Targeted stimuli-responsive drug delivery carrier, and preparation method and application thereof

Abstract

The application discloses a targeted stimulus-responsive drug delivery carrier and a preparation method and application thereof, and relates to the technical field of biological medicine. The drug delivery carrier provided by the application comprises a liposome nano-carrier and a lectin-1 targeting peptide. The lectin-1 targeting peptide can actively target the lectin-1 on the surface of a PDAC cell, realizes precise delivery, efficient targeting and internalization, and significantly improves the drug uptake rate of the cell. The liposome nano-carrier comprises structural phospholipids, a membrane stabilizer and a pegylated lipid. The pegylated lipid contains a GSH response group, can trigger a response under the stimulation of a high GSH environment, realizes intelligent controlled release, realizes rapid and specific release of the drug, improves the curative effect and reduces the systemic toxicity. The nano drug delivery carrier has excellent drug loading and slow-release performance, can overcome multiple physiological barriers such as the dense stroma barrier of PDAC, can efficiently kill tumor cells while inhibiting metastasis, and has extremely high application value.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a targeted, stimulus-responsive drug delivery carrier, its preparation method, and its application. Background Technology

[0002] Pancreatic ductal adenocarcinoma (PDAC) is one of the most malignant tumors, with a five-year survival rate of less than 10%. The primary cause of treatment failure lies in its unique tumor microenvironment (TME)—a fibrotic barrier composed of a dense extracellular matrix (ECM), cancer-associated fibroblasts (CAFs), and sparse blood vessels, which can account for more than 90% of tumor mass. This barrier not only severely hinders the penetration of chemotherapeutic drugs (such as gemcitabine) but also induces strong chemotherapeutic resistance.

[0003] To address these issues, nanomedicine strategies (such as PEGylated liposomes) have been widely adopted, primarily enhancing passive drug accumulation at tumor sites (EPR effect) by prolonging blood circulation time. However, this strategy also suffers from the following major drawbacks: 1) Traditional nanocarriers lack the ability to actively penetrate the dense PDAC matrix, making it difficult for drugs to reach the tumor core; 2) Insufficient specific targeting ability for tumor cells, failing to ensure efficient drug internalization; 3) Lack of controllable drug release, making it difficult to utilize the highly reducing environment within tumor cells for precise, explosive drug release; 4) Limited functionality, failing to inhibit metastasis while killing tumor cells.

[0004] Therefore, how to construct an intelligent delivery platform that can synergistically overcome the multiple physiological barriers of PDAC and effectively integrate multiple functions such as long circulation, matrix penetration, cell targeting, stimulus-response drug release and inhibition of metastasis remains a key issue that current technology urgently needs to address. Summary of the Invention

[0005] This invention aims to address at least one of the technical problems existing in the prior art. To this end, this invention proposes a targeted, stimulus-responsive drug delivery carrier, its preparation method, and its application, aiming to solve the problems of current nanocarriers being unable to actively penetrate multiple physiological barriers of PDAC, having insufficient specific targeting ability, lacking controllable drug release, and having limited functionality.

[0006] An embodiment of a first aspect of the present invention provides a targeted, stimulus-responsive drug delivery carrier, comprising a liposome nanocarrier and a targeting peptide; the targeting peptide includes a lectin-1 targeting peptide, the liposome nanocarrier includes a structural phospholipid, a membrane stabilizer, and a polyethylene glycol-modified lipid linked to an active group; the targeting peptide is chemically coupled to the active group linked to the polyethylene glycol-modified lipid, and the polyethylene glycol-modified lipid contains a glutathione-responsive group.

[0007] The targeted, stimulus-responsive drug delivery carrier according to a first aspect of the present invention has at least the following beneficial effects: The drug delivery carrier provided by the present invention includes a liposome nanocarrier and a lectin-1 targeting peptide. The lectin-1 targeting peptide can actively target the lectin-1 protein on the surface of PDAC cells, achieving precise delivery; the liposome nanocarrier includes structural phospholipids, membrane stabilizers, and polyethylene glycol-modified lipids linked with active groups. The polyethylene glycol-modified lipids contain glutathione responsive groups and can respond to stimulation in a glutathione (GSH) environment, achieving rapid and specific drug release. The novel liposome nanocarrier provided by the present invention has the following beneficial effects: (1) Highly efficient targeting and internalization: By specifically binding the lectin-1 targeting peptide to Plectin-1 on the surface of PDAC cells, active targeting and receptor-mediated endocytosis of drugs are achieved, significantly improving the drug uptake rate of tumor cells.

[0008] (2) Intelligent controlled release: Utilizing the high GSH environment in tumor cells to trigger the response of glutathione response groups, the drug is rapidly and specifically released in target cells, improving efficacy and reducing systemic toxicity.

[0009] (3) Excellent drug loading and sustained release performance: Through optimized formulation, the carrier achieves high drug loading (up to 10% or more) and high encapsulation rate (up to 78.3%); at the same time, it also exhibits excellent sustained release characteristics, with a cumulative release rate of only 10.7% over 16 days, which helps maintain long-term drug concentration at the tumor site.

[0010] (4) It synergistically overcomes multiple physiological barriers such as the dense matrix barrier of PDAC; overcomes the barrier of insufficient uptake by tumor cells through active targeting mediated by lectin-1 targeting peptide; achieves precise and rapid release of drugs in target cells through GSH-responsive drug release; and enhances the penetration ability of dense tumor matrix through optimized particle size and synergistic targeting function (penetration depth reaches 100 μm in 3D tumor sphere model).

[0011] (5) It can effectively kill tumor cells while inhibiting metastasis, and has extremely high practical application value.

[0012] In some embodiments of the present invention, the lectin-1 targeting peptide includes, but is not limited to, the PTP peptide.

[0013] In some embodiments of the present invention, the amino acid sequence of the lectin-1 targeting peptide is lysine-threonine-leucine-leucine-proline-threonine-proline (Lys-Thr-Leu-Leu-Pro-Thr-Pro, KTLLPTP).

