Double-response EGFR targeted paclitaxel liposome based on heat shock protein as well as preparation and application of double-response EGFR targeted paclitaxel liposome

Abstract

The invention relates to a double-response EGFR (epidermal growth factor receptor) targeted paclitaxel liposome based on heat shock protein, which adopts a pH sensitive design and is doped with heat shock protein 90 (HSP90) responsive molecule gambogic acid (GA), the surface of the liposome is modified with an EGFR antibody, and the core of the liposome is packaged with paclitaxel (PTX). According to the double-response EGFR targeted paclitaxel liposome of the heat shock protein, triple negative breast cancer cells are accurately locked through a double targeting strategy, and cell surface specific recognition is achieved through an EGFR antibody; gambogic acid GA responds to HSP90 overexpression in cells to promote drug release; the pH sensitive characteristic ensures that the medicine is quickly released in an acidic tumor microenvironment, the treatment effect is enhanced, and normal cell damage is reduced. According to the invention, the specific quick response release of the medicine is realized, the treatment accuracy and safety are improved, and the preparation method has a remarkable application prospect in the aspect of preparing the medicine for treating the triple-negative breast cancer.

Description

Technical Field

[0001] The present invention relates to the technical field of nano-biomedicine, and in particular to a heat shock protein-based dual-responsive EGFR-targeted paclitaxel liposome and a preparation and application thereof. Background Art

[0002] Breast cancer is one of the most common malignant tumors in women. Its pathogenesis is complex and diverse, involving multiple factors such as genetics, environment, and hormone levels. In recent years, with the deepening of molecular biology research, the role of heat shock proteins (HSPs) in the occurrence, development, and drug resistance of breast cancer has gradually attracted attention. Heat shock proteins are a class of proteins that are highly expressed when cells respond to various stress conditions (such as high temperature, oxidative stress, infection, hypoxia, ischemia, and physical and chemical damage). They are not only involved in cell protection, repair, and survival processes, but are also closely related to tumor cell proliferation, invasion, metastasis, and drug resistance.

[0003] In breast cancer cells, specific types of heat shock proteins, such as HSP70 and HSP90, have been found to be overexpressed, which promote the malignant progression of breast cancer by inhibiting apoptosis, promoting cell migration, and enhancing drug efflux. Therefore, targeted therapeutic strategies against these heat shock proteins, especially inhibiting their activity through small molecule inhibitors, provide new ideas for the treatment of breast cancer.

[0004] Although heat shock proteins have great potential as therapeutic targets for breast cancer, the development of a small molecule inhibitor that can specifically inhibit multiple breast cancer-related heat shock proteins still faces many technical challenges. For example, systemically injected inhibitors need to accurately identify and inhibit target heat shock proteins in breast cancer cells while avoiding adverse effects on similar proteins in normal cells or other key physiological proteins. Summary of the invention

[0005] In order to solve the problems of poor specificity, poor targeting, and high toxicity to normal cells of the above-mentioned heat shock protein inhibitors, the primary purpose of the present invention is to provide a dual-responsive EGFR-targeted paclitaxel liposome based on heat shock protein, thereby improving the specificity and targeting of heat shock protein inhibitors and effectively reducing the toxicity to normal cells.

[0006] Another object of the present invention is to provide a method for preparing the above-mentioned heat shock protein-based dual-responsive EGFR-targeted paclitaxel liposome.

[0007] The present invention is achieved through the following technical solutions:

[0008] 30 mg of dipalmitoyl phospholipids (DPPC), 10 mg of amphiphilic lipid phospholipid polyethylene glycol maleimide (DSPE-PEG 2000-Mal), 20mg gambogic acid (GA) were completely dissolved in 10mL chloroform, the organic solvent was removed by rotary evaporation to form a liposome membrane, and 5mL ultrapure water was added for ultrasonic hydration for 20 minutes to completely dissolve the liposomes in water. 10mg of paclitaxel (PTX) was added, and the cell disruptor was ultrasonically treated for 4 minutes, and then the solution was washed with ultrapure water using a 10kDa, 7500rpm ultrafiltration filter. The unloaded GA, PTX and organic solvent were removed three times to prepare DPG@Lipo. Subsequently, the PBS aqueous solution (0.5mg / mL) of the thiolated EGFR antibody was incubated at room temperature for 8h with a reaction molar ratio of 1:10 to fully react Mal with -SH, and the unbound thiolated EGFR antibody was washed to prepare EDPG@Lipo nanoparticles.

