Preparation method and application of a near-infrared responsive lipid nanoparticle system aggregated with copper sulfide nanoparticles

Abstract

The present invention discloses a preparation method and application of a near-infrared responsive lipid nanoparticle system aggregated with copper sulfide nanoparticles. This method realizes the enhanced permeability and retention of small-sized copper sulfide nanoparticles in tumor therapy and effectively improves the tumor treatment effect. A polypeptide that can respond to transglutaminase overexpressed in tumor sites and undergo crosslinking aggregation is modified on the copper sulfide nanoparticles, and co-assembled with a photosensitizer ICG and a temperature-sensitive liposome to form a nanodelivery system, which enhances its ability to penetrate and aggregate at the tumor site. Meanwhile, it realizes the synergistic tumor treatment effect of PDT and PTT, and can be used for further preparation of drugs for photothermal-photodynamic synergistic tumor therapy.

Description

Technical Field

[0001] The present invention belongs to the technical field of nano drug delivery systems, and particularly relates to a preparation method and application of a lipid nano system in which near-infrared responsive copper sulfide nanoparticles aggregate. Background Art

[0002] Inorganic nanomaterials are materials for PTT that have been studied by researchers earlier. Currently, the inorganic nanomaterials that have been studied more include metal nanoparticles, metal chalcogenide nanoparticles, carbon-based nanomaterials, quantum dots, etc. Among them, copper sulfide has received extensive attention from researchers due to its low cost, good photothermal stability, low cytotoxicity, and controllable particle size and morphology. In addition, transition metal copper is an essential trace element for the human body and is also a bioactive component in the body. In recent years, with the development of nanotechnology and nanomedicine, the biological effects of copper and the physicochemical properties of copper nanoparticles have promoted the development of unique copper-containing bio-nanomaterials. Although the biocompatibility of some copper-based nanoagents has been preliminarily demonstrated, high copper accumulation may cause potential toxicity in the body. Therefore, it is an urgent problem to be solved to ensure that the copper sulfide nanoparticles are small in size and easily cleared by normal tissues while prolonging their retention time at the tumor site.

[0003] Currently, designing a nano system that responds and aggregates at the tumor site while ensuring the penetration ability is an effective method. Utilize the characteristics of the tumor microenvironment such as acidic microenvironment, ROS, specific enzymes, and external stimuli such as temperature, light, etc. to induce the aggregation of particles. Due to its powerful catalytic function and special specificity, enzymes have attracted the attention of researchers in recent years in terms of tumor-specific enzyme-responsive aggregation. Transglutaminase is highly expressed at the tumor site and is closely related to the apoptosis of tumor cells. It can catalyze the amine exchange reaction between the γ-carboxamide group of glutamine and the ε-amino group of lysine, inducing intermolecular or intramolecular cross-linking of proteins. Based on this, relevant polypeptides containing glutamine and lysine can be designed to modify copper sulfide nanoparticles to trigger their in-situ aggregation and retention at the tumor site.

[0004] Photodynamic therapy (PDT) generates singlet oxygen by irradiating a photosensitizer at the tumor site with a laser to oxidize important biomacromolecules and induce apoptosis, which is an emerging cancer treatment method. Compared with traditional treatment methods, it has the advantages of non-invasiveness, lower normal tissue and off-target toxicity. However, its therapeutic effect is limited by the tumor hypoxic microenvironment and the short lifetime of singlet oxygen. PTT is another treatment mode that uses a laser to activate a photothermal reagent to generate heat to eliminate tumors. Combining PDT and PTT can effectively compensate for the effect of photodynamic therapy. On the one hand, PDT can overcome the heat resistance of tumor cells mediated by heat shock proteins. In addition, the photothermal effect in PDT and PTT is usually mild hyperthermia, which can protect adjacent tissues from high-temperature damage. On the other hand, the reactive oxygen species generated by PTT assisting PDT increase the tumor permeability, accelerate the uptake of intracellular drugs, make the cells more sensitive to heat, and thus improve the effect of PTT. Copper sulfide nanoparticles and the photosensitizer ICG can simultaneously produce photothermal and photodynamic therapy effects under 808 nm laser irradiation, and the two can be loaded into a lipid system for the synergistic treatment of tumors. However, there is currently no relevant literature report in this regard. Summary of the Invention

