states of Ce6 and TPZ molecules, and analyzing the self-assembly process through MD simulations, we explored the non-covalent interactions that may dominate supramolecular assembly (see attached diagram). Figure 1 (As shown in the attached image). Taking the C32T64 system as an example, after simulation, it dynamically formed a compact nanoaggregate from its initial random spontaneous configuration, as shown in the attached image. Figure 4 As shown in the figure. The results indicate that the intermolecular hydrogen bonds NH···O=C and OH···O=C are primarily responsible for the internal stability of the structure. Furthermore, π-π interactions and van der Waals interactions between the large planar rings of Ce6 molecules were simulated. Compared to the non-ionized Ce6 system and the ionized TPZ system, Ce6 molecules in the non-ionized system tend to approach and bind tightly to each other through hydrophobic interactions rather than self-assembling with TPZ. This reflects that the ionization of Ce6 molecules in the system may be crucial for the supramolecular assembly of Ce6-TPZ. To evaluate the important role of TPZ, we performed similar ionization and non-ionization operations on the TPZ molecules, and the results are shown in Table 1 and below. Figure 3 As shown in Figure b, with the decrease in the degree of hydrogen bonding between TPZ molecules, the aggregation degree of hydrogen bond distribution decreases significantly, and the H on the amino group of TPZ may form hydrogen bonds with oxygen on other TPZ molecules. Furthermore, when TPZ molecules are in a non-ionized state, NO···HO tends to form hydrogen bonds between TPZ molecules. The different ionization states of the two molecules also have a certain influence on the type of hydrogen bonds during assembly. As shown in the figure, Ce6-TPZ molecules have two main types of hydrogen bonds: NH···O=C( Figure 1 c) and NO···HO( Figure 1 In section d), when TPZ molecules are ionized, NH···O=C seems to be preferred over NO···HO.

[0051] Table 1 Results of hydrogen bonding / molecules in different simulation systems

[0052]

[0053] Note: C represents Ce6. T represents TPZ.

[0054] I - Ionized, N - Non-ionized.

[0055] II-Ce6 and TPZ are ionized.

[0056] IN-Ce6 is ionized, while TPZ is non-ionized.

[0057] NN-Ce6 and TPZ are non-ionized.

[0058] In summary, multiple interactions, including the hydrophobic and π-π interactions of Ce6, and the electrostatic and hydrogen bonding interactions between Ce6 and drugs, provide important driving forces for self-assembly, contributing to the formation of stable nanoassemblies. Therefore, we summarize the rational rules for the assembly of Ce6-based supramolecular structures with hydrophilic drugs: Ce6 can co-assemble with drugs to form nanostructures dominated by hydrogen bonds between the hydrophilic drug and Ce6, and the degree of ionization of the molecules.

[0059] Example 2:

[0060] Example 2 provides a supramolecular assembly method for a hydrophilic drug, wherein the hydrophilic drug is temozolomide (TMZ), the mixing ratio of Ce6 to the hydrophilic drug is 1:1, and other assembly methods are exactly the same as in the example.

[0061] Example 3:

[0062] Example 3 provides a supramolecular assembly method for a hydrophilic drug, wherein the hydrophilic drug is gemcitabine hydrochloride (GEM), the mixing ratio of Ce6 to the hydrophilic drug is 1:1, and other assembly methods are exactly the same as in the example.

[0063] Example 4:

[0064] Example 4 provides a supramolecular assembly method for a hydrophilic drug, wherein the hydrophilic drug is selected as 5-azocytidine (5AC), the mixing ratio of Ce6 to the hydrophilic drug is 1:1, and other assembly methods are exactly the same as in the example.