[0014] Plectin-1 is a protein specifically and highly expressed on the cell membrane of PDAC cells, making it a promising target for precision therapy. Its targeting peptide not only mediates efficient endocytosis but also downregulates Plectin-1 expression, reversing related drug resistance. PTP peptide (KTLLPTP) is a short peptide sequence composed of seven amino acids that exhibits high selective binding to PDAC cells and is a polypeptide sequence that specifically targets Plectin-1. This invention achieves active and efficient drug targeting and receptor-mediated endocytosis through the specific binding of the PTP peptide to Plectin-1 on the surface of PDAC cells, significantly improving drug uptake by tumor cells.

[0015] In some embodiments of the present invention, the liposome nanocarrier includes a structural phospholipid, a membrane stabilizer, and a polyethylene glycol-modified lipid linked with an active group. The targeting peptide is chemically coupled to the active group linked to the polyethylene glycol-modified lipid to form a polyethylene glycol-modified lipid-targeting peptide conjugate. The polyethylene glycol-modified lipid contains a glutathione responsive group.

[0016] The core of the stimulus-responsive drug delivery carrier provided by this invention lies in its composition, specifically comprising structural phospholipids (such as HSPC), membrane stabilizers (such as cholesterol), and a polyethylene glycol-modified lipid-targeting peptide conjugate (such as DSPE-SS-PEG2000-PTP) formed by a targeting peptide and a polyethylene glycol-modified lipid. In this conjugate, polyethylene glycol and the Plectin-1 targeting peptide (such as the PTP peptide) are linked to the lipid (such as DSPE) via a responsive group (such as a disulfide bond) that can be cleaved by the high concentration of glutathione (GSH) within tumor cells. This is the foundation for achieving active targeting and intelligent controlled release.

[0017] In some embodiments of the present invention, the structural phospholipids include, but are not limited to, hydrogenated soybean phosphatidylserine (HSPC), 1,2-distearate-sn-glycerol-3-phosphocholine (DSPC), or dipalmitoylphosphatidylcholine (DPPC).

[0018] In some embodiments of the present invention, the membrane stabilizer includes cholesterol, but is not limited thereto.

[0019] In some embodiments of the present invention, the polyethylene glycol-modified lipid comprises a lipid, polyethylene glycol, and a glutathione-responsive group connecting the two, wherein the polyethylene glycol is terminally attached to an active group.

[0020] In some embodiments of the present invention, the lipids include phospholipids.

[0021] Preferably, the phospholipids include neutral phospholipids.

[0022] Specifically, the phospholipids include distearate, phosphatidylethanolamine (DSPE), 1,2 Dioleoyl SN glycerin 3 Phosphoryl ethanolamine (DOPE), 1,2 Distearate acyl sn Propyltrimethyl 3 Phosphocholine (DSPC), 1,2 Dioleoyl sn glycerin 3 At least one of phosphocholine (DOPC), dilauroyl lecithin (DLPC), hydrogenated soybean lecithin (HSPC), egg yolk lecithin (EPC), hydrogenated soybean phosphatidylethanolamine (HSPE), cephalothorylserine (PS), dipalmitoyl glycerol (DPPG), dioleoyl phosphatidyl glycerol (DOPG), and dipalmitoyl sphingomyelin (DPSP), preferably DSPE.

[0023] In some embodiments of the present invention, the polyethylene glycol includes PEG2000.

[0024] In some embodiments of the present invention, the active group includes a carboxyl group (-COOH), but is not limited thereto.

[0025] In some embodiments of the present invention, the glutathione responsive group includes a disulfide bond (SS) that can be cleaved by glutathione.

[0026] This invention utilizes the high concentration of glutathione (GSH) within tumor cells to design a stimulus-responsive drug delivery system, which can improve treatment specificity. The high GSH concentration within tumor cells triggers disulfide bond breakage, enabling rapid and specific drug release within target cells, thereby improving efficacy and reducing systemic toxicity.

[0027] In some embodiments of the present invention, the polyethylene glycol-modified lipids include at least one of DSPE-SS-PEG2000-COOH, DMG-SS-PEG2000-COOH, PCL2000-SS-PEG2000-COOH, PCL3000-SS-PEG2000-COOH, PLA2000-SS-PEG2000-COOH, OLA-SS-PEG2000-COOH, MTA-SS-PEG2000-COOH, PLGA-SS-PEG2000-COOH, DPPE-SS-PEG2000-COOH, or DOPE-SS-PEG2000-COOH, preferably DSPE-SS-PEG2000-COOH.

[0028] In some specific embodiments of the present invention, the polyethylene glycol-modified lipid is DSPE-SS-PEG2000-COOH. The structural formula of DSPE-SS-PEG2000-COOH is shown below:

[0029] It is a polyethylene glycol (PEG) chain in DSPE-SS-PEG (distearylphosphatidylethanolamine-disulfide bond-polyethylene glycol) with an active carboxyl group (-COOH). DSPE-SS-PEG is composed of three key parts: DSPE (distearylphosphatidylethanolamine), SS (disulfide bond), and PEG (polyethylene glycol). DSPE, a common component of liposome membrane materials, possesses high stability and the ability to encapsulate compounds, effectively protecting them from environmental influences. PEG, a hydrophilic polymer, not only increases the water solubility and stability of nanocarriers but also prevents them from being recognized and cleared by the immune system, thereby improving bioavailability. The disulfide bond (SS) is a dynamic bond that can break and reconnect under specific conditions (such as reducing environments), providing DSPE-SS-PEG with adaptability and reversibility in different environments, enhancing its application flexibility and tunability.

[0030] In some embodiments of the present invention, the targeting peptide is coupled with the polyethylene glycolated lipid to form a polyethylene glycolated lipid-targeting peptide conjugate.

[0031] Specifically, the polyethylene glycol-modified lipid-targeting peptide conjugate includes DSPE-SS-PEG2000-PTP (distearylphosphatidylethanolamine-disulfide bond-polyethylene glycol 2000-PTP peptide).

[0032] The amino group of the targeted peptide PTP of this invention is covalently coupled with the carboxyl group of the polyethylene glycol-modified lipid DSPE-SS-PEG2000-COOH to form the polyethylene glycol-modified lipid-targeted peptide conjugate DSPE-SS-PEG2000-PTP. The specific reaction formula is as follows: Figure 1 As shown in (a).