[0009] The advantages of the present invention are:

[0010] 1. The present invention achieves dual precision targeting of triple-negative breast cancer cells by carefully designing pH-sensitive liposomes as drug carriers and cleverly combining the dual targeting strategy of EGFR antibodies and gambogic acid (GA). The presence of EGFR antibodies ensures that the nanoparticles of the present invention can specifically recognize and bind to the EGFR receptors highly expressed on the surface of breast tumor cells, which is the first level of precision screening.

[0011] 2. The present invention introduces gambogic acid (GA), which further enhances the targeting and specificity of breast cancer treatment. This feature enables the nanoparticles of the present invention to trigger conformational changes of the nanocarrier or directly promote its degradation through the specific binding of GA to HSP90 after reaching the target breast cancer cells, thereby accurately releasing PTX inside the tumor cells. This process not only ensures that the drug can act on tumor cells efficiently and accurately, but also avoids the nonspecific release of the drug in non-target cells, significantly reducing the potential damage to normal cells.

[0012] 3. The pH-sensitive liposomes of the present invention can undergo structural changes in the acidic tumor microenvironment, promoting the rapid release of drugs, while remaining stable under normal physiological pH conditions, further improving the specificity and safety of the treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 This is a SEM characterization diagram of the nanoparticles of the present invention, wherein Figure 1 a is the SEM characterization picture of EDPG@Lipo nanoparticles, Figure 1 b is the SEM characterization image of empty liposomes (EDG@Lipo), scale bar: 200 nm.

[0014] Figure 2Simulate the controlled release of EDPG@Lipo nanoparticles triggered by the acidic environment of tumors.

[0015] Figure 3 In vitro toxicity results of EDPG@Lipo nanoparticles.

[0016] Figure 4 To determine the targeting of EDPG@Lipo nanoparticles, Figure 4 a is the result of MCF-10A cells taking up EDPG@Lipo nanoparticles. Figure 4 b shows the result of MDA-MB-231 cells taking up EDPG@Lipo nanoparticles, scale bar 50 μm.

[0017] Figure 5 Changes in HSP90 expression in MDA-MB-231 cells under different treatments.

[0018] Figure 6 Immunofluorescence staining of HSP90 in tumors after different treatments, scale bar 50 μm.

[0019] Figure 7 H&E staining of tumor sections from different mouse groups, scale bar 50 μm. DETAILED DESCRIPTION

[0020] The present invention will be further described below in conjunction with embodiments.

[0021] Example 1: A method for preparing EDPG@Lipo nanoparticles, comprising the following steps:

[0022] (1) 30 mg of dipalmitoyl phospholipid (DPPC), 10 mg of amphiphilic lipid phospholipid polyethylene glycol maleimide (DSPE-PEG 2000 -Mal), 20mg gambogic acid (GA) were completely dissolved in 10mL chloroform, the organic solvent was removed by rotary evaporation to form a liposome membrane, and 5mL ultrapure water was added for ultrasonic hydration for 20 minutes to completely dissolve the liposome in water. 10mg paclitaxel (PTX) was added, and ultrasonic treatment was performed with a cell disruptor for 4 minutes, and then the obtained solution was washed with ultrapure water using a 10kDa, 7500rpm ultrafiltration filter, and the unloaded GA, PTX and organic solvent were removed three times to prepare DPG@Lipo.

