A liposome hydrogel loaded with heat shock protein inhibitors and its preparation and application

Abstract

The present application provides a liposome hydrogel loaded with heat shock protein inhibitors and a preparation method thereof, aiming at precisely treating breast cancer with high expression of HER2. The surface of the liposome is modified with HER2 antibody and photo-thermal agent indocyanine green, the inside is loaded with trastuzumab, and the heat shock protein inhibitor quercetin is embedded to enhance the treatment effect. By simply mixing the liposome with poly-DL-lactide-polyethylene glycol-poly-DL-lactide aqueous solution, a stable hydrogel drug delivery system is formed, which not only protects the drug but also prolongs its stability. The system realizes the targeted delivery and sustained release of the drug, improves the treatment effect and reduces the side effects, and provides an innovative and efficient solution for breast cancer treatment.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of nanobiomedicine, and particularly relates to a liposome hydrogel loaded with a heat shock protein inhibitor and a preparation and application thereof. BACKGROUND

[0002] In the field of breast cancer treatment, with the deepening understanding of the biological characteristics of tumors, heat shock proteins (HSPs) as a class of key stress response proteins have played an increasingly prominent role in tumor occurrence, development and drug resistance mechanisms. Overexpression of HSPs not only enhances the survival ability of breast cancer cells, but also promotes their invasiveness and drug resistance. Therefore, the development of specific inhibitors targeting HSPs has become a new strategy to improve the treatment effect of breast cancer. However, the research on small molecule inhibitors targeting heat shock proteins in breast cancer is still in its early stages. Despite its great potential, it faces many technical challenges. In particular, how to design and synthesize a small molecule inhibitor that can specifically inhibit breast cancer-related HSPs while ensuring the stability and controllable release of the drug in the body is a key problem in current research.

[0003] As one of the local drug release systems, hydrogels have been widely developed due to their advantages such as long-term concentrated drug release at tumor sites, low systemic drug toxicity, and controlled drug release. However, traditional hydrogels can only release drugs under the passive diffusion of drugs and the degradation of hydrogels, which cannot meet the requirements of controlled release. Therefore, it is necessary to study a hydrogel that can perceive external stimuli to change its internal structure and achieve site-specific, controlled and on-demand drug release. SUMMARY

[0004] The present application relates to the technical field of nanobiomedicine, and particularly relates to a liposome hydrogel loaded with a heat shock protein inhibitor and a preparation and application thereof.

[0005] To achieve the above-mentioned application purposes, the technical solutions adopted by the present application are as follows:

[0006] A preparation scheme of a liposome hydrogel loaded with a heat shock protein inhibitor, comprising the following steps:

[0007] S1: dissolve dipalmitoyl phosphatidylcholine (DPPC), amphiphilic lipid phosphatidylcholine polyethylene glycol maleimide (DSPE-PEG 5000 -Mal) and indocyanine green (ICG) in 15 mL chloroform (CHCl3) at room temperature, rotary evaporate for 8 h to remove the organic solvent, and form a liposome membrane. Transfer the product to 5 mL ultrapure water and ultrasonically hydrate for 20 minutes to completely dissolve the liposome in water.

[0008] S2: adding Trastuzumab and heat shock protein inhibitor molecule Quercetin (Qu) to the end product prepared in step S1, ultrasonic treatment with a cell disrupter for 4 minutes, then washing the obtained solution with ultrapure water using a 10kDa, 7500rpm ultrafiltration filter, repeating the above ultrafiltration washing several times to remove unloaded Qu, Trastuzumab and organic solvents, to prepare pure DTQI@Lips.

[0009] S3: rotating and incubating the DTQI@Lips prepared in step S2 with a thiolated HER2 antibody PBS aqueous solution at room temperature for 8h to allow the Mal to fully react with -SH, washing to remove unbound HER2 antibody, to prepare HDTQI@Lips.

[0010] S4: mixing the HDTQI@Lips prepared in step S3 with a poly-DL-lactide-polyethylene glycol-poly-DL-lactide (PLEL) aqueous solution at a certain mass ratio at room temperature to prepare a HDTQI@Lips-Gels hydrogel system.

[0011] Preferably, the mass ratio of DPPC, DSPE-PEG 5000 The mass ratio between -Mal, ICG is 3:1:2.