[0033] In some embodiments of the present invention, the molar ratio of the structural phospholipid, the membrane stabilizer, and the polyethylene glycol-modified lipid-targeting peptide conjugate is (7.5~9):(0.5~1.5):(0.5~1.5), for example, 8:1:1, 9:0.5:0.5, 7.5:1:1.5, 7.5:1.5:1, or other ratios within the above ranges. Preferably, the molar ratio of the structural phospholipid, the membrane stabilizer, and the polyethylene glycol-modified lipid-targeting peptide conjugate is 8:1:1.

[0034] Specifically, the molar ratio of HSPC, cholesterol, and DSPE-SS-PEG2000-PTP is 8:1:1.

[0035] The present invention sets out specific components and ratios for the drug delivery carrier. These specific ratios are key to achieving high drug loading (12.2%), high encapsulation efficiency (78.3%), and excellent sustained-release properties (cumulative release rate of only 10.7% over 16 days), ensuring the optimal balance between formulation stability, drug loading space, and functional modifications.

[0036] In some embodiments of the present invention, the average particle size of the stimulus-responsive drug delivery carrier is 70-90 nm, preferably 75-85 nm, and more preferably about 80 nm. Exemplarily, it can be 70 nm, 71 nm, 72 nm, 73 nm, 74 nm, 75 nm, 76 nm, 77 nm, 78 nm, 79 nm, 80 nm, 81 nm, 82 nm, 83 nm, 84 nm, 85 nm, 86 nm, 87 nm, 88 nm, 89 nm, or 90 nm, or within any two of the above values. The present invention enhances the penetration ability of the above drug delivery carrier into dense tumor matrix (reaching a penetration depth of 100 μm in a 3D tumor sphere model) through the synergistic effect of optimized particle size (approximately 80 nm) and targeting function.

[0037] In some embodiments of the present invention, the liposome nanocarrier is internally loaded with a drug.

[0038] In some embodiments of the present invention, the drug includes, but is not limited to, gemcitabine.

[0039] In some specific embodiments of the present invention, the present invention provides a targeted, stimulus-responsive drug delivery carrier, the drug delivery carrier comprising a liposomal nanocarrier and a lectin-1 targeting peptide, the liposomal nanocarrier comprising a structural phospholipid, a membrane stabilizer, and a DSPE-SS-PEG; the PEG chain of the DSPE-SS-PEG is linked to an active group, and the lectin-1 targeting peptide is chemically coupled to the active group.

[0040] In some embodiments of the present invention, the lectin-1 targeting peptide includes, but is not limited to, the PTP peptide.

[0041] In some embodiments of the present invention, the amino acid sequence of the lectin-1 targeting peptide is lysine-threonine-leucine-leucine-proline-threonine-proline (Lys-Thr-Leu-Leu-Pro-Thr-Pro, KTLLPTP).

[0042] Figure 1 This is a schematic diagram of one structure of the targeted, stimulus-responsive drug delivery carrier provided by the present invention, wherein (a) is the synthetic route of the peptide-modified stimulus-responsive drug delivery carrier, and (b) is a schematic diagram of one structure. As shown, the structural phospholipid HSPC, the membrane stabilizer cholesterol, and DSPE-SS-PEG2000-COOH form a liposome, and the drug is loaded inside the liposome; the amino group of the targeting peptide PTP and the carboxyl group of DSPE-SS-PEG2000-COOH are coupled by forming a covalent bond to form a polyethylene glycol-modified lipid-targeting peptide conjugate DSPE-SS-PEG2000-PTP.

[0043] The targeted, stimulus-responsive drug delivery carrier provided by this invention has the following beneficial effects: (1) Highly efficient targeting and internalization: By specifically binding the lectin-1 targeting peptide to Plectin-1 on the surface of PDAC cells, active targeting and receptor-mediated endocytosis of drugs are achieved, significantly improving the drug uptake rate of tumor cells.

[0044] (2) Intelligent controlled release: The high GSH concentration in tumor cells triggers the breakage of disulfide bonds, thereby achieving rapid and specific release of drugs in target cells, improving efficacy and reducing systemic toxicity.

[0045] (3) Excellent drug loading and sustained release performance: Through optimized formulation, the carrier achieves high drug loading (up to 10% or more) and high encapsulation rate (up to 78.3%); at the same time, it also exhibits excellent sustained release characteristics, with a cumulative release rate of only 10.7% over 16 days, which helps maintain long-term drug concentration at the tumor site.

[0046] (4) It synergistically overcomes multiple physiological barriers such as the dense matrix barrier of PDAC; overcomes the barrier of insufficient uptake by tumor cells through active targeting mediated by lectin-1 targeting peptide; achieves precise and rapid release of drugs in target cells through GSH-responsive drug release; and enhances the penetration ability of dense tumor matrix through optimized particle size and synergistic targeting function (penetration depth reaches 100 μm in 3D tumor sphere model).

[0047] (5) It can effectively kill tumor cells while inhibiting metastasis, and has extremely high practical application value.

[0048] A second aspect of the present invention provides a method for preparing the above-described targeted, stimulus-responsive drug delivery carrier, comprising the steps of: S100, providing polyethylene glycol-modified lipids, wherein the polyethylene glycol-modified lipids are attached with active groups, and wherein the polyethylene glycol-modified lipids contain glutathione responsive groups; S200. Synthesize the targeting peptide and couple the targeting peptide with the active group on the polyethylene glycol-modified lipid through a chemical bond to form a polyethylene glycol-modified lipid-targeting peptide conjugate. S300: According to a predetermined ratio, the structural phospholipid, membrane stabilizer, and the polyethylene glycol-targeted peptide conjugate are dissolved in an organic solvent, and the solvent is removed by rotary evaporation to form a lipid film. S400: Add a hydration medium to the lipid film, and after preliminary dispersion, form a preliminary dispersion; S500. The preliminary dispersion is subjected to ultrasonic treatment to form nanoliposomes through ultrasonic self-assembly. S600. After purifying the nanoliposomes, the targeted, stimulus-responsive drug delivery carrier is obtained.

