A pH-responsive lyotropic liquid crystal drug delivery system, a preparation method and application thereof

Abstract

The present application relates to a kind of pH response type lyotropic liquid crystal drug delivery system, its preparation method and application.The system includes phospholipid component, inverse liquid crystal induced lipid, organic solvent, pH response gate switch and positively charged therapeutic active substance.The system is flowable liquid before injection, forms lyotropic liquid crystal gel in situ after entering aqueous environment;Under physiological pH condition, pH response gate switch and cationic drug occur electrostatic interaction, so that system maintains inverse hexagonal phase and limits drug release;Under acidic pathological microenvironment, gate switch is protonated and induces system to transform to double-continuous inverse cubic phase, so as to promote sustained drug release.The present application can be used for the local treatment of solid tumor, bacterial infection, chronic inflammation and wound repair and other diseases accompanied by local acidic microenvironment.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically, to a pH-responsive lyotropic liquid crystal drug delivery system, its preparation method, and its applications. Background Technology

[0002] In the course of various diseases such as tumors, infections, inflammation, and tissue damage, lesions often form local pathological microenvironments that differ from normal tissues. Among these, a local acidic microenvironment is one of the more typical and common pathological features. For example, solid tumor tissues often exhibit a weakly acidic microenvironment due to enhanced glycolysis, lactic acid accumulation, and insufficient perfusion in tumor cells. During local bacterial infections, wound infections, or biofilm formation, bacterial metabolites, inflammatory cell infiltration, and tissue hypoxia can also lead to a decrease in local pH. In addition, pathological sites such as chronic inflammation, ischemic injuries, and difficult-to-heal wounds are often accompanied by tissue acidification. Therefore, utilizing the acidic microenvironment at the lesion site to achieve selective drug release is an important strategy for improving local therapeutic effects and reducing systemic toxicity.

[0003] In the treatment of the aforementioned diseases, many therapeutically active substances with positively charged or protonable amino groups have significant application value. For example, cationic antitumor peptides, antimicrobial peptides, aminoglycoside antibiotics, amino-containing small-molecule chemotherapeutic drugs, and some basic proteins can exert therapeutic effects through membrane action, cytotoxicity, antibacterial activity, immunomodulation, or tissue repair. However, these active substances typically suffer from poor in vivo stability, susceptibility to enzymatic degradation or clearance, short local retention time, and rapid diffusion after administration. Especially during local injection, free drugs often undergo burst release, making it difficult to maintain effective drug concentrations at the lesion site for extended periods, while also increasing the risk of toxic side effects after the drug enters systemic circulation.

[0004] To improve drug delivery performance, various local delivery systems have been developed, including liposomes, polymer nanoparticles, hydrogels, microspheres, implants, and in-situ gels. These delivery systems can, to some extent, prolong drug retention time, improve drug stability, or reduce dosing frequency. However, existing delivery systems still have certain shortcomings: First, some carriers mainly rely on passive material degradation or drug concentration gradient diffusion for release, making it difficult to actively adjust the release rate according to changes in the lesion microenvironment; second, some hydrogel or nanoparticle systems still exhibit significant burst release at the initial stage of administration, making it difficult to achieve long-term, stable release; third, although some stimulus-responsive systems can respond to pH, temperature, enzymes, or redox environments, their response mechanisms are usually limited to chemical bond breaking, polymer swelling, or carrier degradation, resulting in insufficient controllability of the release process and limited universality for different types of cationic drugs.

[0005] Therefore, there is an urgent need to develop a new local drug delivery system to fully realize the selective response to the acidic microenvironment of pathology. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides a pH-responsive lyotropic liquid crystal drug delivery system, its preparation method, and its applications.

[0007] According to a first aspect of the present invention, a pH-responsive lyotropic liquid crystal drug delivery system is provided. The drug delivery system is a precursor composition that is in a liquid state before injection and capable of forming a lyotropic liquid crystal gel in situ upon entering an aqueous environment. Based on the total mass of the precursor composition, the precursor composition comprises: 50%–85% carrier matrix mixture; 5%–30% organic solvent; 1%–10% pH-responsive gating switch; and 0.1%–10% therapeutic active substance. The carrier matrix mixture comprises component A and component B, wherein component A is a phospholipid and component B is a... The drug delivery system is equipped with amphiphilic lipid molecules capable of inducing the formation of an antiphase liquid crystal phase; the pH-responsive gating switch is a pH-sensitive molecule containing a protonable anionic group; the therapeutic active substance is a therapeutic molecule carrying a net positive charge under physiological pH conditions; the drug delivery system restricts drug release under physiological pH conditions through electrostatic interaction between the pH-responsive gating switch and the therapeutic active substance, and releases the electrostatic interaction under acidic microenvironment due to protonation of the pH-responsive gating switch, while simultaneously inducing a topological phase transition in the lyotropic liquid crystal gel, thereby promoting the release of the therapeutic active substance.

[0008] Preferably, the mass ratio of component A to component B is 1:5 to 5:1.

[0009] Preferably, the mass ratio of component A to component B is 1:2 to 2:1.

[0010] Preferably, component A is selected from natural phospholipids, synthetic phospholipids, or a combination thereof.

[0011] Preferably, component A is selected from one or more of soybean lecithin, egg yolk lecithin, dipalmitoylphosphatidylcholine, and distearate phosphatidylcholine.

[0012] Preferably, component B is selected from one or more of sorbitan monooleate, glyceryl monooleate, diglyceryl dioleate, and phytanetriol.

[0013] Preferably, the organic solvent is a polar organic solvent that is miscible or partially miscible with water.

[0014] Preferably, the organic solvent is selected from one or more of anhydrous ethanol, dimethyl sulfoxide, N-methylpyrrolidone, propylene glycol, polyethylene glycol, and methanol.

[0015] Preferably, the pH-responsive gating switch is a carboxyl-containing unsaturated fatty acid.

[0016] Preferably, the pH-responsive gating switch is selected from one or more of oleic acid, linoleic acid, and linolenic acid.

[0017] Preferably, the pH-responsive gating switch is oleic acid.

[0018] Preferably, the therapeutic active substance is selected from one or more of cationic amphiphilic polypeptides, small molecule chemical drugs containing protonable amino groups, and therapeutic proteins with an isoelectric point greater than 7.4.

[0019] Preferably, the cationic amphiphilic polypeptide is selected from one or more of the following: Vpr1 protein-derived antitumor peptide (VDAP), bee venom peptide, LL-37, defensin, Xenopus antimicrobial peptide, and LTX-315.

[0020] Preferably, the small molecule chemical drug containing a protonable amino group is selected from one or more of doxorubicin, mitoxantrone, bupivacaine, and aminoglycoside antibiotics.

[0021] Preferably, the therapeutic protein with an isoelectric point greater than 7.4 is selected from one or more of basic fibroblast growth factor and lysozyme.

[0022] Preferably, the precursor composition does not contain an aqueous phase before injection, and after injection into an aqueous environment, the organic solvent diffuses into the surrounding aqueous phase, and the carrier matrix mixture absorbs water and self-assembles to form a lyotropic liquid crystal gel with an ordered nanoporous structure in situ.

[0023] Preferably, the lyotropic liquid crystal gel exhibits an inverted hexagonal phase at pH 7.4.

[0024] Preferably, under pH 7.4 conditions, the pH-responsive gating switch is in a deprotonated state and forms an electrostatic complex with the therapeutic active substance, thereby reducing the cumulative release of the therapeutic active substance within 7 days to less than 10%.

[0025] Preferably, under acidic conditions of pH 5.0 to 6.5, the pH-responsive gating switch undergoes protonation, and the lyotropic liquid crystal gel transforms from an anti-hexagonal phase to a bicontinuous anti-cubic phase.

[0026] Preferably, the bicontinuous antiphase cubic phase is an Im3̄m phase.

[0027] Preferably, under acidic conditions of pH 5.0 to 6.5, the cumulative release of the therapeutic active substance over 7 days is greater than 70%.

[0028] Preferably, the drug delivery system is a local injection drug delivery system.

[0029] Preferably, the drug delivery system is used to deliver therapeutically active substances to lesion sites accompanied by a local acidic microenvironment.

[0030] Preferably, the lesion site accompanied by a local acidic microenvironment is a solid tumor lesion, a bacterial infection lesion, a chronic inflammatory lesion, ischemic tissue, or traumatic tissue.

[0031] Preferably, the therapeutic active substance is a VDAP polypeptide, component A is lecithin, component B is sorbitan monooleate, the pH-responsive gating switch is oleic acid, and the organic solvent includes ethanol and / or dimethyl sulfoxide.

[0032] Preferably, the precursor composition comprises lecithin, sorbitan monooleate, oleic acid, an organic solvent, and a VDAP peptide.