[0005] The technical problem solved by the present invention is to provide a preparation method of a lipid nanoparticle system aggregated with near-infrared-responsive copper sulfide nanoparticles. This method realizes the enhanced permeability and retention of small-sized copper sulfide nanoparticles in tumor treatment and effectively improves the tumor treatment effect. A polypeptide that can respond to transglutaminase (TGase) overexpressed at the tumor site and can crosslink and aggregate is modified on the copper sulfide nanoparticles, and co-assembled with the photosensitizer ICG and thermosensitive liposomes to form a nanodelivery system, enhancing its ability to penetrate and aggregate at the tumor site, and simultaneously achieving the synergistic tumor treatment effect of PDT and PTT, and can be used for further preparation of drugs for the synergistic treatment of tumors by photothermal and photodynamic therapy.

[0006] The present invention adopts the following technical solution to solve the above technical problem. A preparation method of a lipid nanoparticle system aggregated with near-infrared-responsive copper sulfide nanoparticles is characterized in that the specific steps are as follows:

[0007] Step S1: Preparation of copper sulfide nanoparticles functionalized with polypeptide K

[0008] Using copper dichloride dihydrate, thioacetamide and polypeptide K as raw materials, dissolve them in water and heat up to 40-60 °C for reaction. After the reaction is completed, use an ultrafiltration tube to remove unreacted small molecule compounds and polypeptide K and concentrate to obtain a CuS-PepK concentrate for standby. The amino acid sequence of polypeptide K is CRKKKR, as shown in SEQ ID NO.1;

[0009] Step S2: Preparation of copper sulfide nanoparticles functionalized with polypeptide Q

[0010] Using copper chloride dihydrate, thioacetamide, and polypeptide Q as raw materials, dissolve them in water and heat to 40-60 °C for reaction. After the reaction is completed, use an ultrafiltration tube to remove unreacted small molecule compounds and polypeptide Q and concentrate to obtain a CuS-PepQ concentrated solution for standby. The amino acid sequence of polypeptide Q is CRRQQR, as shown in SEQ ID NO.2;

[0011] Step S3: Preparation of temperature-sensitive liposomes containing photosensitizer ICG

[0012] Dissolve cholesterol, dipalmitoyl phosphatidylcholine (DPPC), DSPE-PEG 2k and photosensitizer ICG in chloroform, and then rotary evaporate to remove chloroform to obtain a lipid film, namely the film of Lipo@ICG, and keep it in the dark for standby;

[0013] Step S4: Preparation of a lipid nanosystem with aggregation of near-infrared responsive copper sulfide nanoparticles

[0014] Add the CuS-PepK concentrated solution obtained in step S1 and the CuS-PepQ concentrated solution obtained in step S2 into the film of Lipo@ICG obtained in step S3 respectively, hydrate until the film completely detaches, sonicate, centrifuge to remove unloaded copper sulfide nanoparticles, and extrude using a liposome extruder to obtain a lipid nanosystem.

[0015] Further defined, the specific preparation process of step S1 is: Place 0.1 mmol of copper chloride dihydrate in a 250 mL round-bottom flask, add 100 mL of ultrapure water and stir until fully dissolved, then add 0.01-0.1 mmol of polypeptide K, stir for 30 minutes, add 0.1 mmol of thioacetamide, heat to 50 °C and react for 2-3 hours to obtain a dark brown liquid. Ultrafilter to remove unreacted small molecule compounds and polypeptide K, wash with water 3 times, and concentrate to obtain a CuS-PepK concentrated solution for standby.