[0049] The preparation method provided by this invention is simple: it employs the classic thin-film hydration-ultrasound method, a mature process that is easy to scale up for production, and the formulation quality is stable and controllable. Specifically, the preparation method includes steps such as film formation, hydration, ultrasonic self-assembly, and purification: the above components are dissolved together in an organic solvent, the solvent is removed by rotary evaporation to form a lipid film; a hydration medium is added to hydrate and detach the film, allowing the liposomes to be initially dispersed; the initial dispersion is ultrasonically treated to form nanoliposomes with uniform particle size; finally, filtration purification is performed. This method is mature, reproducible, and a key step in obtaining liposome drug delivery carriers with specific physicochemical properties (e.g., average particle size of approximately 80 nm, PDI < 0.2). The obtained liposome drug delivery carrier has the following characteristics: an average particle size of 70-90 nm (preferably approximately 80 nm), a moderate surface potential, a drug loading capacity of over 12.2%, and an encapsulation efficiency of over 78.3%.

[0050] In some embodiments of the present invention, the polyethylene glycol-modified lipid is DSPE-SS-PEG2000-COOH.

[0051] In some embodiments of the present invention, the lectin-1 targeting peptide includes, but is not limited to, the PTP peptide.

[0052] In some embodiments of the present invention, the amino acid sequence of the lectin-1 targeting peptide is lysine-threonine-leucine-leucine-proline-threonine-proline (Lys-Thr-Leu-Leu-Pro-Thr-Pro, KTLLPTP).

[0053] The target peptide was synthesized according to the above sequence. The amino group of the target peptide was covalently coupled with the carboxyl group of the polyethylene glycol-modified lipid DSPE-SS-PEG2000-COOH to form the polyethylene glycol-modified lipid-target peptide conjugate DSPE-SS-PEG2000-PTP.

[0054] In some embodiments of the present invention, in step S300, the drug to be loaded is dissolved together with structural phospholipids, membrane stabilizers, and polyethylene glycol-modified lipid-targeting peptide conjugates in an organic solvent, and the solvent is removed by rotary evaporation to form a lipid film.

[0055] In some embodiments of the present invention, in step S300, the molar ratio of the structural phospholipid, the membrane stabilizer, and the polyethylene glycol-modified lipid-targeting peptide conjugate is (7.5~9):(0.5~1.5):(0.5~1.5), for example, it can be 8:1:1, 9:0.5:0.5, 7.5:1:1.5, 7.5:1.5:1, or other ratios within the above range. Preferably, the molar ratio of the structural phospholipid, the membrane stabilizer, and the polyethylene glycol-modified lipid-targeting peptide conjugate is 8:1:1.

[0056] Specifically, the molar ratio of HSPC, cholesterol, and DSPE-SS-PEG2000-PTP is 8:1:1.

[0057] Specifically, the organic solvent includes chloroform.

[0058] Specifically, the hydration medium includes ultrapure water.

[0059] Specifically, the conditions for the ultrasonic treatment are: ultrasonic power 30W, treatment time 15 minutes.

[0060] Specifically, the purification step is as follows: the nanoliposomes are filtered through a 0.22 μm filter membrane.

[0061] An embodiment of the third aspect of the present invention provides the application of the above-described targeted stimulus-responsive drug delivery carrier or the method for preparing the above-described targeted stimulus-responsive drug delivery carrier in the preparation of tumor-targeted drug formulations and / or vaccine formulations.

[0062] In some embodiments of the present invention, the pharmaceutical preparation includes a pharmaceutical preparation for treating PDAC.

[0063] In some embodiments of the present invention, the tumor includes pancreatic ductal adenocarcinoma (PDAC).

[0064] This invention confirms that the drug delivery carrier can achieve a drug loading capacity of over 12.2% and an encapsulation efficiency of over 78.3%, and achieves a half-maximal inhibitory concentration (IC50) against PDAC cells in vitro. 50 The concentration of KPC-Luc was 32.8 ng / mL, significantly superior to unmodified liposomes (56.8 ng / mL) and free drug (112 ng / mL). In a mouse model of KPC-Luc orthotopic pancreatic cancer, it inhibited 96.4% of tumor bioluminescent signals (compared to free drug) and completely suppressed lung metastasis, demonstrating excellent antitumor effects and anti-metastatic potential.

[0065] An embodiment of the fourth aspect of the present invention provides a pharmaceutical formulation comprising the above-described targeted stimulus-responsive drug delivery carrier or the targeted stimulus-responsive drug delivery carrier prepared by the above-described preparation method, and a drug loaded within the drug delivery carrier.

[0066] In some embodiments of the present invention, the pharmaceutical preparation includes a pharmaceutical preparation for treating PDAC.

[0067] In some embodiments of the present invention, the drug includes at least one of nucleic acid, plasmid, nucleotide, protein, polypeptide, and small molecule drug.

[0068] Preferably, the pharmaceutical preparation further includes at least one of a pharmaceutically acceptable carrier, excipient, and excipient.

[0069] The term "pharmaceutically acceptable" as used in this invention means, to the extent that reasonable medical judgment allows, that contact with human and animal tissues is suitable without excessive toxicity, irritation, allergic reactions or other problems or complications, and that matches a reasonable benefit / risk ratio.

[0070] The term "pharmaceutically acceptable carrier" as used in this invention refers to a pharmaceutically acceptable material, composition, or medium, such as a liquid or solid filler, diluent, excipient, or solvent-encapsulating material, which participates in carrying or transporting the compounds of this invention from one organ or part of the body to another organ or part of the body. Each carrier must be "acceptable" in the sense that it is compatible with other components of the formulation and harmless to the patient. Examples of materials that can be used as pharmaceutically acceptable carriers include: sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; powdered tragacanth gum; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; polyols such as glycerol, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginate; pyrogen-free water; isotonic saline; Ringer's solution; ethanol; pH buffers; polyesters, polycarbonates, and / or polyanhydrides; and other non-toxic and compatible substances used in pharmaceutical formulations.

[0071] Preferably, the administration method of the pharmaceutical preparation includes at least one of intramuscular injection, intradermal injection, intravenous injection, arterial injection, transdermal absorption, intraperitoneal injection, oral administration, and nasal spray.

[0072] A fifth aspect of the present invention provides a vaccine formulation comprising the above-described targeted stimulus-responsive drug delivery carrier or the targeted stimulus-responsive drug delivery carrier prepared by the above-described preparation method, and a vaccine loaded within the drug delivery carrier.