[0065] Molecular dynamics simulations were performed on the hydrophilic drug supramolecular nanoscale drug delivery systems assembled in Examples 2 to 4 using the simulation method described in Example 1. The results are shown in the appendix. Figure 5 As shown. (Attached) Figure 5In the diagram, a represents the chemical structure and hydrophilicity (LogP) of temozolomide (TMZ), gemcitabine hydrochloride (GEM), and 5-azocytidine (5AC); b represents the particle size distribution and PDI of Ce6-TMZ NPs at different mixing ratios; c represents the particle size distribution and PDI of Ce6-GEM NPs at different mixing ratios; d represents the particle size distribution and PDI of Ce6-5AC NPs at different mixing ratios; e represents the transmission electron microscopy (TEM) image of Ce6-TMZ NPs; f represents the TEM image of Ce6-GEM NPs; g represents the TEM image of Ce6-5AC NPs, with a scale bar of 100 nm; h represents the MD simulation and three-dimensional chemical structure diagram (rod model) of Ce6-TMZ NPs; i represents the MD simulation and three-dimensional chemical structure diagram (rod model) of Ce6-GEM NPs; j represents the MD simulation and three-dimensional chemical structure diagram (rod model) of Ce6-5AC NPs. The concentration of Ce6 was set to 100 μM, and the drug concentration was adjusted accordingly. Hydrogen bonds are represented by red dashed lines.

[0066] from Figure 5 As can be seen, TMZ and Ce6 can assemble into nanoparticles at different mixing ratios. Similar to TPZ, TMZ forms relatively poor nanoparticles at higher molar ratios. The PDI of Ce6-TMZ NPs begins to increase at a 1:2.5 ratio; when the mixing ratio is increased to 1:3, the particle size of Ce6-TMZ NPs increases from ~120 nm to over 300 nm, and the PDI approaches 0.4. We also selected the hydrophilic drug GEM to co-assemble with Ce6. GEM can form nanoparticles with Ce6 at all six mixing ratios. The high ratio of 1:3 shows that the PDI is also higher than the other five mixtures, indicating that a high proportion of hydrophilic drugs is detrimental to Ce6-based supramolecular assembly.

[0067] Furthermore, using the highly hydrophilic drug 5AC and Ce6 in different Ce6 ratios for co-assembly, nanoparticles were formed with 5AC and Ce6 at ratios of 1:0.5 and 1:1. However, when the mixing ratio was 1:2 or higher, DLS could not detect the nanoparticles. This result suggests that Ce6-based supramolecular assembly with hydrophilic drugs will occur within a reasonable range, as more hydrophilic drugs may interfere with self-assembly.

[0068] Based on the above results, the synthesized nanoparticles in an equimolar ratio (Ce6:drug, 1:1), including Ce6-TMZ NPs and Ce6-GEM NPs, were observed using transmission electron microscopy (TEM). As shown in the TEM images, these combinations all formed spherical nanoparticles. MD simulations of Ce6-TMZ NPs, Ce6-GEM NPs, and Ce6-5AC NPs showed that all three hydrophilic drugs could form stable nanostructures with Ce6-based molecules, with hydrogen bonds dominating self-assembly. Among these hydrophilic drugs, 5AC exhibited significantly higher water solubility than TMZ, GEM, and TPZ, indicating that its high water solubility is unfavorable for nanostructure assembly. The hydrogen bonds formed between hydrophilic drug molecules are more conducive to nanostructure formation than other types of hydrogen bonds.

[0069] Furthermore, this assembly mechanism can be applied to more molecular couplings, such as... Figure 6 As shown, a porphyrin derivative a (PPa) containing a carboxylic acid can be co-assembled with TPZ / TMZ / GEM / 5AC to form nanoparticles with a size of 80–200 nm, all with a PDI below 0.25. The STING agonist MSA-2 containing a carboxylic acid can be co-assembled with doxorubicin to form nanoparticles with a size of approximately 100 nm and a PDI of 0.137, as shown in the attached figure. Figure 7 As shown, in the appendix Figure 7 In the diagram, a represents the chemical structures of doxorubicin and MSA-2. b represents the particle size distribution and PDI of the MSA-2 and doxorubicin co-assembled nanoparticles. The mixing ratio was set to 1:1. Hydrogen bonds between doxorubicin and MSA-2 link them together, while hydrophobic forces and π-π stacking interactions dominate the supramolecular assembly. These results demonstrate that the assembly mechanism in this invention can guide a wide range of hydrophilic drug delivery, and even extend to both hydrophobic and hydrophilic drugs. Similar to CTNPs, the supramolecularly assembled NDDSs described above are 100% drug-loaded.