[0012] Preferably, the mass ratio of Trastuzumab: Qu added in step S2 is 1:1.

[0013] Preferably, the concentration of thiolated HER2 antibody PBS aqueous solution in step S3 is 0.5mg / mL.

[0014] Preferably, the molar ratio of DTQI@Lips to thiolated HER2 antibody PBS aqueous solution in step S3 is 1:10.

[0015] Preferably, the mass ratio between HDTQI@Lips and PLEL (20wt%) in step S4 is 1:10.

[0016] Compared with the prior art, the beneficial effects of the present application are as follows:

[0017] The liposome surface in the hydrogel is modified with specific HER2 antibodies, which can accurately identify and target breast cancer cells with high HER2 expression. The liposome is loaded with trastuzumab as a therapeutic drug, and ICG as a photothermal agent, which can cause the ablation of the liposome and trigger drug release. In addition, the liposome is also embedded with heat shock protein responsive molecule quercetin, which can inhibit the heat resistance of tumor cells and promote the heat sensitivity of tumor cells in the heat shock environment inside the cells, thereby enhancing the therapeutic effect of the drug. In addition, the hydrogel and the internal liposome only need to be simply mixed to form a stable drug delivery system. The good phase change (sol-gel) property of the hydrogel enables it to quickly change from a flowing sol state to a solid gel structure at a physiological temperature of 37 DEG C, thereby effectively fixing and encapsulating the internal liposome and the loaded drug, ensuring the stability and durability of the drug delivery system. In addition, the hydrogel matrix provides a good drug protection barrier, further prolonging the stability of the drug, achieving targeted delivery and sustained release of the drug, and improving the therapeutic effect and reducing the toxic side effects. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 Results of particle size and morphology of HDTQI@Lips nanoparticles.

[0019] Figure 2 Results of near-infrared triggered controlled release of Trastuzumab of HDTQI@Lips nanoparticles, Figure 2 a is the cumulative release curve of Trastuzumab of HDTQI@Lips nanoparticles under continuous laser irradiation; Figure 2 b is the Trastuzumab release curve of HDTQI@Lips nanoparticles with / without laser irradiation.

[0020] Figure 3 Results of characterization of HDTQI@Lips-Gels hydrogel, Figure 3 a is the rheological analysis of HDTQI@Lips-Gels hydrogel; Figure 3 b is the SEM characterization result of HDTQI@Lips-Gels hydrogel, scale 5 μm.

[0021] Figure 4 Results of water absorption and swelling properties of HDTQI@Lips-Gels hydrogel system at pH = 6.5.

[0022] Figure 5 Results of HDTQI@Lips release over time in the hydrogel carrier.

[0023] Figure 6The expression levels of HSP70 protein in BT474 cells after different treatments. Groups ①②③④⑤ were treated with PBS, Qu, NIR-Ⅱ, Qu+NIR-Ⅱ and HDTQI@Lips-Gels+NIR-Ⅱ, respectively.

[0024] Figure 7 The results of the cytotoxicity experiment of HDTQI@Lips-Gels hydrogel on tumor cells and normal cells.

[0025] Figure 8 Figure 2 shows the changes in tumor volume over 14 days in mice treated with different treatments.

[0026] Figure 9 The results of H&E staining of tumor masses in mice treated with different treatments. DETAILED DESCRIPTION

[0027] The present invention will be further described below with reference to the embodiments.

[0028] Example 1: A method for preparing HDTQI@Lips-Gels hydrogel, comprising the following steps:

[0029] (1) 30 mg of dipalmitoyl phospholipid (DPPC), 10 mg of amphiphilic lipid phospholipid polyethylene glycol maleimide (DSPE-PEG 5000 -Mal) and 20 mg of indocyanine green (ICG) were completely dissolved in 15 mL of chloroform (CHCl3). The organic solvent was removed, and a liposome membrane was formed by rotary evaporation. 5 mL of ultrapure water was added for 20 minutes of sonication to completely dissolve the liposomes. 10 mg of trastuzumab and 10 mg of the heat shock protein-responsive molecule quercetin (Qu) were added, and the cells were sonicated for 4 minutes using a cell disruptor. The resulting solution was then washed with ultrapure water using a 10 kDa, 7500 rpm ultrafiltration filter. This was repeated three times to remove unloaded Qu, trastuzumab, and the organic solvent, resulting in the preparation of DTQI@Lips.