[0073] In some embodiments of the present invention, the vaccine formulation includes a vaccine formulation for the prevention of PDAC.

[0074] In some embodiments of the present invention, the vaccine formulation further includes an immune adjuvant, such as aluminum hydroxide adjuvant.

[0075] Preferably, the vaccine formulation is at least one of intramuscular liquid injection, intravenous liquid injection, intranasal liquid injection, intradermal liquid injection, or subcutaneous liquid injection.

[0076] Other features and advantages of the invention will be set forth in the description which follows, and will be apparent in part from the description, or may be learned by practicing the invention. The objects and other advantages of the invention may be realized and obtained by means of the structures particularly pointed out in the description, claims, and drawings. Attached Figure Description

[0077] Figure 1 Synthetic pathways and structural diagrams of peptide-modified stimulus-responsive drug delivery carriers provided by the present invention; Figure 2 These are cryo-transmission electron microscopy images of the liposome drug delivery carriers of Examples 1-3 and Comparative Example 1 of the present invention; Figure 3 This is a schematic diagram showing the drug loading and encapsulation efficiency results of the liposome drug delivery carriers in Examples 1-3 and Comparative Example 1 of the present invention. Figure 4 The in vitro drug release curves of the liposome drug delivery carriers of Example 2 and Comparative Example 1 of the present invention are shown. Figure 5 The results of the antitumor toxicity test between the liposome drug delivery carriers of this invention and the control group are shown. Figure 6 This is a comparison of the penetration depth of the liposome drug delivery carriers in 3D tumor sphere models of Examples 1-3 and Comparative Example 1 of the present invention. Figure 7 This is a dynamic monitoring diagram of the growth of mouse tumors in each group of liposome drug delivery carriers and the control group of the present invention; Figure 8 This is a quantitative analysis diagram of the growth of mouse tumors in each group of liposome drug delivery carriers and the control group in this invention; Figure 9 The results of the morphology (a) and weight analysis (b) of mouse ex vivo tumors of each group of liposome drug delivery carriers and the control group in this invention are shown in the figure. Figure 10 To evaluate the anti-transfer effect of the liposome drug delivery carrier of the present invention compared with the control group, (a) stained section analysis and (b) quantitative analysis results; Figure 11 The results show the weight changes of the liposome drug delivery carrier of this invention and the control group. Detailed Implementation

[0078] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0079] In the description of this invention, the terms "one embodiment," "some embodiments," "illustrative embodiment," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of the invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0080] In the description of this invention, unless otherwise stated, the numerical range "a~b" represents a shortened representation of any combination of real numbers between a and b, where a and b are both real numbers. Unless otherwise stated, the various reaction or operation steps may be performed sequentially or not. Preferably, the reaction methods in this invention are performed sequentially.

[0081] As used in this invention, the term "nanocarrier" refers to nanoparticles with a diameter of less than 1000 nanometers (nm). In some embodiments, as defined by the National Science Foundation, the nanoparticles have a diameter of less than 300 nm. In some embodiments, as defined by the National Institutes of Health, the nanoparticles have a diameter of less than 100 nm. In some embodiments, the nanoparticles are microcells, which comprise closed compartments separated from the bulk solution by a microcell membrane, said membrane typically comprising an amphiphilic entity surrounding and enclosing a space or compartment (e.g., defining an interior cavity). In some embodiments, the microcell membrane comprises at least one polymer, such as a biocompatible and / or biodegradable polymer.

[0082] As used in this invention, the term "liposome nanocarrier" includes compositions, formulations, and / or nanomaterials of lipid nanoparticles. In some embodiments, the lipid nanoparticles comprise one or more components, such as compounds, ionizable lipids, sterols, polyethylene glycol-modified lipids, and phospholipids.

[0083] As used in this invention, the terms "polyethylene glycol-modified lipids" or "PEGylated lipids" refer to activated PEG. Lipids are substances that contain one or more active groups.

[0084] The term "peptide" as used herein generally has the art-recognized meaning of a polymer of at least three amino acids. Those skilled in the art will understand that the term "peptide" is intended to be general enough to encompass peptides having the complete sequences enumerated herein, while also encompassing peptides representing functional fragments of such complete peptides (e.g., fragments retaining at least one active component). Furthermore, those skilled in the art will understand that protein sequences can often be substituted with some degree of activity without destroying activity. Therefore, the relevant term "peptide" as used herein encompasses a total sequence identity that retains activity and shares at least about 30 to 40% (typically greater than about 50%, 60%, 70%, or 80%) of another peptide of the same class, and further typically includes at least one region with higher identity (in one or more highly conserved regions, said identity is typically greater than 90% or even 95%, 96%, 97%, 98%, or 99%), generally encompassing any peptide of at least 3 to 4 amino acids and typically up to 20 or more amino acids. Peptides may contain L amino acids, D Amino acids or both, and may contain any of a variety of amino acid modifications or analogs known in the art. Suitable modifications include, for example, terminal acetylation, amidation, methylation, etc.

[0085] Experimental methods in the following examples, unless otherwise specified, are generally performed under standard conditions or as recommended by the manufacturer. Unless otherwise specified, the materials and reagents used in these examples are commercially available.

[0086] Example 1 Preparation of drug delivery carrier PL1G Accurately weigh 5 μmol of HSPC (purchased from Carbohydrate Biotechnology Co., Ltd., catalog number 81000047), 4.5 μmol of cholesterol (purchased from Carbohydrate Biotechnology Co., Ltd., CAS number 57-88-5), 0.5 μmol of DSPE-SS-PEG2000-PTP (purchased from Carbohydrate Biotechnology Co., Ltd., catalog number 81001264), and 2 μmol of gemcitabine (purchased from Huazhong Haiwei (Beijing) Gene Technology Co., Ltd., CAS number 95058-81-4), and dissolve them together in 5 mL of chloroform. The molar percentages of HSPC, cholesterol, and DSPE-SS-PEG2000-PTP, considered as a whole, are 50%, 45%, and 5%, respectively. Evaporate to dryness in a 40°C water bath to form a uniform lipid film. Add 5 mL of ultrapure water to the flask and vortex to allow the film to initially hydrate and detach. The suspension was transferred to centrifuge tubes and placed in an ice-water bath. The tubes were then sonicated at a certain power for 10 minutes using an ultrasonic cell disruptor to obtain a nanoliposome suspension. Finally, the suspension was filtered through a 0.22 μm aqueous syringe filter to obtain a gemcitabine-loaded liposome drug delivery carrier, labeled PL1G, and stored at 4°C for later use.