[0033] Preferably, the therapeutic active substance is doxorubicin, component A is soybean lecithin, component B is diglyceride, the pH-responsive gating switch is oleic acid, and the organic solvent is N-methylpyrrolidone.

[0034] According to a second aspect of the present invention, a method for preparing the pH-responsive lyotropic liquid crystal drug delivery system is provided, comprising the following steps: dissolving component A, component B and a pH-responsive gating switch in an organic solvent to obtain a carrier matrix solution; adding a therapeutic active substance to the carrier matrix solution and mixing them uniformly to obtain a pH-responsive lyotropic liquid crystal precursor composition in a liquid state before injection; wherein, component A is a phospholipid, component B is an amphiphilic lipid molecule capable of inducing the formation of an antiphase liquid crystal phase, the pH-responsive gating switch is a pH-sensitive molecule containing a protonable anionic group, and the therapeutic active substance is a therapeutic molecule carrying a net positive charge under physiological pH conditions.

[0035] Preferably, no aqueous phase is added during the preparation of the carrier matrix solution.

[0036] Preferably, the method of mixing uniformly includes one or more of vortexing, stirring, ultrasonication, and homogenization.

[0037] Preferably, the pH-responsive lyotropic liquid crystal precursor composition can be injected via syringe and gel in situ upon entering an aqueous environment.

[0038] Preferably, the aqueous environment is a physiological buffer solution, tissue fluid, postoperative residual cavity fluid, infection focus fluid, inflammatory exudate, or a combination thereof.

[0039] Preferably, the therapeutic active substance is a VDAP polypeptide, component A is lecithin, component B is sorbitan monooleate, the pH-responsive gating switch is oleic acid, and the organic solvent includes ethanol and / or dimethyl sulfoxide.

[0040] Preferably, the therapeutic active substance is doxorubicin, component A is soybean lecithin, component B is diglyceride, the pH-responsive gating switch is oleic acid, and the organic solvent is N-methylpyrrolidone.

[0041] According to a third aspect of the invention, the pH-responsive lyotropic liquid crystal drug delivery system is provided for use in the preparation of medicaments for treating diseases associated with local acidic microenvironments.

[0042] Preferably, the disease is selected from one or more of solid tumors, bacterial infections, chronic inflammation, ischemic injuries, and trauma repair-related diseases.

[0043] Preferably, the solid tumor is one or more of triple-negative breast cancer, melanoma, liver cancer, pancreatic cancer, lung cancer, and colorectal cancer.

[0044] Preferably, the solid tumor is a solid tumor with a high risk of postoperative residue or recurrence.

[0045] Preferably, the solid tumor is a residual tumor or recurrent tumor in the residual cavity after triple-negative breast cancer surgery.

[0046] Preferably, the drug delivery system is applied to the lesion site or postoperative residual cavity site via local injection.

[0047] Preferably, the drug delivery system restricts the release of therapeutically active substances under physiological pH conditions and promotes the release of therapeutically active substances in the acidic microenvironment at the lesion site.

[0048] Preferably, the therapeutically active substance is a VDAP polypeptide, and the drug is used to inhibit postoperative recurrence of triple-negative breast cancer and / or remodel the tumor immune microenvironment.

[0049] Preferably, the drug can induce immunogenic cell death in tumor cells and increase the concentration of immunogenic cells in tumor tissue. Cell infiltration.

[0050] Preferably, the therapeutic active substance is doxorubicin, and the drug is used for local chemotherapy of solid tumors.

[0051] Preferably, the therapeutically active substance is an antimicrobial peptide or an aminoglycoside antibiotic, and the drug is used to treat local bacterial infections or biofilm infections.

[0052] Compared with the prior art, the present invention has at least the following beneficial effects:

[0053] The pH-responsive lyotropic liquid crystal drug delivery system provided by this invention is a flowable liquid precursor before injection, enabling local drug delivery via a syringe. Upon entering an aqueous environment such as tissue fluid, postoperative residual cavity fluid, infection focus fluid, or inflammatory exudate, the organic solvent diffuses into the surrounding aqueous phase, while the carrier matrix absorbs water and self-assembles, forming an in-situ lyotropic liquid crystal gel reservoir. Therefore, this invention enables the therapeutically active substance to remain locally at the lesion site, reducing rapid drug diffusion and systemic exposure, thus facilitating local, long-lasting, and precise drug delivery.

[0054] This invention establishes a dual-gated release mechanism—"electrostatic locking + topological phase transition"—by introducing a pH-responsive gating switch containing protonable anionic groups, distinct from conventional liquid crystal sustained-release systems. Under normal physiological conditions (pH 7.4), the pH-responsive gating switch is in a deprotonated state, enabling it to form electrostatic interactions with positively charged therapeutic active substances, effectively locking the drug within the liquid crystal gel. Simultaneously, the system forms a relatively dense inverted hexagonal phase structure under these conditions, further restricting drug diffusion and significantly reducing premature drug leakage in normal tissue environments. Taking VDAP-LLC as an example, its cumulative release over 7 days at pH 7.4 is less than 10%, demonstrating that this invention effectively avoids the burst release of cationic drugs and release from non-lesion sites.

[0055] In acidic pathological microenvironments such as solid tumors, bacterial infections, chronic inflammation, ischemic tissue, or traumatic tissue, the pH-responsive gating switch undergoes protonation, weakening or eliminating its electrostatic interaction with cationic drugs. This further induces a topological phase transition in the lyotropic liquid crystal gel from an anti-hexagonal phase to a bicontinuous anti-cubic phase. This phase transition creates three-dimensional interconnected water channels that are more conducive to drug diffusion, thereby promoting the sustained release of therapeutically active substances at acidic lesion sites. Taking VDAP-LLC as an example, its cumulative release over 70% under acidic conditions in 7 days significantly exceeds that under pH 7.4 conditions, indicating that this invention can achieve selective release in response to the acidic microenvironment of the lesion.

[0056] This invention overcomes the problems of rapid diffusion, significant burst release, short local retention time, and high risk of systemic toxicity associated with local administration of free cationic drugs. Free VDAP peptides can release over 90% within hours in in vitro release experiments, making long-term controlled release difficult. This invention, through the synergistic effect of in-situ gel reservoir, electrostatic locking, and acidic phase transition release, maintains low drug release in normal physiological environments and achieves sustained release in acidic lesion environments, thus achieving both long-term controlled release and lesion-selective release. The pH-responsive gating mechanism of this invention is clearly defined, effectively distinguishing it from ordinary liquid crystal delivery systems that rely solely on passive sustained release via a lipid matrix. Comparative results show that ordinary liquid crystal precursors without pH-responsive gating switches such as oleic acid do not show significant differences in release at pH 7.4 and acidic pH conditions, failing to achieve the low release at physiological pH and high release at acidic pH effects achieved by this invention. Therefore, the electrostatic interaction between the pH-responsive gating switch and the cationic therapeutic active substance, and the resulting liquid crystal topological phase transition, are the factors contributing to the intelligent controlled release achieved by this invention.

[0057] This invention demonstrates good therapeutic effects in local antitumor therapy. Taking a 4T1 triple-negative breast cancer recurrence model as an example, VDAP-LLC can gel in situ in the postoperative residual cavity and utilize the acidic characteristics of the residual tumor microenvironment to achieve responsive release. Experimental results show that the VDAP-LLC group can significantly reduce the final tumor weight, with a local tumor inhibition rate of 78.8%, which is significantly better than the free VDAP group and the blank carrier group, indicating that this invention can improve the local therapeutic effect of cationic antitumor peptides. This invention can also induce immunogenic cell death in tumor cells and improve the tumor immune microenvironment. Experimental results show that after VDAP-LLC treatment, the expression of immunogenic cell death-related markers such as HMGB1, HSP70, and CRT in tumor tissue is enhanced, and at the same time... T cells and The significant increase in cell infiltration indicates that the present invention can not only exert a local direct anti-tumor effect, but also enhance the local anti-tumor immune response, which is beneficial to reversing the postoperative tumor immunosuppressive microenvironment.

[0058] This invention exhibits good biocompatibility. Because the delivery system of this invention effectively limits drug leakage under physiological pH conditions and primarily promotes drug release at acidic lesion sites, it reduces the risk of toxic side effects after cationic active substances enter systemic circulation. Animal experiments showed that after local administration of VDAP-LLC, no significant tissue damage was observed in major organs such as the heart, liver, spleen, and kidneys in mice, and no significant abnormalities were observed in white blood cells, red blood cells, liver function, and kidney function-related indicators, indicating that this invention has good systemic biocompatibility.