[0070] Example 5:

[0071] Example 5 presents a performance study of the CTNPs assembled in Example 1, specifically including:

[0072] (1) Evaluation of the critical aggregation concentration (CAC) of CTNPs

[0073] The co-occurrence ratio (CAC) of CT NPs was determined using the pyrene radiation method. 1 μL of 0.1 mM pyrene-acetone solution was added to 999 μL of CT NPs solutions of different concentrations, and the mixture was incubated at 37 °C for 2 h. After incubation, the fluorescence of pyrene was evaluated using a fluorescence spectrophotometer (excitation length 335 nm), and the CAC was calculated.

[0074] (2) Generation and stability of ROS in vitro by CTNPs

[0075] Using DCFH-DA as a probe, the ability of CTNPs to promote ROS generation in vitro was detected. Solutions of different concentrations of CT NPs were incubated with DCFH-DA (10 μm) for 30 min, and the DCFH-DA (10 μm) was pretreated with H2O2. Then, the mixed solution was exposed to a 680 nm laser (0.4 W / cm²). 2 3 min. ROS generation was indicated by measuring the fluorescence of DCF. The stability of CTNPs was evaluated by continuously monitoring the particle size and PDI of CTNPs in aqueous solution for 14 days.

[0076] (3) Drug release from CTNPs

[0077] Drug release from CTNPs was studied in vitro. A 3.5 kDa MWCO aqueous solution of CTNPs was injected into a dialysis chamber (Pierce Chemicals) and then immersed in 1 L of phosphate-buffered saline (PBS) (pH 5.0 and 7.4). The PBS medium was kept at a constant temperature of 37°C with moderate stirring. Samples were collected at different time intervals, and the concentration of TPZ in the samples at different time points was determined by HPLC.

[0078] CT NPs were selected for subsequent biological experiments, and the characterization results are attached. Figure 8 As shown. In the appendix Figure 8 In the figures, a) shows the UV-Vis absorption spectra of free Ce6 + 10% SDS, free TPZ, CT NPs, and CT NPs + 10% SDS; b) shows the fluorescence spectra of free Ce6 + 10% SDS, CT NPs, and CT NPs + 10% SDS. 10% SDS was added to increase the water solubility of Ce6 during optical performance testing. c) shows the stability results of CT NPs after 14 days of continuous monitoring. d) shows the critical aggregation concentration (CAC) of CT NPs. e) shows the in vitro ROS generation promotion results of CT NPs; f) shows the drug release curves of CT NPs at simulated neutral and lysosomal pH conditions.

[0079] From the appendix Figure 8 As can be seen, Ce6 has two main characteristic peaks at 405 nm (Soret band) and 670 nm (q band); while the characteristic peak of CT NPs at 405 nm broadens, and the q band shifts to the near-infrared region by about 10 nm. After adding a surfactant (sodium dodecyl sulfate, SDS) that can disrupt the nanostructure, the Soret band of CT NPs narrows, and the q band shows a blue shift, indicating that Ce6 molecules form j-aggregates in CT NPs. Fluorescence spectroscopy shows that the fluorescence of Ce6 is quenched after the formation of CT NPs and can be recovered in the presence of SDS, and its optical properties prove the successful assembly of Ce6 and TPZ.

[0080] The critical aggregation concentration (CAC) of CT NPs was calculated to be 2.08 μM, as shown in the attached figure. Figure 8 As shown in Figure c, the lower CAC demonstrates that CTNPs can maintain their nanostructure after dilution. The stability of CTNPs was assessed by continuously monitoring their size distribution and PDI for 14 days. (See attached figure) Figure 8 As shown in Figure d, the size and PDI of CTNPs remained within a reasonable range without significant fluctuations over 14 days, indicating that CT NPs can remain stable at room temperature for at least two weeks. The PDT effect of CT NPs was detected by measuring ROS generation. The ROS generated by CT NPs under laser irradiation was concentration-dependent, indicating that CT NPs can trigger the PDT effect and could be applied to cancer treatment.