[0016] Further defined, the specific preparation process of step S2 is: Place 0.1 mmol of copper chloride dihydrate in a 250 mL round-bottom flask, add 100 mL of ultrapure water and stir until fully dissolved, then add 0.01-0.1 mmol of polypeptide Q, stir for 30 minutes, add 0.1 mmol of thioacetamide, heat to 50 °C and react for 2-3 hours to obtain a dark brown liquid. Ultrafilter to remove unreacted small molecule compounds and polypeptide Q, wash with water 3 times, and concentrate to obtain a CuS-PepQ concentrated solution for standby.

[0017] Further defined, the specific preparation process of step S3 is: Dipalmitoyl phosphatidylcholine, cholesterol, photosensitizer ICG, and DSPE-PEG 2kDissolve it in 2 - 3 mL of chloroform solution at a molar ratio of 18:6:1:1.5. After complete dissolution, transfer it to a round - bottom flask, and then form a uniform green film by rotary evaporation at 30 °C using a rotary evaporator. Subsequently, add 2 mL of ultrapure water to it and place it in a water bath at 60 °C for hydration. After complete hydration, transfer the obtained green solution to an ice bath and sonicate for 30 - 60 minutes. Then, extrude the obtained liquid through a polycarbonate membrane filter. Finally, concentrate the extruded sample using an ultrafiltration device to obtain a film of sample Lipo@ICG, and store it in a refrigerator at 4 °C for later use. The molecular weight cut - off of this ultrafiltration device is 30 kDa.

[0018] Further defined, the specific preparation process of step S4 is as follows: Hydrate the obtained Lipo@ICG film with 2 mg / mL CuS - PepQ concentrate and 2 mg / mL CuS - PepK concentrate by incubating at 60 °C for 1 h to mix the copper sulfide nanoparticle solution with the green film - like mixture. After the film is completely hydrated, transfer the mixture to a centrifuge tube and sonicate for 10 - 30 minutes. Then, freeze - thaw the mixed solution for 3 - 5 cycles to promote the loading of CuS - PepQ and CuS - PepK into the liposome. Centrifuge to remove the unloaded CuS - PepQ and CuS - PepK, and extrude the obtained liquid through a polycarbonate membrane filter to obtain the lipid nanosystem Lipo@ICG@CuS - PepQ / K.

[0019] Application of the lipid nanosystem with near - infrared - responsive aggregated copper sulfide nanoparticles in the preparation of a drug for photothermal and photodynamic combined tumor therapy, wherein the lipid nanosystem disintegrates under near - infrared 808 nm laser irradiation to promote the release of copper sulfide nanoparticles, their penetration and enzyme - responsive aggregation and retention at the tumor site, and simultaneously achieve photothermal and photodynamic tumor therapy.

[0020] Compared with the prior art, the present invention has the following advantages and beneficial effects: In the present invention, copper sulfide nanoparticles modified by polypeptide functionalization, photosensitizer ICG, and thermosensitive liposomes are co - assembled to obtain a near - infrared - responsive nanosystem. This nanosystem not only ensures the penetration ability of small - sized copper sulfide nanoparticles in tumor tissues but also prolongs their circulation and retention time in vivo. At the same time, it has both photothermal and photodynamic therapeutic effects and has good application prospects in tumor treatment. Description of the Drawings

[0021] Figure 1 Transmission electron microscope image and Zeta potential image of copper sulfide nanoparticles modified by polypeptide functionalization;

[0022] Figure 2 Transmission electron microscope image of the co - assembled lipid nanosystem;

[0023] Figure 3For in vitro and in vivo penetration;

[0024] Figure 4 For in vitro and in vivo accumulation;

[0025] Figure 5 For tumor treatment effect;

[0026] Figure 6 It is the schematic diagram of the lipid nano - system obtained by co - assembly. ① Penetration + enhanced retention; ② NIR promotes the release of ICG, ICG imaging + PDT; ③ Enzymatic cross - linking of Pep K - Pep Q enhances CuS PTT. Detailed implementation mode