[0059] Furthermore, this invention exhibits good platform applicability. The delivery system of this invention is not limited to a single drug, but is applicable to a variety of therapeutically active substances that carry a net positive charge or contain protonable amino groups under physiological pH conditions, including cationic amphiphilic peptides, amino-containing small molecule chemical drugs, aminoglycoside antibiotics, and therapeutic proteins with an isoelectric point greater than 7.4. Taking doxorubicin as an example, DOX-LLC shows low release at pH 7.4, while release significantly increases under acidic conditions, indicating that this invention can serve as a universal pH-responsive local drug delivery platform.

[0060] In summary, this invention can achieve low viscosity before injection, in-situ gelation after administration, drug locking at physiological pH, and responsive release in an acidic lesion microenvironment. It also has the advantages of long-acting controlled release, enhanced local treatment, reduced systemic toxicity, and platform expansion. It can be widely used in the treatment of diseases accompanied by local acidic microenvironment, such as solid tumors, bacterial infections, chronic inflammation, ischemic tissue, and wound repair. Attached Figure Description

[0061] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this application, illustrate exemplary embodiments of the invention and, together with their description, serve to explain the invention and do not constitute an undue limitation thereof. In the drawings:

[0062] Figure 1 The liquid crystal phase structure characterization diagram and in vitro release curve of VDAP-LLC at pH 7.4 provided for embodiments of the present invention are shown; wherein, Figure 1 A is the small-angle X-ray scattering spectrum of VDAP-LLC under pH 7.4 conditions, showing that it forms a highly ordered anti-hexagonal phase structure; Figure 1 B is the cumulative release curve of VDAP-LLC at pH 7.4, showing that its release is low under physiological pH conditions, indicating that the system is in a drug-locked state.

[0063] Figure 2 The liquid crystal phase structure characterization diagram and in vitro release curve of VDAP-LLC under different pH conditions are provided for embodiments of the present invention; wherein, Figure 2 A is the small-angle X-ray scattering spectrum of VDAP-LLC under acidic conditions at pH 5.0, showing that it forms a bicontinuous anti-cubic phase (Im3m) structure; Figure 2 B is the small-angle X-ray scattering (SAXS) spectrum of VDAP-LLC under weakly acidic conditions at pH 6.5, showing that it undergoes acid-responsive structural rearrangement under these conditions, exhibiting an inverted hexagonal phase (H). II Characteristics of a bicontinuous anti-cubic phase (Im3m) mixed or transitional phase; Figure 2C represents the cumulative release curves of VDAP-LLC at pH 7.4, pH 6.5, and pH 5.0. The curves show that VDAP-LLC releases slowly at pH 7.4, exhibits a sustained release trajectory at pH 6.5 (weakly acidic), with a release rate faster than at pH 7.4 but slower than at pH 5.0. The release amount increases significantly at pH 5.0 (acidic), indicating that the system can undergo structural transformation and open release channels in response to acidic conditions.

[0064] Figure 3 This is a diagram showing the detection results of VDAP-LLC-induced immunogenic cell death in tumor cells provided in an embodiment of the present invention; wherein, Figure 3 This image shows the immunohistochemical staining and quantitative analysis results of immunogenic cell death-related markers such as HMGB1, HSP70, and CRT in tumor tissues from different treatment groups.

[0065] Figure 4 This is a graph showing the detection results of the effect of VDAP-LLC on cytotoxic T cell infiltration in tumor tissue, provided in an embodiment of the present invention; wherein, Figure 4 Showing tumor tissue from different treatment groups Flow cytometry results and quantitative analysis of cell infiltration.

[0066] Figure 5 This is a diagram illustrating the antitumor effect of VDAP-LLC in a 4T1 triple-negative breast cancer recurrence model provided in this embodiment of the invention; wherein, Figure 5 A represents a representative photograph of the resected recurrent tumor in each treatment group at the experimental endpoint. Figure 5 B represents the quantitative analysis results of the final tumor weight for each treatment group. Figure 5 C represents the statistical results of the relative tumor inhibition rate of each treatment group.

[0067] Figure 6 This is a histopathological analysis diagram of the major organs after local administration of VDAP-LLC, provided in an embodiment of the present invention; wherein, Figure 6 The H&E staining results of heart, liver, spleen and kidney tissues of mice in different treatment groups are shown to evaluate the systemic biocompatibility and major organ safety of VDAP-LLC.

[0068] Figure 7 This is a hematological and serum biochemical index analysis chart provided in an embodiment of the present invention after local administration of VDAP-LLC; wherein, Figure 7 The results of the detection of indicators such as white blood cell count, red blood cell count, aspartate aminotransferase, alanine aminotransferase, blood urea nitrogen and creatinine in mice in different treatment groups are shown to evaluate the effects of VDAP-LLC on bone marrow function, liver function and kidney function.

[0069] Figure 8 The diagram shows the macroscopic injection properties and in-situ gelation performance of the VDAP-LLC liquid crystal precursor provided in the embodiments of the present invention; wherein, Figure 8 The VDAP-LLC liquid crystal precursor was shown to be able to be ejected via an injection needle and form a lyotropic liquid crystal gel in situ after injection into pH 7.4 PBS buffer.

[0070] Figure 9 Small-angle X-ray scattering characterization of VDAP-LLC at pH 7.4, provided for embodiments of the present invention; wherein, Figure 9 A shows the characteristic diffraction peaks of the reverse hexagonal phase of VDAP-LLC at pH 7.4. Figure 9 B shows a comparison of the small-angle X-ray scattering spectra of VDAP-LLC and Blank LLC, indicating that the liquid crystal lattice shrinks after VDAP is loaded.

[0071] Figure 10 These are in-situ gelation and polarized light microscopy images of high-drug-load VDAP-LLC provided in embodiments of the present invention; wherein, Figure 10 A is a macroscopic photograph of VDAP-LLC with high drug loading after in-situ gelation. Figure 10 Image B is a polarized light microscope image of high-drug-load VDAP-LLC at pH 7.4, showing its anisotropic liquid crystal structure. Figure 10 C is a polarized light microscope image of VDAP-LLC with high drug loading under acidic pH conditions, showing that its birefringence is weakened or disappears, indicating that the system has undergone a liquid crystal phase transition.

[0072] Figure 11 The in vitro cumulative release curves of VDAP-LLC provided in this embodiment of the invention are shown in the figures at pH 7.4 and acidic pH 5.0. Figure 11 The results showed that VDAP-LLC had a low release rate at pH 7.4, but a significantly increased release rate at acidic pH 5.0, indicating that it has a pH-responsive release characteristic.

[0073] Figure 12 The small-angle X-ray scattering spectrum of VDAP-LLC under acidic pH 5.0 conditions provided in this embodiment of the invention; wherein, Figure 12 The presence of bicontinuous reversed cubic phase characteristic diffraction peaks in VDAP-LLC under acidic pH 5.0 conditions indicates that the system undergoes a topological phase transition in an acidic microenvironment.

[0074] Figure 13 The in vitro cumulative release curves of the VDAP liquid crystal precursor without a pH-responsive gating switch, provided as a comparative example of the present invention, are shown in the figure at pH 7.4 and acidic pH 6.5. Figure 13The results showed that the release of the comparative system without oleic acid did not differ significantly under different pH conditions, indicating that the pH-responsive gating switch plays an important role in achieving acidic response release.

[0075] Figure 14 The in vitro release curve of the free VDAP solution provided as a comparative example of the present invention; wherein, Figure 14 The results show that free VDAP is rapidly released in the early stages of release, indicating that free drugs exhibit a significant burst release phenomenon, making it difficult to achieve long-term controlled release.

[0076] Figure 15 The in vitro cumulative release curves of DOX-LLC under pH 7.4 and acidic pH 6.5 conditions provided for embodiments of the present invention are shown; wherein, Figure 15 The results showed that DOX-LLC release was low at pH 7.4, but significantly increased at acidic pH 6.5, indicating that the delivery system of the present invention is applicable to small molecule drugs containing protonable amino groups.

[0077] Figure 16 The figure shows the effect of different oleic acid contents on the in-situ gelation properties and in vitro release behavior of VDAP-LLC liquid crystal precursors.

[0078] Figure 17 The figure shows the effect of different mass ratios of component A and component B on the hydration synergistic properties and in vitro release behavior of VDAP-LLC liquid crystal precursors. Component A is lecithin, and component B is SMO. Figure 17 The in-situ gelation of VDAP-LLC liquid crystal precursor in aqueous medium is shown when the mass ratio of component A to component B is 1:5, 1:2, 2:1 and 5:1, respectively, as well as the cumulative VDAP release curves of each ratio system under pH 7.4 and pH 6.5 conditions. Detailed Implementation

[0079] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. It should be understood that the following embodiments are only for illustrating the technical solutions of the present invention and should not be construed as limiting the scope of protection of the present invention. Equivalent substitutions or conventional adjustments made by those skilled in the art to the types of components, proportions, preparation conditions, dosage, detection methods, or indications of the present invention without departing from the concept of the present invention should all fall within the scope of protection of the present invention.