[0081] It is hypothesized that changes in pH modulate the ionization of Ce6 and TPZ, disrupting the nanoparticle structure and triggering drug release. Therefore, the drug release of CTNPs was investigated by adjusting the acidic conditions of PBS. (See attached image.) Figure 8 As shown in Figure f, CTNPs remain stable at neutral pH, releasing the drug in a burst manner during the first few hours, then gradually reaching a plateau. Drug degradation results indicate that CTNPs release the drug in acidic pH microenvironments (such as lysosomes).

[0082] (4) Cell culture and cytotoxicity test

[0083] Mouse breast cancer 4T1 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum and 1% antibiotics, and incubated at 37°C, 10% humidity, and 5% CO2. 4T1 cells were then seeded into 96-well plates and cultured overnight at a cell density of 5 × 10⁶ cells per well. 3 Cells were treated with different concentrations of Ce6, TPZ, a mixture of free Ce6 / TPZ, and CTNPs for 24 hours, followed by washing twice with PBS and then replacing with fresh culture medium. The laser-treated group was exposed to a 680nm laser (0.4w / cm²). 2 After incubating for 1 minute, incubate for another 24 hours. Then add MTT solution (3 mg / mL) and incubate with the cells for 4 hours. Dissolve the cells in DMSO and measure cell viability by detecting the OD value at 490 nm using a microplate reader.

[0084] (5) Cellular uptake of CTNPs

[0085] Four T1 cells were seeded in four-well culture dishes and incubated overnight, with a cell density of 5 × 10⁶ cells per well. 4Cells were then treated with 10 μM CTNPs for 2 h and 4 h, respectively. After washing twice with PBS, the nuclei and lysosomes were stained with Hoechst 33342 (2 μg / mL) and Lysotracker green for 10 min, respectively. Cellular uptake of CTNPs was observed using a confocal laser scanning microscope (CLSM).

[0086] (6) Photodynamic assessment of CTNPs at the cellular level

[0087] To evaluate the ability of CT NPs to promote ROS generation at the cellular level, 4T1 cells were seeded at a density of 5 × 10⁶ cells / cells. 4 Cells were cultured overnight in 4-well culture dishes until fully adhered. They were then treated with 10 μM CT NPs for 4 hours and exposed to a 680 nm laser (0.4 w / cm²). 2 Cells were stained with DCFH-DA (10 μM) for 40 min. Subsequently, the cell nuclei were stained with Hoechst 33342 for 10 min. The DCF signal, representing the production of ROS, was observed by CLSM.

[0088] (7) Protein immunoblotting assay

[0089] The expression of hypoxia-inducible factor 1 (HIF-1α) and α-enolase (ENO1) in 4T1 cells was detected by Western blotting. Total protein was extracted using a whole-cell lysis kit (Keygen Biotech, Nanjing, China). Proteins were separated on a 10% polyacrylamide gel and transferred to nitrocellulose membranes by wet transfer. The membranes were incubated overnight with HIF-1α monoclonal antibody, ENO-1 monoclonal antibody, and actin monoclonal antibody. Detection and visualization were performed using a high-sensitivity chemiluminescent substrate (Beyotime, USA) combined with goat anti-rabbit secondary antibody.

[0090] The results of the studies in (4)-(7) are attached. Figure 9 As shown, in the appendix Figure 9 In the figures, a represents the uptake and distribution of CT NPs in 4T1 cells. The scale bar is 20 μm. b represents ROS production by Ce6-mediated PDT in 4T1 cells, with a scale bar of 40 μm. c represents the quantitative results of ROS production, p < 0.001. d represents the detection of hypoxia-related proteins by Western blotting, - indicating no laser irradiation and + indicating laser irradiation. e represents the in vitro antitumor effect of CT NPs and the control group. f represents the combination index (CI) between PDT and chemotherapy, with a CI below 1 considered synergistic. g represents the synergistic effect of Ce6 and TPZ in CT NPs, indicating a stronger antitumor effect.