[0027] The above content of the present invention will be further described in detail through the following examples. However, it should not be understood that the scope of the above - mentioned subject matter of the present invention is limited to the following examples. All technologies implemented based on the above content of the present invention belong to the scope of the present invention. Example 1

[0028] Preparation of polypeptide - functionalized copper sulfide nanoparticles:

[0029] Put 0.1 mmol of copper (II) chloride dihydrate into a 250 - mL round - bottom flask, add 100 mL of ultrapure water and stir until completely dissolved. Then add 0.01 mmol of polypeptide K, stir for 30 minutes, add 0.1 mmol of thioacetamide, heat up to 50 °C and react for 2 hours to obtain a dark - brown liquid. Ultra - filter to remove unreacted small - molecule compounds and polypeptide K, wash with water 3 times, and concentrate to obtain a CuS - PepK concentrated solution for standby. The amino - acid sequence of polypeptide K is CRKKKR, as shown in SEQ ID NO.1.

[0030] Put 0.1 mmol of copper (II) chloride dihydrate into a 250 - mL round - bottom flask, add 100 mL of ultrapure water and stir until completely dissolved. Then add 0.01 mmol of polypeptide Q, stir for 30 minutes, add 0.1 mmol of thioacetamide, heat up to 50 °C and react for 2 hours to obtain a dark - brown liquid. Ultra - filter to remove unreacted small - molecule compounds and polypeptide Q, wash with water 3 times, and concentrate to obtain a CuS - PepQ concentrated solution for standby. The amino - acid sequence of polypeptide Q is CRRQQR, as shown in SEQ ID NO.2. Example 2

[0031] Preparation of thermosensitive liposomes containing photosensitizer ICG:

[0032] Put dipalmitoylphosphatidylcholine, cholesterol, photosensitizer ICG and DSPE - PEG 2kDissolve it in 3 mL of chloroform solution at a molar ratio of 18:6:1:1.5. After complete dissolution, transfer it to a round-bottom flask, and then rotate and evaporate it at 30 °C using a rotary evaporator to form a uniform green film. Subsequently, add 2 mL of ultrapure water to it and place it in a water bath at 60 °C for hydration. After complete hydration, transfer the obtained green solution to an ice bath and ultrasonicate it for 30 minutes. Then, extrude the obtained liquid through polycarbonate membrane filters with pore sizes of 400 nm, 200 nm, and 100 nm. Finally, concentrate the extruded sample using an ultrafiltration device to obtain a film of sample Lipo@ICG, and store it in a refrigerator at 4 °C for later use. The molecular weight cut-off of this ultrafiltration device is 30 kDa (Millipore). Example 3

[0033] Preparation of near-infrared responsive nanosystem:

[0034] Hydrate the film of Lipo@ICG by incubating it with 2 mg / mL CuS-PepQ concentrate and 2 mg / mL CuS-PepK concentrate at 60 °C for 1 h to mix the copper sulfide nanoparticle solution with the green film-like mixture. After complete hydration of the film, transfer the mixture to a centrifuge tube and ultrasonicate it for 10 minutes. Then, freeze-thaw the mixed solution for 3 cycles to promote the loading of CuS-PepQ and CuS-PepK into the liposomes. Centrifuge to remove the unloaded CuS-PepQ and CuS-PepK, and extrude the obtained liquid through polycarbonate membrane filters with pore sizes of 400 nm, 200 nm, and 100 nm to obtain the lipid nanosystem Lipo@ICG@CuS-PepQ / K. Example 4

[0035] In vitro and in vivo penetration:

[0036] Construct tumor spheroids using A375 cells. The specific steps are as follows: Add 50 μL of DMEM medium containing 1 wt% agarose to a 96-well plate and let it cool and solidify. Culture 1×10 4 ~1×10 7 A375 cells per well for 4 - 5 days to obtain 3D cell spheroids. Incubate the liposomes loaded with CuS-K / Q-FITC with the cell spheroids for 4 hours, then observe the fluorescence distribution using a laser confocal microscope and perform quantitative analysis using Image J software.