[0080] Unless otherwise stated, all "%" in the following examples refer to mass percentages. The pH 7.4 in the following examples is used to simulate a normal physiological environment, and the acidic pH is used to simulate the pathological acidic microenvironment of solid tumors, localized bacterial infections, chronic inflammation, ischemic tissue, or traumatic tissue. The preferred pH range is pH 5.0–6.5, more preferably pH 5.0 or pH 6.5.

[0081] The pH-responsive lyotropic liquid crystal drug delivery system of this invention is a low-viscosity liquid precursor before injection. Upon injection into an aqueous environment, the organic solvent diffuses outward, the lipid matrix absorbs water and undergoes self-assembly, forming an in-situ lyotropic liquid crystal gel reservoir. Under physiological pH conditions, this gel reservoir achieves drug locking through electrostatic interactions between a pH-responsive gating switch and the cationic therapeutic active substance. In an acidic microenvironment, the electrostatic interactions are released due to protonation of the pH-responsive gating switch, simultaneously inducing a topological phase transition in the lyotropic liquid crystal system, thereby achieving intelligent responsive release at the lesion site.

[0082] Example 1: Preparation and Injection Molding Performance of VDAP-LLC Liquid Crystal Precursor

[0083] This embodiment illustrates the preparation method of the pH-responsive lyotropic liquid crystal drug delivery system of the present invention when using VDAP peptide as a cationic antitumor peptide, and its properties of being liquid before injection and gelling in situ upon contact with water. The amino acid sequence of the VDAP peptide is: IIRILQQLLFIHFRIGCRHSRIGVTRQRRARNG, molecular weight: 3985, purity: > 95% (98%), isoelectric point: 12.58, net charge at pH 7.4: 8.1; supplier: Zhejiang Paipai Biotechnology Co., Ltd. In the formulation screening of the present invention, lecithin, sorbitan monooleate, ethanol, and DMSO constitute the basic liquid crystal precursor phase, wherein the mass ratio of lecithin, sorbitan monooleate, ethanol, and DMSO is 35:35:10:10. While maintaining the internal proportions of the above basic liquid crystal precursor phase, different mass percentages of oleic acid are added to obtain VDAP-LLC liquid crystal precursors with different oleic acid contents. The oleic acid acts as a pH-responsive gating switch, and its content is calculated according to the following formula: OA content = ,in, Indicates the mass of oleic acid. This indicates the total mass of the basic liquid crystal precursor phase, which consists of lecithin, sorbitan monooleate, ethanol, and DMSO.

[0084] In this embodiment, an oleic acid content of approximately 5 wt% was selected as a representative formulation, and the components were weighed according to the table below:

[0085]

[0086] Among them, lecithin is a phospholipid component A, SMO is an amphiphilic lipid component B that can induce the formation of an antiphase liquid crystal phase, oleic acid is a pH-responsive gate switch containing a carboxyl group, and VDAP peptide is a therapeutically active substance with a net positive charge under physiological pH conditions.

[0087] 1.2 Preparation method

[0088] Weigh 350 mg of lecithin, 350 mg of SMO, and 50 mg of oleic acid and place them in a clean glass bottle. Add 200 mg of ethanol / DMSO mixed solvent and vortex at room temperature to fully dissolve the lecithin, SMO, and oleic acid, resulting in a clear or nearly clear lipid matrix solution.

[0089] Subsequently, 10 mg of VDAP peptide was weighed and added to the above lipid matrix solution. The mixture was then vortexed to ensure that the VDAP peptide was uniformly dissolved or dispersed in the system, thus obtaining the VDAP-LLC liquid crystal precursor.

[0090] The obtained VDAP-LLC liquid crystal precursor was a homogeneous, low-viscosity, and flowable liquid before injection, which could be smoothly ejected through a 26G injection needle. After the VDAP-LLC liquid crystal precursor was injected into pH 7.4 PBS buffer, the liquid precursor rapidly absorbed water and solidified in situ, forming a lyotropic liquid crystal gel reservoir with a certain degree of elasticity and intact morphology. The results are as follows... Figure 8 As shown, the delivery system of the present invention has good injectability and in-situ gelation performance.

[0091] 1.3 Technical Effect Analysis

[0092] This embodiment demonstrates that the system of the present invention can maintain a low-viscosity liquid state before injection, and form a local gel reservoir through organic solvent diffusion and lipid self-assembly after entering an aqueous environment.

[0093] Example 2: H+ of VDAP-LLC at pH 7.4 Ⅱ Phase structure and drug locking effect

[0094] The delivery system of this invention can form a dense anti-hexagonal phase structure under physiological pH conditions and reduce drug leakage through the electrostatic interaction between oleic acid and cationic drugs.

[0095] 2.1 Sample Preparation

[0096] The VDAP-LLC liquid crystal precursor was prepared according to the method in Example 1. 100 μL of the VDAP-LLC liquid crystal precursor was injected into pH 7.4 PBS buffer and incubated at 37°C for 24 h to allow it to fully absorb water and form a gel in situ. The gel sample was then subjected to small-angle X-ray scattering (SAXS) detection. Before testing, the gel sample after 24 h of incubation was sealed between two layers of Kapton film to form a thin-layer sample.

[0097] A blank liquid crystal gel, Blank LLC, without VDAP, was used as a control. Its composition was the same as in Example 1, except that the VDAP peptide was not added. Blank LLC was also injected into pH 7.4 PBS buffer and incubated at 37°C for 24 h to form a gel. The gel was then sealed between two Kapton films in the same manner to form a thin-layer sample for SAXS detection.

[0098] 2.2 SAXS Characterization

[0099] Small-angle X-ray scattering (SAXS) was performed using a Xeuss 3.0 C small-angle X-ray scattering system (Xenocs, France). A Cu Kα X-ray source was used with a wavelength λ = 0.154 nm; the sample-to-detector distance was 575 mm; and the exposure time was 10 min. The resulting two-dimensional scattering map was azimuth-averaged using the Xenocs / Xeuss system's software to obtain a one-dimensional scattering curve. Figure 1 A and Figure 9 As shown in Figure A, under pH 7.4 conditions, the VDAP-LLC gel exhibits multiple ordered diffraction peaks, the peak positions of which conform to the anti-hexagonal phase H. Ⅱ The characteristic peak ratio of the phase is 1:√3:2, which corresponds to the crystal planes (10), (11), and (20). According to the SAXS diffraction peaks, the lattice parameter of VDAP-LLC at pH 7.4 is approximately 50.0 Å.

[0100] like Figure 9 As shown in Figure B, compared to Blank LLC, the lattice parameter of VDAP-LLC shrinks from approximately 55.1 Å to approximately 50.0 Å, indicating that the introduction of the VDAP peptide can induce liquid crystal lattice shrinkage. Combined with the physicochemical characteristics of VDAP peptide carrying a net positive charge under physiological pH conditions and oleic acid being deprotonated and negatively charged at pH 7.4, it can be explained that an electrostatic complex is formed between the VDAP peptide and oleic acid. This electrostatic complex further compresses the lipid lattice, maintaining a dense H+ lattice in the system. Ⅱ Phase structure.

[0101] 2.3 Release experiment at pH 7.4

[0102] The VDAP-LLC liquid crystal precursor prepared in Example 1 was injected into pH 7.4 PBS release medium to allow it to gel in situ. The release system was kept at a constant temperature of 37°C, and samples were taken at preset time points, with an equal volume of fresh pH 7.4 PBS release medium added. The VDAP peptide content in the release medium was quantitatively determined by high-performance liquid chromatography (HPLC). Specifically, an Ultimate 3000 HPLC system (Thermo Fisher Scientific, USA) was used, with an Agilent ZORBAX300SB-C18 column (4.6 × 150 mm, 3.5 μm), and a column temperature of 30°C. The mobile phase was an acetonitrile / water mixture containing 0.01% TFA, with a volume ratio of acetonitrile to water of 33:67. Isocratic elution was used at a flow rate of 1.0 mL / min, and the UV detection wavelength was 220 nm. A standard curve was established based on VDAP standards, and the concentration of VDAP in the release medium at each time point was calculated using the standard curve. The cumulative release rate of VDAP was then calculated.

[0103] The cumulative release rate is calculated using the following formula: Cumulative release rate = Cumulative VDAP mass released at each time point / Initial total VDAP mass × 100%.