[0023] (2) The DPG@Lipo prepared in step (1) was mixed with a thiolated EGFR antibody PBS aqueous solution (0.5 mg / mL) at a reaction molar ratio of 1:10 and incubated with rotation at room temperature for 8 h to allow Mal to fully react with -SH, and the unbound thiolated EGFR antibody was washed away to prepare EDPG@Lipo nanoparticles.

[0024] Example 2: SEM characterization experiment of EDPG@Lipo nanoparticles

[0025] According to the preparation method of EDPG@Lipo nanoparticles in Example 1, empty liposomes loaded with PTX were prepared. The EDPG@Lipo nanoparticles and empty liposomes prepared in Example 1 were subjected to morphological examination by field emission scanning electron microscopy (SEM). The silicon wafer was divided into irregular small silicon wafers of suitable size using a diamond scriber, and the silicon wafer was ultrasonically cleaned with anhydrous ethanol and then dried with N2. After the sample was treated to an appropriate concentration, the EDPG@Lipo nanoparticles and the empty liposome suspension were carefully added dropwise to the smooth surface of the silicon wafer after the above-mentioned cutting, and the silicon wafer was carefully shaken to make the sample flat and spread out and then dried overnight, so that the treated silicon wafer with the sample was fixed above the scanning electron microscope sample stage under the action of conductive glue, and the sample on the sample stage was treated with a vacuum evaporator for metal film spraying, and the SEI mode was selected, and the voltage was 3kV. The morphological characteristics of EDPG@Lipo nanoparticles and empty liposomes were observed in the field of view.

[0026] Field emission scanning electron microscopy (SEM) was used to examine the morphology of EDPG@Lipo nanoparticles and empty liposomes. Figure 1 As shown in the figure, both have regular shapes and smooth spherical vesicle structures. Among them, the particle size of EDPG@Lipo nanoparticles is about 160nm ( Figure 1 b), slightly larger than the liposomes without PTX loading, with a particle size of about 120 nm ( Figure 1 a). This is because the introduction of PTX increases the volume inside the liposome vesicles, resulting in an increase in the overall size of the nanoparticles.

[0027] Example 3: Simulation of controlled release of EDPG@Lipo nanoparticles in the acidic environment of tumors

[0028] The prepared EDPG@Lipo nanoparticles were dissolved in 10 mL of PBS buffer solution (pH = 5.5, pH = 6.5 and pH = 7.4), transferred to a dialysis bag (MWCO = 3500Da), immersed in 40 mL of the same PBS release medium, placed in a 37°C constant temperature oscillating box to start the release experiment, and 4 mL of the release solution was taken out at a specific time point, and an equal volume of fresh release solution was added at the same time. The release solution was freeze-dried after being obtained, and its release efficiency at different periods was tested by re-dissolving it in DMF.

[0029] like Figure 2As shown, the release amount of EDPG@Lipo nanoparticles in PBS (pH=7.4) within 30 hours is less than 20%, indicating that it has good stability and can reduce the nonspecific release of drugs in normal tissues. When the experimental result of the release rate of EDPG@Lipo nanoparticles in a pH5.5 environment reached 50% within 24 hours, this data preliminarily confirmed that the nanoparticles have pH sensitivity. Further, under pH6.5 conditions, which is closer to the lysosome environment in tumor cells, the nanoparticles can release up to 80% of PTX within 24 hours, which fully proves that EDPG@Lipo nanoparticles can accurately respond to acidic environments and achieve efficient targeted release of drugs. In view of the fact that the liposomes of the present invention are constructed of pH-sensitive materials, they can cleverly utilize the weak acidic characteristics of microenvironments such as tumor tissues and lysosomes in tumor cells. When the EGFR antibody on the surface of EDPG@Lipo nanoparticles specifically recognizes and binds to tumor cells, this acidic environment becomes a trigger point, prompting the liposome membrane to undergo structural changes and then fuse with the tumor cell membrane. This process not only ensures that the drug PTX can be directly and efficiently delivered into tumor cells, but also significantly improves the drug's targeting and therapeutic effects.