[0037] Inject 10 - 30 μL of Lipo@CuS-K / Q-FITC into the in-situ tumors of tumor-bearing mice using an automatic syringe. After 2 hours, remove the tumors, embed them with OCT to make frozen sections, observe the fluorescence distribution on the sections using a laser confocal microscope, and remove the background signal of the tissue with the untreated tumor tissue as a reference. The area with the largest coverage of the fluorescence signal in the tissue section is used as the representative picture for determining the penetration distance. Quantitative analysis is performed using Image J software. Example 5

[0038] In vitro and in vivo accumulation:

[0039] A375 cells were incubated with Lipo@CuS-K, Lipo@CuS-Q, and Lipo@CuS-K&Q for 6 hours respectively, washed with PBS, and then collected. Next, they were fixed with glutaraldehyde at room temperature for 1 hour. After sample treatment, observation and photography were carried out by TEM.

[0040] CuS-K nanoparticles were labeled with HS-PEG2000-FITC, CuS-Q nanoparticles were labeled with HS-PEG2000-RB, and liposomes were labeled with DiI. An automatic syringe was used to inject 10 - 30 μL of DiI-Lipo@CuS-K / Q-FITC in situ into tumor-bearing mice. After 2 hours, the tumors were removed, embedded with OCT to make frozen sections, and the co-localization distribution of the two fluorescences was observed under a laser confocal microscope. Example

[0041] Tumor treatment:

[0042] 1×10 7 A375 cells were subcutaneously inoculated into the right back of each BALB / c female nude mouse. When the tumor volume reached about 100 mm 3 , the mice were randomly divided into four groups: PBS, Lipo@ICG@CuS-K (5 mg / kg CuS, 0.92 mg / kg ICG), Lipo@ICG@CuS-Q (5 mg / kg CuS, 0.92 mg / kg ICG), and Lipo@ICG@CuS-K&Q (5 mg / kg CuS, 0.92 mg / kg ICG). The above-mentioned nanoparticles were injected into the tail vein of the mice. After 24 hours of injection, they were irradiated with 808 nm near-infrared laser for 10 minutes (1.0 W / cm 2 ). The tumor volume and mouse body weight were measured every other day. After 14 days of treatment, the mice were euthanized, and each tumor tissue and organ were taken out to make tissue sections for immunohistochemical analysis.

[0043] The above examples describe the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above examples. What is described in the above examples and the specification only illustrates the principles of the present invention. Without departing from the scope of the principles of the present invention, the present invention will have various changes and improvements, and these changes and improvements all fall within the scope of protection of the present invention.

[0044] SEQUENCE LISTING

[0045] <110> Henan Normal University

[0046] <120> Preparation method and application of a near-infrared responsive lipid nanoparticle system aggregated with copper sulfide nanoparticles