[0104] like Figure 1 As shown in Figure B, the cumulative release of VDAP-LLC over 7 days at pH 7.4 was 6.72%, which is less than 10%. This result indicates that under normal physiological pH conditions, the system of this invention is in a "locked-in" or "silent" state, which can effectively reduce premature leakage of cationic drugs.

[0105] 2.4 Technical Effect Analysis

[0106] This embodiment combines Figure 1 and Figure 9 Evidence: Under pH 7.4 conditions, oleic acid, after deprotonation, forms an electrostatic complex with VDAP peptides, inducing or maintaining dense H+. Ⅱ It uses an anti-hexagonal phase and reduces the cumulative release over 7 days to less than 10%.

[0107] Example 3: Topological phase transition and response release of VDAP-LLC under acidic pH conditions

[0108] The delivery system of this invention can break electrostatic lock-in in acidic pathological microenvironments and is powered by H. Ⅱ The phase transitions to a bicontinuous antiphase cubic phase, thereby promoting drug release.

[0109] 3.1 Sample Preparation

[0110] VDAP-LLC liquid crystal precursors were prepared according to the method in Example 1. 100 μL of the VDAP-LLC liquid crystal precursor was injected into acidic buffer solutions at pH 5.0 and / or pH 6.5, and incubated at 37°C to allow for in-situ gelation and full response to the acidic environment. Gel samples were then used for SAXS characterization and in vitro release experiments.

[0111] 3.2 SAXS characterization under acidic conditions

[0112] like Figure 2 A and Figure 12 As shown, under acidic pH conditions, the SAXS diffraction peaks of the VDAP-LLC gel changed significantly, with the H peaks at the original pH 7.4 condition decreasing. Ⅱ The phase characteristic peaks weaken or disappear, and characteristic diffraction peaks consistent with the bicontinuous anti-cubic phase appear. The diffraction peak ratio under these acidic conditions conforms to the characteristic peak ratio of the Im3̄m bicontinuous anti-cubic phase, i.e., √2:√6:√8:√12. Based on the SAXS diffraction peaks, the lattice parameter under acidic conditions is approximately 141.9 Å.

[0113] The above results indicate that VDAP-LLC transforms from a dense, anti-hexagonal phase to a bicontinuous, anti-cubic phase with three-dimensional interconnected water channels under acidic conditions. This structural change facilitates the opening of drug diffusion channels, thereby promoting the release of VDAP peptides.

[0114] 3.3 Release experiment under acidic conditions

[0115] The VDAP-LLC liquid crystal precursor was injected into release media with pH 7.4 and acidic pH, respectively, and in vitro release experiments were conducted at 37°C. The release experiment method was the same as in Example 2.

[0116] like Figure 2 B and Figure 11 As shown, the release rate of VDAP-LLC under acidic pH conditions is significantly higher than that under pH 7.4 conditions. The cumulative release over 7 days is less than 10% under pH 7.4 conditions, while it exceeds 70% under acidic pH conditions. This result demonstrates that the system of this invention exhibits significant pH-differentiated release capability.

[0117] 3.4 Mechanism Analysis

[0118] At pH 7.4, the oleic acid carboxyl group is deprotonated and becomes negatively charged, then electrostatically recombines with the positively charged VDAP peptide, confining the drug within the liquid crystal gel and maintaining the system's dense H₂O. ⅡPhase structure. Under acidic conditions, the oleic acid carboxyl group undergoes protonation, reducing or eliminating its negative charge, thus weakening or dissolving the electrostatic interaction between oleic acid and the VDAP polypeptide. Simultaneously, the protonation of oleic acid alters the critical molecular packing parameters of the system, causing the liquid crystal structure to change from H... Ⅱ The phase transition is an Im3̄m bicontinuous cubic phase, forming three-dimensional interconnected water channels that promote sustained drug release.

[0119] 3.5 Technical Effect Analysis

[0120] This embodiment combines Figure 2 , Figure 11 and Figure 12 This demonstrates that the system of the present invention does not rely solely on material degradation or passive diffusion for drug release, but rather achieves release in response to acidic microenvironments through a dual mechanism of "electrostatic lock-out + topological phase transition to open channels".

[0121] Example 4: Preparation and Phase Retention Capacity of High Drug Loading VDAP-LLC

[0122] The delivery system of this invention retains injection gelation capability and pH-responsive phase change capability even after increasing the cationic peptide drug loading.

[0123] 4.1 Preparation method

[0124] The preparation method is basically the same as in Example 1, except that the amount of VDAP polypeptide added is increased from 10 mg to 20 mg, while the other components and preparation conditions remain unchanged.

[0125] Specifically, 350 mg of lecithin, 350 mg of SMO and 50 mg of oleic acid were weighed and added to 200 mg of ethanol / DMSO mixed solvent, and mixed thoroughly to obtain a lipid matrix solution; then 20 mg of VDAP peptide was added and vortexed until a homogeneous liquid precursor was formed to obtain a high drug loading VDAP-LLC liquid crystal precursor.

[0126] 4.2 In-situ gelation and observation under polarizing light microscope

[0127] High-drug-load VDAP-LLC liquid crystal precursor was injected into pH 7.4 PBS, and the results showed that it could still be successfully injected and gelled in situ. Figure 10 As shown in Figure A.

[0128] Further phase changes were observed using a polarizing microscope. For example... Figure 10 As shown in Figure B, under pH 7.4 conditions, the high-drug-load VDAP-LLC exhibits significant birefringence and a focal conic texture, indicating that the system possesses an anisotropic liquid crystal structure, consistent with H. Ⅱ Phase characteristics. For example... Figure 10As shown in Figure C, under acidic pH conditions, the birefringence of the sample is significantly reduced or disappears, exhibiting optical isotropic characteristics, suggesting that the system is composed of anisotropic H... Ⅱ Phase-to-phase optical isotropic cubic intermediate phase transition.

[0129] 4.3 Technical Effect Analysis

[0130] This embodiment demonstrates that even when the amount of VDAP added is increased to twice that of Example 1, the system of the present invention still maintains good injectability, in-situ gelation properties, and pH-responsive phase change capabilities, indicating that the delivery system of the present invention has a certain tolerance for cationic peptides.

[0131] Comparative Example 1: 0% OA-based liquid crystal carrier control group

[0132] This comparative example illustrates the gelation properties and release behavior of the basic liquid crystal carrier when oleic acid is not introduced as a pH-responsive gating switch.

[0133] The preparation method of this comparative example is basically the same as that of Example 1, except that oleic acid is not added, and the mass ratio of lecithin, SMO, ethanol, and DMSO in the basic liquid crystal precursor phase is maintained at 35:35:10:10. Specifically, 350 mg of lecithin and 350 mg of SMO are dissolved in 200 mg of ethanol / DMSO mixed solvent, mixed evenly, and then 10 mg of VDAP peptide is added to obtain an oleic acid-free VDAP liquid crystal precursor. This comparative example serves as a control group for the 0% OA basic liquid crystal carrier and is used to evaluate the liquid crystal phase structure and in vitro release behavior of the basic liquid crystal carrier without the introduction of pH-responsive gating oleic acid. Compared with the oleic acid-containing VDAP-LLC liquid crystal precursor, the internal proportions of the basic liquid crystal precursor phase in this comparative example are maintained, except that the oleic acid functional gating component is not added.

[0134] 1.2 Release Experiment

[0135] Following the methods of Examples 2 and 3, ordinary VDAP liquid crystal precursors without oleic acid were injected into release media with pH 7.4 and acidic pH, respectively, and the VDAP release behavior was detected.

[0136] like Figure 13 As shown, the release of ordinary liquid crystal precursors without oleic acid does not differ significantly under pH 7.4 and acidic pH conditions, and cannot achieve the low release at physiological pH and high release at acidic pH characteristics exhibited by VDAP-LLC of this invention.

[0137] 1.3 Technical Effect Analysis

[0138] This comparative example demonstrates that oleic acid is not a common lipid adjuvant, but rather a key component in achieving pH-responsive gating in the system of this invention. At pH 7.4, oleic acid electrostatically recombines with the VDAP peptide through deprotonation of its carboxyl group, thereby locking the drug in place. Under acidic conditions, oleic acid protonates and induces a liquid crystal phase transition, thus promoting drug release. Without oleic acid, the system cannot form an effective electrostatic / phase transition dual gating mechanism.

[0139] Comparative Example 2: Free VDAP solution

[0140] The delivery system of this invention has the advantages of avoiding burst release and achieving long-term local release compared to free cationic peptides.

[0141] 2.1 Sample Preparation

[0142] Weigh the same amount of VDAP peptide as in Example 1 and dissolve it directly in PBS buffer to obtain a free VDAP solution.

[0143] 2.2 In vitro release experiment

[0144] The free VDAP solution was placed in a release device, and a release experiment was conducted at 37°C. The release medium, sampling time points, and detection methods were consistent with those in Examples 2 and 3.