[0030] (2) The DTQI@Lips prepared in step (1) was mixed with a thiolated HER2 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. The unbound HER2 antibody was then washed away to prepare HDTQI@Lips.

[0031] (3) Poly (DL-lactide)-poly (ethylene glycol)-poly (DL-lactide) (PLEL) was completely dissolved in ultrapure water, and HDTQI@Lips and PLEL (20 wt%) were mixed uniformly at a mass ratio of 1:10 at room temperature to prepare the HDTQI@Lips-Gels hydrogel system.

[0032] Example 2: Particle size and morphology characterization of HDTQI@Lips nanoparticles

[0033] The HDTQI@Lips prepared in step (2) of Example 1 was diluted 10-fold with distilled water, and the particle size was measured using a laser dynamic light scattering nanoparticle size analyzer. Concurrently, the HDTQI@Lips was diluted 35-fold with distilled water, and 20 μL was added dropwise to a transmission electron microscope copper grid. After 1 hour, the sample was carefully blotted dry from one side of the copper grid with filter paper and stained with phosphomolybdic acid for 3 seconds before scanning under a transmission electron microscope.

[0034] from Figure 1 It can be seen that the HDTQI@Lips nanoparticles are spherical and about 100 nm in size.

[0035] Example 3: Controlled Release of Trastuzumab Triggered by HDTQI@Lips Nanoparticles

[0036] 1 mL of HDTQI@Lips solution was placed in a dialysis bag (3500 Da) to measure the release of trastuzumab from HDTQI@Lips. The dialysis bag was immersed in 20 mL of PBS (pH = 7.4) and the dialysis bag was irradiated with NIR-II (1064 nm, 1.0 W cm -2 After laser irradiation for 5 min, 400 μL of release medium was removed and replaced with an equal volume of fresh PBS. The release of trastuzumab was detected by high performance liquid chromatography (HPLC) at 280 nm, and the cumulative release percentage was calculated as follows: E r (%) = F t / F 100 ×100%.

[0037] Due to the photothermal effect of ICG, DPPC can be heated and melted, prompting the effective release of Trastuzumab from the ruptured liposomes. Under continuous laser irradiation, the release process of Trastuzumab from HDTQI@Lips nanoparticles was accelerated, and the cumulative release rate could reach 83% ( Figure 2 a), indicating that HDTQI@Lips has thermoresponsiveness. In addition, the drug release of HDTQI@Lips nanoparticles with and without laser irradiation was measured, with the infrared light switched on and off every 10 minutes. The results are shown in Figure 2.Figure 2 As shown in b, the drug release rate of Trastuzumab under laser irradiation was 60%, while no release of Trastuzumab was detected without laser irradiation, which further demonstrated that HDTQI@Lips has thermosensitive responsiveness.

[0038] Example 4: SEM characterization experiment of HDTQI@Lips-Gels hydrogel system

[0039] The HDTQI@Lips-Gels prepared in Example 1 was analyzed in a rheometer for gelation strength and gelation time at 37°C. The specific operation was as follows: the Thermo Scientific rheometer was turned on, the instrument was preheated, and the oscillation mode was set to 37°C. The distance between the parallel plate and the rotor of the rheometer was adjusted to 1 nm. 200 μL of HDTQI@Lips-Gels solution was dropwise added to the parallel plates of the rheometer to avoid bubble generation. The changes in storage modulus (G') and loss modulus (G") over time within 1 minute were recorded.

[0040] The HDTQI@Lips-Gels prepared in Example 1 were suspended in a 37°C water bath until a gel was formed. The hydrogel system was then frozen and broken in liquid nitrogen at -196°C. The surface of the sample slices was coated with gold before scanning electron microscopy imaging, and the morphology of the HDTQI@Lips-Gels was observed by scanning electron microscopy.

[0041] like Figure 3 As shown in Figure 1a, the initial spring modulus (G') and viscous modulus (G") are very low, and the values ​​of G' and G" fluctuate greatly, indicating that the gel has not yet formed and the system is still in an aqueous solution state. Subsequently, at 33 s, G' is significantly greater than G", and the subsequent curve trend gradually stabilizes, indicating that the hydrogel has good hydrogel phase transition (sol-gel) properties and can be used to prepare injectable drug delivery vehicles. Figure 3 b is a scanning electron microscope image of the HDTQI@Lips-Gels material prepared in Example 1 of the present invention. It can be seen that the material exhibits a typical hydrogel three-dimensional network structure under the scanning electron microscope, which fully proves that the HDTQI@Lips-Gels with the expected structure was successfully prepared by the method described in Example 1. The three-dimensional network structure inside it is clearly visible, showing a highly cross-linked state and a porous structure.