[0091] The intracellular distribution of CTNPs was investigated by observing the intrinsic fluorescence of Ce6. (See attached image.) Figure 9 As shown in Figure a, cellular uptake of CTNPs is time-dependent, with Ce6 mainly distributed in the cytoplasm. Fluorescent staining of lysosomes revealed good co-localization of the fluorescent region of Ce6 with lysosomes, suggesting that the acidic pH of lysosomes may promote drug release.

[0092] DCFH-DA was used as a fluorescent probe to assess ROS production of CTNPs in 4T1 cells. (See attached image) Figure 9 As shown in Figure b, PBS-treated cells did not exhibit any DCF fluorescence signal even after laser irradiation, indicating that laser cannot trigger ROS generation in the absence of a photosensitizer. In the CT NPs treatment group containing a photosensitizer, ROS generation significantly increased after laser irradiation, while the corresponding group without laser treatment showed only a weak DCF signal. Quantitative results are attached. Figure 9 As shown in Figure c, the intracellular ROS generated after laser irradiation with CT NPs was 13.8 times higher than that without laser treatment, demonstrating that CT NPs can promote intracellular ROS generation. Ce6, as a photosensitizer, absorbs light energy and excites ambient oxygen to generate ROS. This invention hypothesizes that PDT (Protein-Dependent Transformation) consumes oxygen, leading to increased cellular hypoxia. To verify this result, we detected the expression of hypoxia-related proteins using Western blotting. Figure 9 As shown in Figure d, the expression of HIF-1α and ENO1 in cells treated with laser by CT NPs was significantly upregulated compared to the CT NPs group without laser treatment, indicating that PDT can induce cellular hypoxia. These results demonstrate that CT NPs can effectively trigger the PDT effect under laser irradiation, thereby consuming intracellular oxygen and inducing intracellular microenvironment hypoxia.

[0093] TPZ is a hypoxia-activated prodrug that can be activated into hydroxyl radicals and benzotriazine radicals (BTZ) under hypoxic conditions, leading to DNA structural damage and inducing apoptosis. The efficacy of TPZ depends on the degree of hypoxia, and Ce6 PDT can consume oxygen to create a hypoxic intracellular environment, thereby activating TPZ and enhancing antitumor effects through synergistic action. First, we used the MTT assay to evaluate the cytotoxicity of CT NPs on 4T1 cells. 4T1 cells were incubated with different concentrations of free photosensitizer (Ce6), free chemotherapeutic agent (TPZ), free mixed drug (Ce6+TPZ), and CT NPs, with a parallel control group receiving laser treatment. Results are attached. Figure 9As shown in Figure e, free Ce6 exhibits almost no cytotoxicity without laser irradiation, while it displays significant phototoxicity under laser irradiation, indicating that PDT is highly effective in cancer treatment. Free TPZ shows anti-tumor efficacy at high concentrations, while CTNPs are even more effective, suggesting that nanoparticle formulations can enhance the therapeutic effect of TPZ. The free mixed drug group shows similar inhibitory effects on tumor cells as free TPZ, and exhibits stronger cytotoxic activity after laser irradiation. Notably, the CTNPs+L group shows the most effective anti-tumor efficacy against 4T1 cells, indicating a potential synergistic effect between PDT and chemotherapy in CTNPs. Further calculations showed that the combination index (CI) of PDT and chemotherapy at different concentrations was less than 1, indicating a synergistic effect of Ce6 and TPZ under laser irradiation, as shown in the attached figure. Figure 9 As shown in Figure f. Therefore, Ce6-mediated PDT and TPZ can exert a "1+1>2" effect on cancer cells, as shown in the attached figure. Figure 9 As shown in Figure g, Ce6-mediated PDT consumes oxygen, making the intracellular microenvironment more hypoxic; TPZ is more effectively converted into TBZ free radicals and hydroxyl free radicals in the hypoxic microenvironment, thereby achieving a higher anti-cancer effect.