[0087] Example 2 Preparation of drug delivery carrier PL2G The difference between this embodiment and Example 1 is that: HSPC is 5.6 μmol, cholesterol is 3.9 μmol, and DSPE-SS-PEG2000-PTP is 0.5 μmol. Taking HSPC, cholesterol, and DSPE-SS-PEG2000-PTP as a whole, the molar percentage of HSPC is 56%, the molar percentage of cholesterol is 39%, and the molar percentage of DSPE-SS-PEG2000-PTP is 5%. The resulting gemcitabine-loaded liposomal drug delivery carrier is labeled PL2G.

[0088] The rest is the same as in Example 1, and will not be repeated here.

[0089] Example 3 Preparation of drug delivery carrier PL3G The difference between this embodiment and Example 1 is that: HSPC is 6.5 μmol, cholesterol is 3 μmol, and DSPE-SS-PEG2000-PTP is 0.5 μmol. Considering HSPC, cholesterol, and DSPE-SS-PEG2000-PTP as a whole, the molar percentage of HSPC is 65%, the molar percentage of cholesterol is 30%, and the molar percentage of DSPE-SS-PEG2000-PTP is 5%. The resulting gemcitabine-loaded liposomal drug delivery carrier is labeled PL3G.

[0090] The rest is the same as in Example 1, and will not be repeated here.

[0091] Comparative Example 1: Preparation of the drug delivery carrier L2G The difference between this comparative example and Example 2 is that it uses DSPE-SS-PEG2000-COOH (purchased from Carbohydrate Biotechnology Co., Ltd., catalog number Z0031337-2000) without PTP peptide conjugation. The resulting gemcitabine-loaded liposomal drug delivery carrier is labeled L2G.

[0092] The rest is the same as in Example 2, and will not be repeated here.

[0093] Test Example 1 Performance Characterization The particle size and zeta potential of the liposome drug delivery carriers obtained in Examples 1-3 and Comparative Example 1 were determined using a dynamic light scattering instrument. The specific steps were as follows: A 1.0 mL sample solution of liposome drug delivery carrier with a concentration of 0.01 mg / mL and no bubbles was placed in a four-sided transparent quartz cuvette under constant temperature (25℃) and fixed scattering angle (90°) conditions. The hydrodynamic diameter and particle size distribution of the nanoparticles were determined by detecting the fluctuation of the scattered light intensity over time and analyzing the autocorrelation function.

[0094] The results are shown in Table 1. The average particle size was 78.8 ± 3.2 nm, the polydispersity index (PDI) was less than 0.2, and the zeta potential was -5.1 ± 0.8 mV (Table 1).

[0095] Table 1. Particle size, potential, and dispersion coefficient of liposome drug delivery carriers with different formulations

[0096] The liposome drug delivery carriers of Examples 1-3 and Comparative Example 1 were observed by cryo-transmission electron microscopy, and the results are as follows: Figure 2 As shown in the figure, the liposome drug delivery carrier is spherical or near-spherical in shape and has a uniform morphology. Figure 2 ).

[0097] The drug loading and encapsulation efficiency of the liposome drug delivery carriers in Examples 1-3 and Comparative Example 1 were determined by ultraviolet spectrophotometry. The specific steps were as follows: Using a Shimadzu UV-2600 ultraviolet spectrophotometer, 0.7 mL of a bubble-free sample solution with a liposome drug delivery carrier concentration of 0.01 mg / mL was placed in a slit quartz cuvette, and the intensity of the characteristic absorption peak (269 nm) of gemcitabine was measured. The results are as follows: Figure 3After plotting the gemcitabine standard curve, an appropriate amount of lyophilized liposomes was reconstituted in DMSO, and the drug content was determined. The calculated maximum drug loading was 12.2%, and the maximum encapsulation efficiency was 78.3%. Figure 3 ).

[0098] Test Example 2: In vitro drug release behavior study In vitro drug release experiments were conducted using dialysis. Equal volumes of PL2G liposomes (prepared in Example 2) and unmodified L2G liposomes (prepared in Comparative Example 1) were placed separately in PBS release medium containing 10 mM GSH (simulating the intracellular environment of tumor cells). The mixture was kept at 37°C with constant shaking. Samples were taken at different time points, and equal volumes of fresh release medium were added to determine the cumulative drug release rate. The results are as follows: Figure 4 The results showed that the cumulative release rate of PL2G over 16 days was 10.7%, significantly lower than that of L2G (29.8%). Figure 4 This demonstrates that PTP modification and optimized particle size together endow it with excellent sustained-release properties.

[0099] Test Example 3: In vitro cytotoxicity and targeting validation To evaluate the killing effect and targeting of the liposome drug delivery carrier of the present invention on PDAC cells, a systematic in vitro cell experiment was conducted.

[0100] Cell culture: The human pancreatic cancer cell line PANC-1 (highly expressing Plectin-1) was selected. All cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS) in a constant temperature incubator at 37°C and 5% CO2.

[0101] Dose-response (CCK-8) assay: Cell viability was assessed using the CCK-8 assay. PANC-1 cells in logarithmic growth phase were cultured at 5 × 10⁶ cells per well. 3 Cells were seeded at a density of [number] cells / well in 96-well plates and cultured for 24 hours to allow cell adhesion. The medium was then replaced with different concentrations (20 ng / mL to 100 ng / mL) of free gemcitabine, unmodified L2G liposomes (Comparative Example 1), and modified peptides PL1G, PL2G, and PL3G (Examples 1-3). After 72 hours of further culture, 10 μL of CCK-8 solution was added to each well and incubated for 2 hours. The absorbance (OD) of each well was measured at 450 nm using a microplate reader. Cell viability (%) = (OD value of experimental group - OD value of blank well) / (OD value of control group - OD value of blank well) × 100%. A dose-response curve was fitted using GraphPad Prism software, and the half-maximal inhibitory concentration (IC50) was calculated. 50 ).