[0042] Example 5: Swelling properties of HDTQI@Lips-Gels hydrogel system

[0043] To study the swelling properties of HDTQI@Lips-Gels hydrogel system at different time points, 1 mL of HDTQI@Lips-Gels hydrogel was placed in a 50 mL centrifuge tube, the mass of the centrifuge tube was weighed as W0, the mass of the centrifuge tube and the hydrogel was weighed as W1, 30 mL of PBS was added to the centrifuge tube to completely immerse the hydrogel, and the centrifuge tube was shaken at 37°C. Every certain time, the PBS in the centrifuge tube was removed through a 1 mm diameter hole in the middle of the bottle cap, the total mass of the centrifuge tube and the hydrogel was weighed as W2, and the swelling rate was calculated according to the formula: (W2-W0) / (W1-W0) x 100%.

[0044] The weight analysis method was used to evaluate the swelling properties of the hydrogel system. As shown in Figure 4 When pH = 6.5, the HDTQI@Lips-Gels hydrogel system showed strong water absorption and swelling properties, and reached the maximum swelling value at about 16 h. The acidic microenvironment of the tumor provided ideal swelling conditions for the HDTQI@Lips-Gels hydrogel system, and when the hydrogel system was implanted into the tumor tissue, the internal DPPC would quickly respond to the acidic environment, promoting the swelling process of the hydrogel, and this time window also provided favorable conditions for the sustained and stable release of the internal drugs Trastuzumab and heat shock protein inhibitor Qu.

[0045] Example 6: In vitro release of HDTQI@Lips-Gels hydrogel system HDTQI@Lips

[0046] To study the drug release of the hydrogel, 5 mL of PBS (pH = 6.5) was slowly added to the prepared HDTQI@Lips-Gels, and incubated at 37°C in a 100 rpm shaker. At the predetermined time, 400 μL of release medium was taken out and replaced with the same volume of fresh PBS. The release of Trastuzumab was detected by high performance liquid chromatography (HPLC) at 280 nm, and the cumulative release percentage was calculated according to the formula: E r (%) = F t / F 100 x 100%.

[0047] On the basis of Example 4 which confirmed that the HDTQI@Lips-Gels hydrogel system prepared by the present application had good phase transition properties at 37°C, the in vitro release of HDTQI@Lips-Gels was studied in a simulated tumor acidic microenvironment (PBS, pH = 6.5). The results are shown in Figure 5As shown, HDTQI@Lips can be slowly and continuously released from the hydrogel, so it is faster in the first 24h of release, and then slower, and after 96h, the cumulative release rate is about 78%. The above experimental results show that the hydrogel system of the present application can slowly release HDTQI@Lips, and then release Trastuzumab from the liposomes under NIR-Ⅱ light, and the hydrogel system has a sustained and stable drug release capacity.

[0048] Example 7: Protein analysis of HSP70 in tumor cells after different treatment methods

[0049] BT474 cells (1 x 10 5 were inoculated into a six-well plate and pre-cultured for 24h. Then the culture medium was replaced with PBS, Qu, NIR-Ⅱ, Qu+NIR-Ⅱ and HDTQI@Lips-Gels+NIR-Ⅱ (10nM) for incubation for 24h. After the culture of the cells, the cells were washed with PBS for 2 times, and the total protein was extracted by lysing the cells, and the protein concentration was determined by BCA kit. Equal amounts of protein samples were subjected to 10% SDS-PAGE electrophoresis, and then transferred to PVDF membrane. The successfully transferred PVDF membrane was blocked with 5% (w / v) skimmed milk powder (1 x TBST) at room temperature for 1h, washed with 1 x TBST containing 0.1% Tween-20 for 3 times, and incubated with the first antibody at 4℃ overnight. After washing with 1 x TBST to remove the unbound first antibody, the second antibody was incubated at room temperature for 2h, and then washed again and developed by ECL.