[0030] Example 4: Cytotoxicity of EDPG@Lipo Nanoparticles

[0031] After the triple-negative breast cancer cells MDA-MB-231 and human normal breast cells MCF-10A were cultured to the logarithmic growth phase, they were seeded into 96-well plates and adhered to the wall for 24 hours. They were divided into 4 groups: PBS, PTX, DPG@Lipo and EDPG@Lipo. The above sample solutions diluted with complete medium at the same concentration of 10μg / mL were added to the wells and incubated at 37°C for 24 hours. After the incubation, 10μL CCK8 solution was added to each well, incubated for another 3 hours, and the absorbance at 450nm was measured with an enzyme reader.

[0032] like Figure 3As shown in the figure, the cell death in the control group was negligible. For the PTX group, most of the tumor cells were killed due to the broad-spectrum anticancer effect of PTX, and it also had the ability to kill normal breast cells. For the DPG@Lipo group, since the encapsulation material is pH-sensitive, when in an acidic tumor microenvironment, the liposome lysis is triggered to release the drug, and the cell viability is further reduced; while for normal cells, the neutral environment does not trigger the liposome lysis, and no obvious cytotoxicity is shown to normal cells. It is worth noting that for the EDPG@Lipo group, compared with other groups, due to the targeting of EGFR, it showed the most effective cytotoxicity to MDA-MB-231 cells. This is because the EDPG@Lipo group can deliver the drug directly into breast cancer cells and enhance the efficacy of the drug; and by targeting EGFR on the surface of triple-negative breast cancer cells, the EDPG@Lipo group may be able to bypass certain drug resistance mechanisms, thereby killing breast cancer cells more effectively. In addition, for normal cells, EDPG@Lipo nanoparticles are not cytotoxic, indicating that EDPG@Lipo nanoparticles can effectively attack cancer cells while reducing damage to normal cells, ensuring the safety of treatment.

[0033] Example 5: Targeting of EDPG@Lipo Nanoparticles

[0034] After the triple-negative breast cancer cells MDA-MB-231 and human normal breast cells MCF-10A were cultured to the logarithmic growth phase, they were inoculated in confocal dishes and cultured at 37°C for 24 hours to adhere to the wall. Then, 10 μg / mL of EDPG@Lipo nanoparticle solution of the same concentration diluted with complete culture medium and labeled with DiD was added, and the incubation continued for 4 hours. The cells were rinsed three times with PBS (0.1M, pH7.4), 1 mL of 4% paraformaldehyde was added, and the cells were fixed in the dark for 10 minutes. The 4% paraformaldehyde was discarded and Hoechst33258 cell nuclear staining solution was added for staining for 15 minutes. After the staining was completed, the cells were rinsed twice with PBS, and observed and images were collected using a laser confocal microscope.

[0035] DiD-labeled EDPG@Lipo nanoparticles were added to triple-negative breast cancer cells MDA-MB-231 and human normal breast cells MCF-10A. Figure 4 Confocal imaging results showed that almost no fluorescence was observed in normal breast cells ( Figure 4 a), indicating that EDPG@Lipo nanoparticles were not taken up or internalized by these cells. This selective uptake mechanism ensures the targeting and safety of EDPG@Lipo nanoparticles during treatment and reduces the potential impact on non-target cells. However, significant red fluorescence was observed in triple-negative breast cancer cells ( Figure 4b) This is because the EDPG@Lipo nanoparticles are modified with EGFR antibodies, which have a high affinity for the EGFR protein on the surface of triple-negative breast cancer cells and have the ability to recognize and bind. Therefore, EDPG@Lipo nanoparticles achieve precise positioning and targeted delivery of triple-negative breast cancer cells by specifically recognizing and binding to EGFR.