[0094] (8) Evaluation of tumor distribution of CTNPs

[0095] First, a 4T1 mouse model was established. Balb / c mice (6-8 weeks old, female) were purchased from Vital River Laboratory Animal Technology Co., Ltd. (Shanghai, China). All animal experiments were conducted strictly in accordance with the regulations of the Animal Use and Conservation Management Advisory Committee of Shanghai Jiao Tong University. All animals were housed under pathogen-free conditions and acclimatized to their environment for at least 5 days before the start of the experiment. 4 × 10⁴ mice were subcutaneously injected into the lower right back of the mice. 5 A mouse subcutaneous breast cancer model was established using 4T1 cells (50 μL PBS containing 10% matrix gel).

[0096] NIRF imaging was used to assess the distribution and intratumoral accumulation of CTNPs in 4T1 tumor-bearing mice to further determine the time point for laser therapy. Free Ce6 and CTNPs (10 mg / kg) were injected intravenously into 4T1 tumor-bearing mice (n=6). The distribution of free Ce6 and CTNPs was assessed by quantitative analysis of Ce6 fluorescence signals at different time points in the tumor site. For in vitro imaging of major organs and tumors, another group of tumor-bearing mice (n=6) had their major organs collected 6 hours after injection of Ce6 and CTNPs and used for NIRF imaging. Tumor tissue was further prepared into frozen sections, and nanoparticle accumulation at the tumor tissue level was observed using laser confocal microscopy.

[0097] The results are attached. Figure 10 As shown. In the appendix Figure 10In the figure, a shows the tumor distribution of CT NPs and free Ce6 in 4T1 tumor-bearing mice; b shows the quantitative fluorescence data of figure a; c shows the in vitro imaging of major organs and tumors in mice after injection of free Ce6 and CTNPs; d shows the quantitative fluorescence analysis of in vitro imaging; e shows the tissue distribution of Ce6 and CTNPs in frozen sections of tumors, with a scale bar of 25 μm.

[0098] The tumor distribution of CTNPs was assessed by monitoring the pre-existing fluorescence of Ce6 using NIRF, as shown in the attached figure. Figure 10 As shown in Figure a, free Ce6 accumulated at the tumor site 2 hours after injection and then rapidly dissipated. In contrast, CTNPs began to accumulate gradually 2 hours after injection, reaching their peak level at 6 hours, and then gradually faded. Quantitative results of fluorescence signal (see attached figure). Figure 10 (b) shows that, compared with free Ce6, the nanoparticle formulation has a longer accumulation time and residence time at the tumor site, indicating that CT NPs can effectively accumulate at the tumor site due to the passive targeting effect of the nanoparticles. Six hours after injection, in vitro imaging of the major organs and tumor tissues of tumor-bearing mice was performed, and the results are shown in the attached figure. Figure 10 As shown in C and D, free Ce6 significantly accumulates in both the liver and kidneys and is excreted via the kidneys. In contrast, CTNPs show similar accumulation in the liver but are less distributed in the kidneys, indicating slower clearance of CTNPs in vivo. Furthermore, frozen section tissue-level imaging results indicate that CTNPs accumulate more in the tumor 6 hours after injection (see attached image). Figure 10 (As shown in e). Animal imaging experiments showed that this nanoformulation can improve the tissue distribution, tumor accumulation, and retention of free drugs in mice.

[0099] (9) In vivo anti-tumor therapeutic effect

[0100] 4T1 tumor-bearing mice were randomly divided into 5 groups (n=6) to evaluate the in vivo antitumor efficacy of CTNPs. When the tumor volume reached 50 mm², the tumor was divided into 5 groups. 3 Dosing began around 2:00 PM. CT NPs (10 mg / kg) and corresponding control materials were administered intravenously, once every two days. Six hours after administration, based on tumor distribution results, the tumors were irradiated with a 680 nm laser (0.4 W / cm²). 2 5 min. The five groups are annotated as follows: PBS, free Ce6+TPZ with or without laser treatment (Mix / Mix+L), CTNPs with or without laser treatment (CT NPs / CTNPs+L). Body weight and tumor size were monitored twice weekly. Tumor volume was calculated using the formula: length × width × width / 2.