[0145] like Figure 14 As shown, free VDAP diffuses rapidly in the early stages of release, with more than 90% of the amount released within a few hours, exhibiting a significant burst release effect.

[0146] 2.3 Technical Effect Analysis

[0147] Free VDAP lacks a local gel reservoir structure and pH-responsive gating mechanism, making it difficult to maintain effective drug concentrations at the lesion site and potentially leading to systemic exposure and toxic side effects. The VDAP-LLC of this invention, through in-situ gelation, electrostatic locking, and acidic phase transition release mechanisms, can significantly reduce burst release and achieve long-lasting controlled release.

[0148] Example 5: Preparation and pH-responsive release of DOX-LLC liquid crystal precursor

[0149] The delivery system of this invention is applicable not only to cationic peptides, but also to small molecule chemical drugs containing protonable amino groups.

[0150] 5.1 Raw Material Composition

[0151] Doxorubicin hydrochloride (DOX) was used as a model small molecule drug. A DOX-LLC liquid crystal precursor was prepared according to the following components:

[0152] Component A is soybean lecithin;

[0153] Component B is diglyceride (GDO).

[0154] The pH-responsive gating switch is oleic acid;

[0155] The organic solvent is N-methylpyrrolidone (NMP);

[0156] The therapeutically active substance is doxorubicin hydrochloride (DOX).

[0157] The mass ratio of soybean lecithin to GDO is 1:1, and the amount of oleic acid used is about 5% of the total mass of the system.

[0158] 5.2 Preparation method

[0159] 350 mg of soybean lecithin, 350 mg of GDO, and 50 mg of oleic acid were added to 200 mg of NMP and vortexed at room temperature until fully dissolved to obtain a lipid matrix solution. Then, 10 mg of doxorubicin hydrochloride (DOX) was pre-dissolved in a small amount of NMP and added to the lipid matrix solution, mixed thoroughly to obtain the DOX-LLC liquid crystal precursor. The obtained DOX-LLC liquid crystal precursor was injected into pH 7.4 and pH 5.0 buffer solutions, respectively, and liquid crystal gels formed in situ in both cases.

[0160] 5.3 pH Response Release Experiment

[0161] In vitro release experiments were conducted according to the methods of Examples 2 and 3. Figure 15 As shown, DOX-LLC also exhibits significant pH-responsive release characteristics: at pH 7.4, the cumulative release over 48 hours is less than 15%; at pH 6.5, the cumulative release over 48 hours reaches more than 60%.

[0162] 5.4 Technical Effect Analysis

[0163] DOX molecules contain protonable amino groups and can exhibit cationic characteristics under certain pH conditions. This embodiment illustrates that the system of the present invention can be extended to small molecule chemical drugs containing protonable amino groups, and can similarly achieve gated drug release effects at low physiological pH and high acidic pH.

[0164] Example 6: Antitumor effect of VDAP-LLC in a 4T1 triple-negative breast cancer recurrence model after surgery

[0165] The delivery system of this invention demonstrates its effectiveness in local treatment of acidic residual cavities after solid tumor surgery.

[0166] 6.1 Establishment of animal models

[0167] Female BALB / c mice aged 6–8 weeks and weighing 18–20 g were selected and acclimatized in an SPF-grade animal facility. An orthotopic breast cancer model was established using 4T1-Luc triple-negative breast cancer cells from a commercial cell bank. Before inoculation, 4T1-Luc cells were digested, centrifuged, and resuspended in sterile PBS or serum-free culture medium to adjust the cell suspension concentration. Subsequently, cells were inoculated into each mouse... A 4T1-Luc in situ breast cancer model was established by injecting a cell suspension at a dose of 100 μL into the fat pad of the fourth mammary gland in mice. After the tumor grew to the predetermined volume, the in situ tumor was incompletely surgically removed under aseptic conditions, leaving a small amount of residual tumor tissue or residual tumor cells to create a local lesion environment that simulates the residual cavity after clinical solid tumor surgery.

[0168] 6.2 Grouping and Dosing

[0169] After surgery, the mice were randomly divided into the following groups, with 8 mice in each group, i.e., n=8:

[0170] PBS group;

[0171] Blank LLC group, i.e., blank liquid crystal precursor group without VDAP;

[0172] Free VDAP group, i.e., free VDAP peptide group;

[0173] VDAP-LLC group.

[0174] Before administration, corresponding formulations were prepared. The Free VDAP group used a free VDAP peptide solution with a VDAP concentration of 10 mg / g; the VDAP-LLC group used the VDAP-LLC liquid crystal precursor prepared in Example 1, in which the equivalent concentration of VDAP was 10 mg / g; the Blank LLC group used a blank liquid crystal precursor without VDAP, whose liquid crystal matrix composition and concentration corresponded to those of the VDAP-LLC group, but without the VDAP peptide.

[0175] Following tumor resection, the appropriate preparation was injected locally into the postoperative residual cavity, and a second local administration was performed on the 7th postoperative day, for a total of two local administrations. The administration methods for each group were as follows: PBS group received local injection of PBS; Blank LLC group received local injection of blank liquid crystal precursor without VDAP; Free VDAP group received local injection of free VDAP peptide solution at a VDAP dose of 15 mg / kg; and VDAP-LLC group received injection of the VDAP-LLC liquid crystal precursor prepared in Example 1, with an equivalent VDAP dose of 15 mg / kg.

[0176] When administering medication, the actual dosage volume for each experimental animal is calculated based on its body weight on the day of administration and the concentration of the therapeutic active substance in the formulation to be administered, ensuring that the dose of the therapeutic active substance received by each animal is consistent per unit body weight. The dosage volume may vary between different experimental animals due to differences in body weight, and between different administration groups due to differences in the concentration of the therapeutic active substance in the formulation or the dosage, but all are controlled within the permissible local dosage volume range for animal experiments.

[0177] For the PBS group and Blank LLC group, which do not contain therapeutically active substances, the administration volume can be determined according to the same or equivalent administration volume as the VDAP-LLC group. Specifically, the Blank LLC group was given a blank liquid crystal precursor with the same or equivalent molecular weight as the VDAP-LLC group, but without the VDAP peptide. After injection, the VDAP-LLC liquid crystal precursor gels in situ in the aqueous environment of the postoperative residual cavity, forming a local drug reservoir, thereby achieving continuous release of VDAP in the postoperative lesion area.

[0178] 6.3 Evaluation of antitumor efficacy

[0179] Postoperatively, the mice were regularly observed for their general condition, and the volume of locally recurrent tumors was measured. At the end of the experiment, the mice were sacrificed, and the recurrent tumor tissue was dissected and weighed. The tumor inhibition rate was calculated using the following formula: Tumor inhibition rate = (average tumor weight of control group − average tumor weight of treatment group) / average tumor weight of control group × 100%.

[0180] like Figure 5 As shown, the PBS group and Blank LLC group showed significant local tumor recurrence; the Free VDAP group had a certain tumor-suppressing effect, but the efficacy was limited; the VDAP-LLC group showed a significant reduction in terminal tumor weight and a local tumor inhibition rate of 78.8%, indicating that the VDAP-LLC of the present invention can significantly inhibit the recurrence of triple-negative breast cancer after surgery.

[0181] 6.4 Technical Effect Analysis

[0182] This embodiment illustrates that VDAP-LLC can form a liquid crystal gel reservoir in situ in the postoperative residual cavity and release VDAP peptides in response to the acidic pathological microenvironment of the postoperative residual cavity, thereby prolonging the local drug retention time, reducing burst release, and improving the local anti-tumor effect.

[0183] Example 7: VDAP-LLC induces immunogenic cell death and remodels the tumor immune microenvironment

[0184] The VDAP-LLC of this invention not only has a direct tumor-suppressing effect, but can also induce immunogenic cell death and enhance anti-tumor immune responses.

[0185] 7.1 ICD Marker Detection

[0186] A 4T1 triple-negative breast cancer recurrence model was established and treated with medication according to the method in Example 6. At the experimental endpoint, tumor tissues from each group were collected for immunohistochemical staining to detect immunogenic cell death-related markers, including HMGB1, HSP70, and CRT. Specifically, in the immunohistochemical staining, tissue sections were 4 μm thick, treated with EDTA antigen retrieval solution (pH 9.0) for antigen retrieval, blocked, and then incubated overnight at 4°C with primary antibodies for CRT, HMGB1, and HSP70, respectively. Detection was then performed using the HRP-DAB colorimetric system, with counterstaining with hematoxylin.