[0050] The expression level of HSP70 in BT474 cells under different treatments was detected by Western blot analysis with GADPH as an internal control. As shown in Figure 6 Compared with the control group, the expression level of HSP70 in the NIR-Ⅱ treatment group was significantly higher, and the expression of HSP70 after Qu+NIR-Ⅱ treatment was significantly reduced, which indicated that Qu could effectively inhibit the increase of HSP70 content caused by photothermal, and reduce the heat resistance of tumor cells. Notably, the expression level of HSP70 after treatment with HDTQI@Lips-Gels+NIR-Ⅱ was also reduced, almost the same as the Qu+NIR-Ⅱ treatment group, indicating that the HDTQI@Lips-Gels hydrogel system did not affect the activity of Qu, further indicating that the prepared material of the present application can stably release Qu in the in vivo environment, effectively target and inhibit the expression of HSP70, so as to realize the precise regulation of photothermal stress response.

[0051] Example 8: Cytotoxicity test of HDTQI@Lips-Gels hydrogel system

[0052] HER2-positive breast cancer cells BT474 and normal human breast cells MCF-10A were cultured to the logarithmic growth phase and seeded into 96-well plates. After 24 hours of adherence, the cells were divided into five groups: PBS, Trastuzumab, PLEL, Qu+NIR-II, and HDTQI@Lips-Gel+NIR-II. Each well was then incubated at 37°C for 24 hours with 10 nM of the same concentration of the above sample solutions diluted in complete culture medium. After incubation, 10 μL of CCK8 solution was added to each well, followed by an additional 3-hour incubation. The absorbance at 450 nm was measured using a microplate reader.

[0053] like Figure 7 As shown, cell death in the control group was negligible. In the trastuzumab group, due to the broad-spectrum anticancer effect of trastuzumab, most tumor cells were killed, and it also had the ability to kill normal breast cells. In the PLEL group, due to the good biocompatibility of PLEL, it did not produce cytotoxicity to tumor cells or normal cells. In the Qu+NIR-Ⅱ group, because Qu inhibited the development of heat resistance in tumor cells stimulated by near-infrared irradiation, it produced some cytotoxicity to tumor cells, while not showing significant cytotoxicity to normal cells. Notably, the HDTQI@Lips-Gel+NIR-Ⅱ group showed the most effective cytotoxicity against BT474 cells compared with the other groups due to its HER2 targeting. It also had good biocompatibility with normal cells, demonstrating the specificity and safety of HDTQI@Lips-Gel+NIR-Ⅱ treatment.

[0054] Example 9: In vivo antitumor effect of HDTQI@Lips-Gels liposome-hydrogel system

[0055] Twenty-five healthy female nude mice (BALB / c Nude) were selected and BT474 cells were subcutaneously injected into the right forelimb area to prepare tumor-bearing mice. 3 For the experiment, mice were randomly divided into 5 groups (n=5): (1) PBS; (2) NIR-Ⅱ; (3) Qu+NIR-Ⅱ; (4) HDTQI@Lips+NIR-Ⅱ; (5) HDTQI@Lips-Gels+NIR-Ⅱ (500μg / mL, 100μL). Drugs were injected every two days. 30 minutes after the injection, 0.6W / cm 2 Tumors were irradiated with a 1064 nm laser for 10 minutes. Tumors were removed from the mice 14 days after treatment and the mice were sacrificed. Tumor sections were stained with hematoxylin and eosin (H&E) to further assess the therapeutic effects.

[0056] During treatment, every 2 days according to the formula: width 2X length X pi / 6, record the tumor volume of each group, draw the function of time. As shown in Figure 8 The tumor volume of PBS control group and NIR-II laser group increased rapidly, indicating that NIR light irradiation alone had little effect on tumor inhibition. The tumor volume growth rate of Qu+NIR-II group slowed down to some extent, indicating that Qu had a certain tumor inhibition effect. In addition, the tumor growth rate of HDTQI@Lips+NIR-II treatment group slowed down. First, the HER2 antibody on the surface of the liposome accurately recognized and targeted HER2 high-expressing breast cancer cells, and the irradiation of near-infrared light excited the photothermal effect of ICG, which made the liposome rupture and release the internal loaded drug, and played a therapeutic effect; at the same time, Qu can inhibit the heat resistance of tumor cells produced by photothermal therapy, and the comprehensive effect slows down the tumor growth rate. Due to the local intelligent release and sustained release effect of the hydrogel, the tumor growth rate of HDTQI@Lips-Gels+NIR-II group further slowed down, and on the basis of the treatment effect of HDTQI@Lips+NIR-II, the hydrogel outside HDTQI@Lips played a good sustained and stable drug release effect, thereby providing longer treatment time in the tumor tissue site, and the tumor treatment effect was the best. In addition, as shown in Figure 9 The H&E staining results also confirmed that, compared with the control group, although the HDTQI@Lips+NIR-II treatment group showed tumor cell necrosis results, the HDTQI@Lips-Gels+NIR-II group showed the most severe tumor cell necrosis and the best treatment effect, further indicating that the HDTQI@Lips-Gels liposome hydrogel system integrated photothermal therapy, heat shock protein inhibition, tumor targeting and stable release treatment effect.