[0102] The results are as follows Figure 5As shown, dose-response experiments confirmed the superior efficacy of PL2G. PL2G showed an effect on the IC50 concentration of PANC-1 cells. 50 The concentration was 32.8 ng / mL, significantly lower than unmodified L2G liposomes (56.8 ng / mL) and free gemcitabine (112 ng / mL), and also lower than PL1G (53.3 ng / mL) and PL3G (60.3 ng / mL). Figure 5 This highlights the enhanced cytotoxicity resulting from the optimized formulation (combining PTP targeting and GSH-responsive release).

[0103] Test Example 4: 3D Tumor Sphere Penetration Experiment To simulate the dense microenvironment of tumors in vivo and to evaluate the penetration ability of liposomes, a 3D tumor sphere experiment was conducted.

[0104] Tumor spheroid culture: PANC-1 tumor spheroids were cultured using an ultra-low adsorption 96-well plate method. Single-cell suspensions were seeded at a density of 1000 cells per well into 96-well plates pre-coated with 1.5% agarose to prevent cell adhesion. 100 μL of complete culture medium containing 10% FBS was added to each well. The culture plates were incubated at 37°C in a 5% CO2 incubator, with half of the culture medium replaced every 2–3 days. After approximately 7 days, regular spherical tumor spheroids with a diameter of approximately 300–400 μm were formed.

[0105] Penetration assay: Well-grown tumor spheres were transferred to confocal culture dishes. The tumor spheres were treated with DiI-labeled PL2G, PL1G, PL3G (PTP-modified liposomes of different particle sizes), and L2G, respectively. After incubation for 24 hours, the spheres were gently washed three times with PBS, fixed with 4% paraformaldehyde, stained with Hoechst 33342, and finally mounted with antifluorescent quenching mounting medium.

[0106] Results and Analysis: Z-stack scanning was performed using laser confocal microscopy, and the intensity distribution of fluorescence signals along the radial direction of the tumor sphere was analyzed using ImageJ software. The results are as follows: Figure 6 As shown, PL2G exhibited the deepest penetration depth, reaching approximately 100 μm, followed by PL1G. In contrast, PL3G (approximately 142 nm in diameter) and unmodified L2G both showed limited penetration, with fluorescence signals primarily enriched at the periphery of tumor spheres. This result highlights the crucial synergistic role of optimal particle size (approximately 80 nm) and active targeting (PTP peptide) in enhancing tumor tissue penetration. Smaller particle size facilitates diffusion within a dense matrix, while PTP peptide-mediated specific binding to tumor cells may further promote liposome delivery to deeper tumor spheres through a "bind-internalization-release" cycle.

[0107] Test Example 5: Evaluation of in vivo antitumor pharmacodynamics and anti-metastatic effects (KPC-Luc mouse model) To comprehensively evaluate the antitumor efficacy, anti-metastatic ability, and biosafety of the liposome drug delivery carrier of this invention in vivo, an orthotopic PDAC mouse model was established, and systematic pharmacodynamic studies were conducted. All animal experiments were approved by the Institutional Animal Care and Use Committee of Southern University of Science and Technology.

[0108] Animal model establishment and experimental grouping: 6-8 week old C57BL / 6 mice were selected and a spontaneous, highly invasive PDAC in situ model was established by in situ injection of KPC-Luc (derived from genetically engineered mice carrying luciferase reporter genes, KrasG12D / +; Trp53R172H / +; Pdx1-Cre) pancreatic cancer cells. After the tumors grew to a detectable fluorescence signal (about 7 days after inoculation), the tumor-bearing mice were randomly divided into 6 groups (n=5 per group): (1) Saline control group; (2) Free gemcitabine group; (3) PTP-modified liposome groups of different particle sizes PL1G (Example 1) and PL3G (Example 3); (4) Unmodified liposome group L2G (Comparative Example 1); (5) PL2G (Example 2), the optimal formulation of this invention. All treatment groups were administered gemcitabine via tail vein injection at an equivalent dose of 10 mg / kg, given every 3 days for a total of 5 doses. During the experiment, mouse weight and activity levels were monitored regularly, and tumor growth and metastasis were dynamically monitored using an in vivo imaging system.

[0109] Dynamic monitoring of antitumor efficacy: The growth of tumors in each group of mice was dynamically monitored using a small animal in vivo imaging system (IVIS). For example... Figure 7 As shown, throughout the entire treatment cycle (days 7, 10, 13, and 16), the PL2G treatment group exhibited the best tumor growth inhibition effect. Compared with the Saline control group, the bioluminescent signal in the tumor region of the PL2G group was almost completely inhibited. Quantitative analysis showed ( Figure 8 On day 16 of treatment, the tumor bioluminescence intensity in the PL2G group decreased by 96.4% compared to the free gemcitabine group and by 95.2% compared to the non-targeted liposome (L2G) group. This result confirms that PTP peptide modification, through active targeting mediated by the Plectin-1 receptor, significantly enhances drug accumulation at the tumor site, thereby achieving remarkable therapeutic effects.

[0110] Ex vivo tumor morphology and weight analysis: After treatment, mice were euthanized and tumor tissue was dissected for ex vivo analysis. Figure 9As shown in Figures ab, the PL2G group exhibited the smallest tumor volume and weight among all treatment groups, with clearly defined areas of necrosis and apoptosis. This indicates that the sustained-release properties of PL2G can maintain a sustained effective drug concentration within the tumor, while avoiding the problems of insufficient efficacy due to rapid metabolism of free drugs and toxicity accumulation due to nonspecific distribution.

[0111] Evaluation of Anti-metastatic Efficacy: Lung metastasis in pancreatic cancer is a common and fatal clinical complication. This invention systematically evaluated the inhibitory effect of different treatment groups on lung metastasis through gross observation of ex vivo lung tissue and analysis of hematoxylin and eosin (H&E) stained sections. Figure 10 As shown in Figures ab, the incidence of lung metastasis was 60% in the saline group, 40% in the free gemcitabine group, and 20% in both the PL3G and L2G groups. Notably, no lung metastases were observed in either the PL1G or PL2G groups. This result fully demonstrates that the PTP-targeting liposomes provided in this invention, especially the optimized PL2G, can not only effectively inhibit the growth of in situ tumors, but also effectively block distant metastasis of PDAC through its deep penetration ability and regulation of the tumor microenvironment (such as inhibiting epithelial-mesenchymal transition, EMT), exhibiting strong anti-metastatic potential.