[0036] Example 6: Changes in HSP90 expression in MDA-MB-231 cells before and after EDPG@Lipo nanoparticle treatment

[0037] MDA-MB-231 cells (1×10 5 / well) were inoculated into six-well plates for pre-culture for 24 hours. Then, the culture medium was replaced with PBS, GA and EDPG@Lipo (10μg / mL) and incubated for 24 hours. After the cells were cultured, they were washed twice with PBS, the cells were lysed to extract total protein, and the protein concentration was determined using a BCA kit (Biyuntian, China). Equal amounts of protein samples were taken and subjected to 10% SDS-PAGE electrophoresis and transferred to PVDF membranes. The successfully transferred membrane was blocked with 5% (w / v) skim milk powder (prepared with 1×TBST) at room temperature on a shaker for 1 hour, washed three times with 1×TBST containing 0.1% Tween-20, incubated with primary antibody overnight in a 4°C refrigerator, washed with 1×TBST to remove unbound primary antibody, incubated with secondary antibody at room temperature for 2 hours, washed again and developed with ECL.

[0038] As an HSP90 responsive molecule, GA can sensitively sense and respond to HSP90 overexpressed in tumor cells. It is a potential HSP90 inhibitor that can bind to the ATP binding pocket at the N-terminus of HSP90 and block the ATP-HSP90 interaction, thereby interfering with the normal function of HSP90 and inhibiting the overexpression of anti-apoptotic proteins (such as Akt), reducing the heat resistance of tumor cells. The present invention uses Western blot analysis and uses GADPH as an internal reference to detect the HSP90 expression level of MDA-MB-231 cells under different treatments. Figure 5As shown in the figure, the expression level of HSP90 after treating MDA-MB-231 cells with different methods, in the samples using PBS as the control group, the expression level of HSP90 showed a significantly increased state, which reflects the expression level under basal or non-intervention conditions. In contrast, when the samples were treated with GA, the expression level of HSP90 decreased significantly. This change highlights the inhibitory effect of GA on HSP90 expression. It is worth noting that when EDPG@Lipo nanoparticles were introduced, the expression level of HSP90 also decreased significantly, and the degree of decrease was almost the same as that of the group treated with GA alone, verifying the effectiveness of EDPG@Lipo nanoparticles as a drug delivery system. It also further showed that after loading GA, the nanoparticles did not affect the biological activity of GA, thereby ensuring that GA can maintain its original ability to inhibit HSP90 expression in EGFR-targeted paclitaxel liposomes. In summary, Figure 5 The experimental results not only intuitively demonstrated the changes in HSP90 expression levels under different treatment conditions, but also profoundly revealed the potential of EDPG@Lipo nanoparticles in the field of drug delivery and their efficient delivery capabilities without interfering with GA activity.

[0039] Example 7: In vivo anti-tumor evaluation of EDPG@Lipo nanoparticles

[0040] (1) Immunofluorescence labeling

[0041] Female BALB / c mice were purchased from Shanghai Slake Laboratory Animals. 100 μL PBS containing MDA-MB-231 cells (2×10 6 / mL), a tumor model was established. When the tumor volume reached about 50 mm 3 Mice bearing MDA-MB-231 tumors were randomly divided into groups (n=5): PBS, GA and EDPG@Lipo, and 10 mg / kg of the above treatment drugs were injected into the tail vein every 2 days. After 14 days of treatment, tumor masses were collected and tumor sections were stained with mouse anti-HSP90 primary antibody and FITC-labeled goat anti-mouse secondary antibody. Finally, tumor sections were further stained with Hoechst33342 and imaged on a confocal laser scanning microscope.

[0042] The expression level of HSP90 in tumors after different treatments was evaluated by immunofluorescence staining. Figure 6As shown, for the PBS group, a significant increase in HSP90 expression was detected at the tumor site. In contrast, a much lower HSP90 expression level was observed in the GA group, indicating that HSP90 expression was inhibited by GA molecules. At the same time, the HSP90 expression level in the EDPG@Lipo group was also relatively low, which was consistent with the results of HSP90 protein expression levels in vitro, indicating that the EDPG@Lipo nanoparticles did not affect the inhibitory effect of GA on HSP90.