[0057] The above describes the present application exemplarily, which is more specific and detailed, but it cannot be understood as the limitation of the scope of the present application. Those skilled in the art should understand that the technical solutions of the present application can be modified or replaced equivalently without departing from the purpose and scope of the present application, which should be covered in the scope of the claims of the present application.

Claims

1. A liposome hydrogel loaded with a heat shock protein inhibitor, characterized in that, The liposome hydrogel loaded with heat shock protein inhibitors comprises a hydrogel carrier, the inside of the hydrogel is loaded with liposomes, the surface of the liposomes is modified with specific HER2 antibodies and ICG as a photothermal agent, the inside of the liposomes is loaded with trastuzumab as a therapeutic drug and a heat shock protein responsive molecule quercetin; The hydrogel carrier is poly-DL-lactide-polyethylene glycol-poly-DL-lactide.

2. A method of preparing a liposome hydrogel loaded with a heat shock protein inhibitor, characterized in that, The preparation method comprises the following steps: S1: Dipalmitoyl phosphatidylcholine DPPC, amphiphilic lipid phosphatidylcholine polyethylene glycol maleimide DSPE-PEG 5000 -Mal, indocyanine green ICG was dissolved in 15 mL chloroform CHCl3, rotary evaporation for 8 h to remove organic solvent, form liposome membrane, the product was transferred to 5 mL ultrapure water and ultrasonic hydration for 20 minutes, so that the liposomes were completely dissolved in water; S2: adding trastuzumab and heat shock protein inhibitor molecule quercetin to the final product prepared in step S1, ultrasonic treatment for 4 minutes by using a cell disruptor, then washing the obtained solution with ultrapure water by using a 10kDa, 7500rpm ultrafiltration filter, repeating the above ultrafiltration washing several times to remove unloaded Qu, trastuzumab and organic solvents, and preparing pure DTQI@Lips; S3: rotating and incubating the DTQI@Lips prepared in step S2 with a thiolated HER2 antibody PBS aqueous solution at room temperature for 8h, so that Mal fully reacts with -SH, and then washing to remove unbound HER2 antibody, thereby preparing HDTQI@Lips; S4: mixing the HDTQI@Lips prepared in step S3 with a poly-DL-lactide-polyethylene glycol-poly-DL-lactide PLEL aqueous solution at a certain mass ratio under room temperature conditions to prepare a HDTQI@Lips-Gels hydrogel system.

3. The preparation method according to claim 2, characterized in that: The DPPC:DSPE-PEG in step S1 5000 The mass ratio between -Mal:ICG is 3:1:

2.

4. The preparation method according to claim 2, characterized in that The mass ratio of trastuzumab and Qu added in step S2 is 1:

1.

5. The preparation method according to claim 2, characterized in that: The concentration of the thiolated HER2 antibody PBS aqueous solution in step S3 is 0.5mg / mL.

6. The preparation method according to claim 2, characterized in that: The molar ratio of the DTQI@Lips and the thiolated HER2 antibody PBS aqueous solution in step S3 is 1:

10.

7. The preparation method according to claim 2, characterized in that: The mass ratio between HDTQI@Lips and PLEL in step S4 is 1:

10.

8. The preparation method according to claim 2, characterized in that: The concentration of PLEL in step S4 is 20wt%.

9. Use of the liposome hydrogel loaded with heat shock protein inhibitors according to claim 1 in the preparation of anti-HER2 positive breast cancer drugs.

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