[0112] Systemic biosafety assessment: Throughout the treatment period, the present invention closely monitored changes in mouse body weight as a preliminary indicator of systemic toxicity. For example... Figure 11 As shown, mice in the free gemcitabine group experienced significant weight loss in the later stages of treatment, suggesting some systemic toxicity. In contrast, the weight of mice in the PL2G treatment group remained stable throughout the experiment, showing no significant difference from the saline control group, indicating good biocompatibility and safety.

[0113] The above is a detailed description of the preferred embodiments of this application. However, this application is not limited to the above embodiments. Those skilled in the art can make various equivalent modifications or substitutions without departing from the spirit of this application. All such equivalent modifications or substitutions are included within the scope defined by the claims of this application.

Claims

1. A targeted, stimulus-responsive drug delivery carrier, characterized in that, The invention includes liposome nanocarriers and targeting peptides; the targeting peptides include lectin-1 targeting peptides, and the liposome nanocarriers include structural phospholipids, membrane stabilizers, and polyethylene glycol-modified lipids linked with active groups; the targeting peptides are chemically coupled to the active groups linked to the polyethylene glycol-modified lipids, and the polyethylene glycol-modified lipids contain glutathione responsive groups.

2. The targeted, stimulus-responsive drug delivery carrier according to claim 1, characterized in that, The lectin-1 targeting peptide includes the PTP peptide.

3. The targeted, stimulus-responsive drug delivery carrier according to claim 1, characterized in that, The targeting peptide is coupled with the polyethylene glycol-modified lipid to form a polyethylene glycol-modified lipid-targeting peptide conjugate, wherein the molar ratio of the structural phospholipid, the membrane stabilizer, and the polyethylene glycol-modified lipid-targeting peptide conjugate is (7.5~9):(0.5~1.5):(0.5~1.5); And / or, the average particle size of the stimulus-responsive drug delivery carrier is 70-90 nm.

4. The targeted, stimulus-responsive drug delivery carrier according to claim 1, 2, or 3, characterized in that, Including at least one of (a1) to (a8): (a1) The structural phospholipids include at least one of hydrogenated soybean phosphatidylserine, dipalmitoylphosphatidylcholine, or 1,2-distearate-sn-glycerol-3-phosphocholine; (a2) The membrane stabilizer includes cholesterol; (a3) The polyethylene glycol-modified lipids include lipids, polyethylene glycol, and glutathione responsive groups connecting the two, wherein the polyethylene glycol has an active group attached to its end; (a4) When (a3) ​​is included, the active group includes a carboxyl group; (a5) When (a3) ​​is included, the glutathione responsive group includes a disulfide bond that can be cleaved by glutathione; (a6) The PEGylated lipids include at least one of DSPE-SS-PEG2000-COOH, DMG-SS-PEG2000-COOH, PCL2000-SS-PEG2000-COOH, PCL3000-SS-PEG2000-COOH, PLA2000-SS-PEG2000-COOH, OLA-SS-PEG2000-COOH, MTA-SS-PEG2000-COOH, PLGA-SS-PEG2000-COOH, DPPE-SS-PEG2000-COOH, or DOPE-SS-PEG2000-COOH; (a7) The liposome nanocarrier is internally loaded with a drug; (a8) When (a7) is included, the drug includes gemcitabine.

5. The targeted, stimulus-responsive drug delivery carrier according to claim 2, characterized in that, The drug delivery carrier includes a liposome nanocarrier and a lectin-1 targeting peptide. The liposome nanocarrier includes a structural phospholipid, a membrane stabilizer, and DSPE-SS-PEG. The PEG chain of the DSPE-SS-PEG is linked to an active group, and the lectin-1 targeting peptide is chemically coupled to the active group.

6. A method for preparing a targeted, stimulus-responsive drug delivery carrier as described in any one of claims 1-5, characterized in that, Including the following steps: A polyethylene glycol-modified lipid is provided, wherein the polyethylene glycol-modified lipid is attached with an active group and the polyethylene glycol-modified lipid contains a glutathione responsive group; A targeted peptide is synthesized, and the targeted peptide is chemically coupled to the active group on the polyethylene glycol-modified lipid to form a polyethylene glycol-modified lipid-targeted peptide conjugate. According to a predetermined ratio, the structural phospholipid, membrane stabilizer, and the polyethylene glycol-targeted peptide conjugate are dissolved in an organic solvent, and the solvent is removed by rotary evaporation to form a lipid film. A hydration medium is added to the lipid film, and after preliminary dispersion, a preliminary dispersion is formed; The preliminary dispersion was subjected to ultrasonic treatment, and nanoliposomes were formed through ultrasonic self-assembly. After purification, the nanoliposomes were obtained as the targeted, stimulus-responsive drug delivery carrier.

7. The preparation method according to claim 6, characterized in that, The drug to be loaded, along with structural phospholipids, membrane stabilizers, and polyethylene glycol-modified lipid-targeting peptide conjugates, are dissolved in an organic solvent. The solvent is then removed by rotary evaporation to form a lipid film.

8. The use of a method for preparing a targeted stimulus-responsive drug delivery carrier as described in any one of claims 1-5 or as described in any one of claims 6-7 in the preparation of tumor-targeted drug formulations and / or vaccine formulations.

9. A pharmaceutical preparation, characterized in that, Includes a targeted stimulus-responsive drug delivery carrier as described in any one of claims 1-5 or a targeted stimulus-responsive drug delivery carrier prepared by the preparation method described in any one of claims 6-7, and a drug loaded inside the drug delivery carrier.

10. A vaccine formulation, characterized in that, Includes a targeted stimulus-responsive drug delivery vehicle as described in any one of claims 1-5 or a targeted stimulus-responsive drug delivery vehicle prepared by the preparation method described in any one of claims 6-7, and a vaccine loaded inside the drug delivery vehicle.

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