[0043] (2) H&E staining

[0044] Female BALB / c mice were purchased from Shanghai Slake Laboratory Animals. 100 μL PBS containing MDA-MB-231 cells (2×10 6 / mL), a tumor model was established. When the tumor volume reached about 50 mm 3 Mice bearing MDA-MB-231 tumors were randomly divided into groups (n=5): PBS, PTX and EDPG@Lipo, and 10 mg / kg of the above therapeutic drugs were injected into the tail vein every 2 days. After 14 days of treatment, the tumor masses were collected for H&E staining.

[0045] Tumor sections were stained with hematoxylin and eosin (H&E) to further evaluate the therapeutic effect of EDPG@Lipo nanoparticles. Compared with the PTX group, the EDPG@Lipo group showed the most severe tumor damage and necrosis ( Figure 7 ), this is because the EGFR antibody modified on the surface of EDPG@Lipo nanoparticles has better targeting, and the incorporation of GA and the synergistic treatment of the internally loaded PTX drug show the most significant tumor growth inhibition effect.

[0046] The above is an exemplary description of the present invention, which is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. Those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution of the present invention, which should be included in the scope of the claims of the present invention.

Claims

1. A dual-responsive EGFR-targeted paclitaxel liposome based on heat shock protein, characterized in that: The main body of the heat shock protein-based dual-responsive EGFR-targeted paclitaxel liposome is a pH-sensitive liposome, and the heat shock protein 90-responsive molecule gamboge acid GA is incorporated into the liposome, the outer surface of the liposome is modified with EGFR antibody, and the inner core of the liposome encapsulates paclitaxel PTX.

2. The method for preparing the heat shock protein-based dual-responsive EGFR-targeted paclitaxel liposome according to claim 1, characterized in that: The preparation method is: Dipalmitoyl phospholipid DPPC, amphiphilic lipid phospholipid polyethylene glycol maleimide DSPE-PEG 2000 -Mal and gambogic acid GA were completely dissolved in 10 mL of chloroform at a certain mass ratio, and the organic solvent was removed by rotary evaporation to form a liposome membrane, and 5 mL of ultrapure water was added for ultrasonic hydration for 20 minutes to completely dissolve the liposome in water; then paclitaxel PTX was added, and ultrasonic treatment was performed with a cell disruptor for 4 minutes, and then the obtained solution was washed with ultrapure water with a 10 kDa, 7500 rpm ultrafiltration filter, and the unloaded GA, PTX and organic solvent were removed three times to prepare DPG@Lipo; Subsequently, the prepared DPG@Lipo and the thiolated EGFR antibody PBS aqueous solution were incubated with rotation at room temperature for 8 h at a certain reaction molar ratio to allow Mal to fully react with -SH, and the unbound thiolated EGFR antibody was washed away to prepare EDPG@Lipo nanoparticles.

3. The preparation method according to claim 2, characterized in that: The DPPC, DSPE-PEG 2000 The reaction mass ratio of -Mal, GA, and PTX is 3:1:2:

1.

4. The preparation method according to claim 2, characterized in that: The concentration of the thiolated EGFR antibody PBS aqueous solution is 0.5 mg / mL.

5. The preparation method according to claim 2, characterized in that: The reaction molar ratio of the DPG@Lipo to the thiolated EGFR antibody PBS aqueous solution is 1:

10.

6. Use of the heat shock protein-based dual-responsive EGFR-targeted paclitaxel liposome as claimed in claim 1 in the preparation of a drug for treating triple-negative breast cancer.

7. The use of the heat shock protein-based dual-responsive EGFR-targeted paclitaxel liposome in the preparation of a drug for treating triple-negative breast cancer according to claim 6, characterized in that: The dual-responsive EGFR-targeted paclitaxel liposomes precisely target the EGFR receptors on the surface of breast cancer and intelligently respond to the release of drugs PTX and GA in the acidic microenvironment of breast cancer tumors.

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