[0101] At the endpoint of the antitumor experiment, blood was collected from each mouse in EDTA-anticoagulated vessels for hematological analysis, and whole blood cell components were compared. Liver function was assessed by measuring serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP) levels. Kidney function was assessed by measuring uric acid (UA) and creatinine (CRE) levels. Additionally, tumors and major organs from each group were fixed in 4% (w / v) paraformaldehyde for 48 hours and then embedded in paraffin. The paraffin-embedded tumors and organs were sectioned into 10 nm thick sections and stained with hematoxylin and eosin (H&E).

[0102] The process of animal experiments is as follows: Figure 11 As shown, in the appendix Figure 11 In the example, 'a' represents the establishment of a subcutaneous 4T1 breast tumor model and the subsequent treatment of different groups. The laser (680nm) power was set to 0.4W / cm². 2 Irradiation time: 5 min; b represents the tumor at 0.4 W / cm². 2 Results of laser exposure for 5 minutes using an infrared thermal imaging camera; c shows tumor growth curves in different treatment groups; d shows optical photographs of tumors in different groups at the experimental endpoint; e shows changes in body weight of mice in different experimental groups; f shows optical photographs of lung metastases in different groups; g shows H&E staining of liver and lung metastases in different treatment groups, with black arrows indicating micrometastases, and a scale bar of 100 μm.

[0103] As attached Figure 11 As shown in Figure a, all materials were injected via the tail vein, administered every two days for a total of five doses. Based on the tumor distribution results, 680nm laser irradiation (0.4W / cm²) was performed 6 hours after administration. 2 5 min. The five treatment groups were PBS, Mix / Mix+L, and CTNPs / CT NPs+L, respectively. Tumor temperature monitored by an infrared thermal imaging camera showed that laser dose-induced hyperthermia was relatively low, with tumor tissue temperature approximately 39°C (see attached). Figure 11 (b) This demonstrates that subsequent laser treatment will primarily rely on PDT.

[0104] The tumor volume change curve is shown in the attached figure. Figure 11 As shown in Figure c, due to the high malignancy of 4T1 breast cancer, neither the PBS nor the Mix group showed significant anti-tumor effects, and the tumor volume rapidly increased to 1000 mm within two weeks. 3The above indicates that CT NPs showed superior efficacy compared to the Mix group, suggesting that nanoparticles can enhance the in vivo antitumor effect of TPZ. After combination with PDT, the antitumor effect of Mix+L was superior to both the Mix group and CT NPs, indicating that CT NPs combined with PDT can effectively inhibit tumor growth. CT NPs+L exhibited the strongest antitumor effect, demonstrating that CT NPs combined with PDT improved the chemotherapy effect of TPZ and synergistically inhibited tumor development. Tumor images at the endpoint also support that CT NPs+L can maximally inhibit tumor growth (see attached image). Figure 11 (as shown in d).

[0105] Histological examination showed that the tumor cells in the CTNPs+L treatment group had significantly sparse nuclei, further demonstrating that CTNPs+L can effectively inhibit tumor growth, as shown in the appendix. Figure 12 As shown.

[0106] As attached Figure 11 As shown in Figure e, mice in all treatment groups did not experience significant weight loss during treatment, indicating that the material did not cause significant acute toxicity.

[0107] It has been reported that 4T1 breast cancer cells can spontaneously metastasize to the lungs and liver; therefore, the anti-metastatic effect of this treatment was also evaluated in this invention. (See attached document.) Figure 11 As shown in f and g, significant micrometastatic nodules were observed in the lungs of mice treated with PBS, Mix, Mix+L, and CTNPs. In contrast, no significant metastatic nodules were found in the lungs of mice treated with CTNPs+L, which may be related to the potential immune response induced by phototherapy. No macroscopically visible metastatic nodules were found in the liver. Pathological analysis of lung and liver tissue sections was then performed. Significant micrometastases were observed in the lungs and liver tissues of mice treated with PBS, Mix, Mix+L, and CTNPs, while no significant metastases were found in the lungs and liver of mice treated with CTNPs+L. This indicates that CTNPs+L treatment has a good anti-metastatic effect. The above animal experimental results demonstrate that CTNPs have a synergistic anti-tumor effect of PDT and hypoxia-activated chemotherapy, effectively inhibiting primary tumor growth and distant metastasis.