[0187] Stained tissue sections were digitized using a digital section scanner, such as the 3D HiStech Pannoramic Midi scanner. The expression levels of each biomarker were analyzed using ImageJ software. Each group of samples was segmented using a consistent fluorescence intensity threshold or positive signal threshold, and the positive signal area was expressed as the proportion of the total tissue area to the positive signal area, i.e., the positive area fraction.

[0188] like Figure 3 As shown, compared with the PBS group, Blank LLC group and Free VDAP group, the tumor tissue of the VDAP-LLC group showed a significant increase in ICD-related markers such as HMGB1, HSP70 and CRT, indicating that VDAP-LLC can induce immunogenic cell death in tumor cells.

[0189] 7.2 Cell infiltration detection

[0190] Tumor tissues from each group were collected, and single-cell suspensions were prepared by enzymatic digestion. Flow cytometry was used to detect the presence of single cells in the tumor tissues. Cells and Cell ratio.

[0191] like Figure 4 As shown, cytotoxicity in tumor tissues of the VDAP-LLC group The number of cells increased significantly within the tumor parenchyma. Cell infiltration was approximately 11 times that of the Blank LLC group, indicating that the VDAP-LLC of this invention can enhance cytotoxic T cell infiltration and activation, and reverse the postoperative tumor immunosuppressive microenvironment.

[0192] 7.3 Technical Effect Analysis

[0193] This embodiment illustrates that the VDAP-LLC of the present invention releases VDAP polypeptide in response to an acidic microenvironment, which can induce immunogenic cell death in tumor cells and further enhance... Cellular infiltration and IFN-γ expression reshape the tumor immune microenvironment.

[0194] Example 8: In vivo biosafety evaluation of VDAP-LLC

[0195] The delivery system of this invention reduces system toxicity through local locking and acid-response release mechanisms.

[0196] 8.1 Sample collection and testing methods

[0197] Mice were treated according to the method in Example 6. On day 28 post-treatment, peripheral blood and major organs were collected from mice in each group. The major organs included the heart, liver, spleen, lungs, and kidneys.

[0198] Blood samples were collected on day 28 post-treatment via the orbital venous plexus / orbital sinus. A portion of the collected whole blood was used directly for routine blood tests, including white blood cell count (WBC), red blood cell count (RBC), and hemoglobin (HGB). The remaining blood sample was centrifuged at 2000 g for 10 min to separate serum, which was then used for serum biochemical analysis. Serum biochemical indicators included liver function indicators (alanine aminotransferase (ALT) and aspartate aminotransferase (AST)) and kidney function indicators (blood urea nitrogen (BUN) and creatinine (CREA)). The routine blood tests were performed using an animal blood analyzer, while the serum biochemical indicators were detected using a fully automated biochemical analyzer.

[0199] Mice were sacrificed on day 28 post-treatment, and tumor tissue and major organs such as the heart, liver, spleen, lungs, and kidneys were dissected. The obtained tissue samples were fixed in 10% neutral buffered formalin, dehydrated routinely, embedded in paraffin, and sectioned to a thickness of 4 μm. The tissue sections were stained with hematoxylin and eosin (H&E) and then examined under a light microscope to observe the histopathological changes of each major organ, in order to evaluate the systemic toxicity of each treatment regimen.

[0200] 8.2 Experimental Results

[0201] like Figure 6 As shown, the major organ structures of mice in the VDAP-LLC group remained intact, with no obvious necrosis, inflammatory cell infiltration, or tissue damage. Figure 7 As shown, no significant abnormalities were observed in blood and biochemical indicators such as WBC, RBC, AST, ALT, BUN, and CREA in the VDAP-LLC group mice, indicating that the VDAP-LLC of this invention did not cause significant bone marrow suppression, liver function damage, or kidney function damage.

[0202] 8.3 Technical Effect Analysis

[0203] This embodiment illustrates that the VDAP-LLC of the present invention can effectively reduce premature leakage of VDAP peptides under normal physiological pH conditions and is mainly released at acidic lesion sites, thereby improving system safety while maintaining local anti-tumor efficacy.

[0204] Example 9: Effect of different oleic acid contents on the phase state and release behavior of VDAP-LLC

[0205] This embodiment is used to further investigate the effect of oleic acid content on the in-situ gelation properties and pH-responsive release behavior of VDAP-LLC liquid crystal precursor, and to illustrate the regulatory role of oleic acid as a pH-responsive component on the release behavior of the system.

[0206] Following the method of Example 1, VDAP-LLC liquid crystal precursors with oleic acid contents of 2%, 5%, and 10% were prepared. The total amounts of lecithin, SMO, VDAP, and organic solvent were kept essentially consistent, and the total mass of the precursors in each group was made consistent by adjusting the amount of oleic acid and the mass of the lipid groups. The formulation with 5% oleic acid content was the same as described in Example 1. The amount of VDAP in each formulation was kept consistent; while maintaining the relative mass ratios of lecithin, SMO, ethanol, and DMSO in the basic liquid crystal precursor phase, different mass percentages of oleic acid were added to prepare VDAP-LLC liquid crystal precursors with different oleic acid contents. The oleic acid content was adjusted according to… Calculate, where W 0A For oleic acid quality, W basal This refers to the total mass of all components of the basic liquid crystal precursor, excluding oleic acid.

[0207] VDAP-LLC liquid crystal precursors with oleic acid contents of 2% and 10% were injected into aqueous media, and their in-situ gelation behavior was observed. Simultaneously, the in vitro release behavior of VDAP was detected under pH 7.4 and pH 6.5 conditions. The results are as follows: Figure 16 As shown, VDAP-LLC liquid crystal precursors with oleic acid contents of 2% and 10% can both form gels in situ in an aqueous environment, indicating that the system still has good hydration gelation ability within this oleic acid content range.

[0208] In the VDAP-LLC system with 2% oleic acid content, VDAP release was relatively slow at pH 7.4, with a cumulative release rate of approximately 15% by day 7. However, at pH 6.5, VDAP release accelerated significantly, with a cumulative release rate exceeding 40% on day 1, exceeding 50% on day 2, and approximately 60% by day 7. These results indicate that the VDAP-LLC system with lower oleic acid content can still maintain the pH-responsive release characteristics of slow release under neutral conditions and accelerated release under acidic conditions.

[0209] For the VDAP-LLC system with 10% oleic acid, VDAP release was increased compared to the 2% oleic acid group at pH 7.4, with a cumulative release rate of over 20% on day 1 and approximately 35% on day 7. However, at pH 6.5, VDAP release accelerated significantly, with a cumulative release rate of over 90% on day 1 and approaching or reaching 95% on day 3. These results indicate that increasing the oleic acid content enhances the release response under acidic conditions and also increases the basal release level at pH 7.4.

[0210] Based on the results of Example 1 with an oleic acid content of 5%, it can be seen that changes in oleic acid content can regulate the pH-responsive release behavior of the VDAP-LLC system. When the oleic acid content is 2%, the system exhibits good sustained-release performance at pH 7.4, but the release is relatively low under acidic conditions. When the oleic acid content is increased to 10%, the release is significantly enhanced at pH 6.5, but the release also increases accordingly at pH 7.4. The formulation with an oleic acid content of 5% shows a good balance between stable sustained release under neutral conditions and responsive release under acidic conditions.

[0211] Therefore, the content of oleic acid as a pH-responsive component can be adjusted within the range of 2% to 10% to obtain VDAP-LLC systems with different release intensities; wherein, the oleic acid content is preferably about 5%, which can take into account the in-situ gelling, retention under neutral conditions and response release capacity under acidic pH conditions.

[0212] Example 10: Effect of different component A / component B ratios on the performance of VDAP-LLC

[0213] Based on the 1:1 A:B formulation described in Example 1, the ratio of component A to component B was further adjusted to prepare VDAP-LLC liquid crystal precursors with A:B ratios of 1:5, 1:2, 2:1, and 5:1, respectively. The effects of different A:B ratios on gelation properties and in vitro VDAP release behavior were investigated. Component A was lecithin, component B was SMO, and the amounts of oleic acid and VDAP remained consistent.

[0214] Take 0.5g of each proportion of VDAP-LLC liquid crystal precursor and place them in sample vials. Add 5ml of pH 7.4 PBS buffer as a hydration medium and incubate at 37℃ to allow the liquid crystal precursor to fully absorb water and form gels in situ. After hydration, invert the sample vials and observe whether each group of samples can form gels, whether the gels remain intact, and whether there is obvious phase separation or structural inhomogeneity.

[0215] The hydration-gelling behavior, VDAP release behavior at pH 7.4, and VDAP release behavior at pH 6.5 were measured for each group of samples. The results are shown in the figure. Different mass ratios of component A and component B have a significant impact on the hydration-gelling behavior and VDAP release behavior of the liquid crystal precursor.

[0216] like Figure 17 As shown in the figure, the hydration results of component A into gels indicate that systems with a mass ratio of component A to component B of 1:5, 1:2, 2:1, and 5:1 can all undergo hydration and form gel-like systems in an aqueous environment. Among these, the gel appearance of the A:B=1:5 group after hydration is relatively heterogeneous, with noticeable phase separation or structural inhomogeneity in some areas. The gels formed after hydration of the A:B=1:2, 2:1, and 5:1 groups are more complete and have a more uniform appearance, indicating that the systems within the above ratio ranges all possess in-situ gelation ability, with the gel integrity being relatively better in the A:B=1:2 to 5:1 range.

[0217] At pH 7.4, all sample ratios exhibited relatively slow VDAP release behavior. The A:B=1:5 group showed the fastest release rate, with a cumulative release rate of approximately 18% by day 5; the A:B=1:2 group showed a cumulative release rate of approximately 9% by day 5; the A:B=2:1 group showed a cumulative release rate of approximately 8% by day 5; and the A:B=5:1 group showed the slowest release, with a cumulative release rate of approximately 1% by day 5. These results indicate that under neutral physiological pH conditions, as the relative content of component A increases, the system's retention effect on VDAP is enhanced, thus reducing the VDAP release rate.

[0218] Under acidic conditions at pH 6.5, the VDAP release rate of each ratio of samples was significantly higher than that under pH 7.4, indicating that the VDAP-LLC system exhibits acidic pH-responsive release characteristics. Specifically, the A:B=1:5 group showed the fastest release under acidic conditions, with a cumulative release rate approaching or exceeding 90% on day 1 and over 90% on day 2; the A:B=1:2 group showed a cumulative release rate of approximately 60% on day 1 and approximately 70% on day 3; the A:B=2:1 group showed a relatively gradual release, with a cumulative release rate of approximately 55% on day 5; and the A:B=5:1 group showed the slowest release, with a cumulative release rate of approximately 45% on day 5.

[0219] Based on the combined results of hydration gelation properties and pH-responsive release, it can be seen that when the mass ratio of component A to component B is within the range of 1:5 to 5:1, the system can form a hydration gel-like VDAP-LLC system, exhibiting differentiated release behavior: slow release at pH 7.4 and accelerated release at pH 6.5 (acidic condition). Specifically, the A:B=1:5 group showed the most rapid release under acidic conditions, but the gel appearance uniformity was relatively poor, and the release was also relatively fast at pH 7.4; the A:B=5:1 group had better gel integrity, but the overall release rate was lower; the A:B=1:2 and A:B=2:1 groups showed a good balance between gel integrity, slow release capacity under neutral conditions, and acidic responsive release capacity.

[0220] Therefore, the preferred mass ratio of component A to component B is 1:2 to 2:1. Within this preferred range, the VDAP-LLC liquid crystal precursor can balance injectability, in-situ gelation, gel stability, pH-responsive release, and sustained drug release performance.

[0221] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A pH-responsive lyotropic liquid crystal drug delivery system, characterized in that, The drug delivery system is a precursor composition that is in a liquid state before injection and can form a lyotropic liquid crystal gel in situ after entering an aqueous environment. The precursor composition comprises, by weight of the total mass of the precursor composition: Carrier matrix mixture 50%–85%; Organic solvents 5%–30%; pH-responsive gating switch 1%–10%; Therapeutic active substances: 0.1%–10%; The carrier matrix mixture includes component A and component B, wherein component A is a phospholipid and component B is an amphiphilic lipid molecule capable of inducing the formation of an antiphase liquid crystal phase; The pH-responsive gating switch is a pH-sensitive molecule containing a protonable anion group; The therapeutic active substance is a therapeutic molecule that carries a net positive charge under physiological pH conditions.

2. The pH-responsive lyotropic liquid crystal drug delivery system according to claim 1, characterized in that, The mass ratio of component A to component B is 1:5 to 5:1, preferably 1:2 to 2:

1. Under physiological pH conditions, the drug delivery system restricts drug release through electrostatic interaction between the pH-responsive gating switch and the therapeutic active substance. In an acidic microenvironment, the electrostatic interaction is released due to protonation of the pH-responsive gating switch, which simultaneously induces a topological phase transition in the lyotropic liquid crystal gel, thereby promoting the release of the therapeutic active substance.

3. The pH-responsive lyotropic liquid crystal drug delivery system according to claim 1 or 2, characterized in that, Component A is selected from one or more of soybean lecithin, egg yolk lecithin, dipalmitoylphosphatidylcholine, and distearate phosphatidylcholine; Component B is selected from one or more of sorbitan monooleate, glyceryl monooleate, glyceryl dioleate, and phytanetriol.

4. The pH-responsive lyotropic liquid crystal drug delivery system according to claim 2, characterized in that, The organic solvent is a polar organic solvent that is miscible or partially miscible with water, preferably selected from one or more of anhydrous ethanol, dimethyl sulfoxide, N-methylpyrrolidone, propylene glycol, polyethylene glycol, and methanol.

5. The pH-responsive lyotropic liquid crystal drug delivery system according to claim 2, characterized in that, The pH-responsive gating switch is an unsaturated fatty acid containing a carboxyl group, preferably selected from one or more of oleic acid, linoleic acid, and linolenic acid, and more preferably oleic acid.

6. The pH-responsive lyotropic liquid crystal drug delivery system according to claim 2, characterized in that, The therapeutic active substance is selected from one or more of cationic amphiphilic polypeptides, small molecule chemical drugs containing protonable amino groups, and therapeutic proteins with an isoelectric point greater than 7.4; preferably, the cationic amphiphilic polypeptide is selected from one or more of VDAP, melipotaneous bee venom peptide, LL-37, defensin, Xenopus xenograft antimicrobial peptide, and LTX-315; the small molecule chemical drug containing protonable amino groups is selected from one or more of doxorubicin, mitoxantrone, bupivacaine, and aminoglycoside antibiotics; and the therapeutic protein with an isoelectric point greater than 7.4 is selected from one or more of basic fibroblast growth factor and lysozyme.

7. The pH-responsive lyotropic liquid crystal drug delivery system according to claim 2, characterized in that, The precursor composition is free of aqueous phase before injection, and after injection into an aqueous environment, the organic solvent diffuses into the surrounding aqueous phase, and the carrier matrix mixture absorbs water and self-assembles to form a lyotropic liquid crystal gel with an ordered nanoporous structure in situ. Preferably, under pH 7.4 conditions, the lyotropic liquid crystal gel is in an inverted hexagonal phase, and the cumulative release of the therapeutic active substance within 7 days is less than 10%. Under acidic conditions of pH 5.0 to 6.5, the lyotropic liquid crystal gel transforms from the inverted hexagonal phase to a bicontinuous inverted cubic phase, preferably to the Im3μm phase, and the cumulative release of the therapeutic active substance within 7 days is greater than 70%.

8. The pH-responsive lyotropic liquid crystal drug delivery system according to claim 2, characterized in that, The drug delivery system is any one of the following: (1) The therapeutically active substance is a VDAP polypeptide, component A is lecithin, component B is sorbitan monooleate, the pH-responsive gating switch is oleic acid, and the organic solvent includes ethanol and / or dimethyl sulfoxide; or, (2) The therapeutic active substance is doxorubicin, component A is soybean lecithin, component B is diglyceride, the pH-responsive gating switch is oleic acid, and the organic solvent is N-methylpyrrolidone.

9. A method for preparing a pH-responsive lyotropic liquid crystal drug delivery system according to any one of claims 1 to 8, characterized in that, Includes the following steps: Component A, component B, and a pH-responsive gating switch are dissolved in an organic solvent to obtain a carrier matrix solution; the therapeutic active substance is added to the carrier matrix solution and mixed evenly to obtain a pH-responsive lyotropic liquid crystal precursor composition that is in a liquid state before injection. The preparation method does not involve adding an aqueous phase during the preparation process; The method of achieving uniform mixing includes one or more of vortexing, stirring, ultrasonication, and homogenization. The pH-responsive lyotropic liquid crystal precursor composition can be injected via syringe and gelled in situ upon entering an aqueous environment.

10. The use of the pH-responsive lyotropic liquid crystal drug delivery system according to any one of claims 1 to 8 in the preparation of a medicament for treating diseases accompanied by a local acidic microenvironment, characterized in that, The disease is selected from one or more of solid tumors, bacterial infections, chronic inflammation, ischemic injuries, and trauma repair-related diseases; preferably, the solid tumor is one or more of triple-negative breast cancer, melanoma, liver cancer, pancreatic cancer, lung cancer, and colorectal cancer; more preferably, the drug delivery system is applied to the lesion site or postoperative residual cavity site by local injection.

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