[0108] Furthermore, the biocompatibility of all therapeutic formulations was evaluated in both histopathological and hematological aspects, and the results are attached. Figure 13 As mentioned above, in the appendix Figure 13In the table, a represents the histopathological results of mice in different treatment groups, with a scale bar of 100 μm; b represents the hematological and biochemical test results of mice in different treatment groups, abbreviated as: WBC - white blood cells, RBC - red blood cells, PLT - platelets, MCHC - mean corpuscular hemoglobin, HGB - hemoglobin, ALP - alkaline phosphatase, AST - aspartate aminotransferase, TP - total protein, ALB - albumin, CRE - creatinine, UA - uric acid. Spleen images of different treatment groups at the experimental endpoint are attached. Figure 14 As shown.

[0109] From the appendix Figure 13 and 14 The results showed no obvious pathological abnormalities in the H&E staining of major organs, including metastatic organs (liver, lung) or excretory organs (kidney). Except for the spleen, there were no significant differences in organ coefficients among the different treatment groups. The 4T1 breast cancer mouse model spontaneously induced spleen swelling; the CTNPs+L group had a lower spleen organ coefficient and a smaller spleen, indicating that CTNPs+L can reduce systemic inflammation.

[0110] The CT NPs+L group mice had the lowest white blood cell count among all experimental groups, indicating the lowest systemic inflammation compared to other groups. Other blood-related parameters remained within the normal range compared to normal mice. Furthermore, the levels of biochemical parameters were similar to those in normal mice, further demonstrating that our treatment did not induce significant toxicity. These results indicate that CT NPs have good biocompatibility and are a potential photodynamic chemotherapeutic agent.

[0111] In summary, this invention selects a photosensitizer (Ce6) as the core and forms CT NPs through supramolecular co-assembly with a hydrophilic drug (TPZ). The CT NPs were characterized, and their supramolecular assembly process was studied using MD simulations. Based on this, a widely applicable self-assembly mechanism is summarized to guide the supramolecular assembly of structurally similar drugs such as Ce6 with hydrophilic drugs. Using Ce6 and TPZ-assembled NPs as representatives, the in vivo and in vitro antitumor capabilities of CT NPs were further evaluated. The results show that the assembly method in this invention can significantly improve the in vivo bioavailability of hydrophilic drugs and effectively inhibit the growth and metastasis of breast cancer through a good synergistic effect. The supramolecular nanoparticle assembly method in this invention can significantly improve the drug loading and tumor targeting of hydrophilic drugs and is simple to prepare. This will provide a new model for developing easily manufactured and multifunctional NDDS and a reference for broader hydrophilic drug delivery.

[0112] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents.

Claims

1. A method for assembling a hydrophilic drug supramolecular nanoscale drug delivery system, characterized in that, Includes the following steps, S1: The hydrophilic drug is premixed with the photosensitizer Ce6 to obtain a premix; wherein the hydrophilic drug includes teirazamine, and the molar ratio of the photosensitizer to teirazamine is 1:2; S2: Add the premix from step S1 to deionized water under ultrasonic conditions; S3: The system obtained in step S2 is subjected to sonication for 10 seconds, and then the system is vortexed for 5 seconds to finally obtain supramolecular self-assembled nanomedicine.

2. A method for assembling a hydrophilic drug supramolecular nanoscale drug delivery system, characterized in that, Includes the following steps, S1: The hydrophilic drug is premixed with the photosensitizer Ce6 to obtain a premix; wherein the hydrophilic drug includes temozolomide, gemcitabine hydrochloride or 5-azocytidine; the molar ratio of photosensitizer to hydrophilic drug is 1:1; S2: Add the premix from step S1 to deionized water under ultrasonic conditions; S3: The system obtained in step S2 is subjected to sonication for 10 seconds, and then the system is vortexed for 5 seconds to finally obtain supramolecular self-assembled nanomedicine.

3. A hydrophilic drug supramolecular nanoscale delivery system assembled using the assembly method described in claim 1 or 2.

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