[0047] <160> 2

[0048] <170> PatentIn version 3.3

[0049] <210> SEQ ID NO:1

[0050] <211> 6

[0051] <212> PRT

[0052] <213> artificial sequence

[0053] <400> SEQ ID NO:1

[0054] CRKKKR

[0055] <210> SEQ ID NO:2

[0056] <211> 6

[0057] <212> PRT

[0058] <213> artificial sequence

[0059] <400> SEQ ID NO:2

[0060] CRRQQR

Claims

1. A preparation method of a near-infrared responsive lipid nanoparticle system aggregated by copper sulfide nanoparticles, characterized in that The specific steps are as follows: Step S1: Preparation of copper sulfide nanoparticles functionalized with polypeptide K Using copper chloride dihydrate, thioacetamide, and polypeptide K as raw materials, dissolve them in water and heat up to 40 - 60 °C for reaction. After the reaction is completed, use an ultrafiltration tube to remove unreacted small molecule compounds and polypeptide K and concentrate to obtain a CuS-PepK concentrate for standby. The amino acid sequence of polypeptide K is CRKKKR, as shown in SEQ ID NO.1; Step S2: Preparation of copper sulfide nanoparticles functionalized with polypeptide Q Using copper chloride dihydrate, thioacetamide, and polypeptide Q as raw materials, dissolve them in water and heat up to 40 - 60 °C for reaction. After the reaction is completed, use an ultrafiltration tube to remove unreacted small molecule compounds and polypeptide Q and concentrate to obtain a CuS-PepQ concentrate for standby. The amino acid sequence of polypeptide Q is CRRQQR, as shown in SEQ ID NO.2; Step S3: Preparation of temperature-sensitive liposomes containing photosensitizer ICG Dissolve cholesterol, dipalmitoylphosphatidylcholine, DSPE-PEG 2k and the photosensitizer ICG in chloroform, and then rotary evaporate to remove chloroform to obtain a lipid film, namely the film of Lipo@ICG, and keep it in the dark for standby; Step S4: Preparation of a lipid nanoparticle system with near-infrared responsive aggregation of copper sulfide nanoparticles Add the CuS-PepK concentrate obtained in step S1 and the CuS-PepQ concentrate obtained in step S2 into the film of Lipo@ICG obtained in step S3 respectively, hydrate until the film completely detaches, sonicate, and centrifuge to remove unloaded copper sulfide nanoparticles, and extrude using a liposome extruder to obtain a lipid nanoparticle system.

2. The preparation method of the near-infrared responsive lipid nanoparticle system aggregated with copper sulfide nanoparticles according to claim 1, characterized in that The specific preparation process of step S1 is as follows: Place 0.1 mmol of copper chloride dihydrate in a 250 mL round-bottom flask, add 100 mL of ultrapure water and stir until fully dissolved, then add 0.01 - 0.1 mmol of polypeptide K, stir for 30 minutes, add 0.1 mmol of thioacetamide, heat up to 50 °C and react for 2 - 3 hours to obtain a dark brown liquid. Ultrafilter to remove unreacted small molecule compounds and polypeptide K, wash with water 3 times, and concentrate to obtain a CuS-PepK concentrate for standby.

3. The preparation method of the near-infrared responsive lipid nanoparticle system aggregated with copper sulfide nanoparticles according to claim 1, characterized in that The specific preparation process of step S2 is as follows: Place 0.1 mmol of copper chloride dihydrate in a 250 mL round-bottom flask, add 100 mL of ultrapure water and stir until fully dissolved, then add 0.01 - 0.1 mmol of polypeptide Q, stir for 30 minutes, add 0.1 mmol of thioacetamide, heat up to 50 °C and react for 2 - 3 hours to obtain a dark brown liquid. Ultrafilter to remove unreacted small molecule compounds and polypeptide Q, wash with water 3 times, and concentrate to obtain a CuS-PepQ concentrate for standby.

4. The preparation method of the near-infrared responsive lipid nanoparticle system aggregated with copper sulfide nanoparticles according to claim 1, characterized in that The specific preparation process of step S3 is as follows: Dissolve dipalmitoyl phosphatidylcholine, cholesterol, photosensitizer ICG, and DSPE-PEG 2k in a chloroform solution at a molar ratio of 18:6:1:1.5 in 2 - 3 mL, and after complete dissolution, transfer it to a round-bottom flask. Then, form a uniform green film by rotary evaporation at 30 °C using a rotary evaporator. Subsequently, add 2 mL of ultrapure water and place it in a water bath at 60 °C for hydration. After complete hydration, transfer the obtained green solution to an ice bath and sonicate for 30 - 60 minutes. Then, extrude the resulting liquid through a polycarbonate membrane filter. Finally, concentrate the extruded sample using an ultrafiltration device to obtain a film of sample Lipo@ICG, and store it in a refrigerator at 4 °C for later use. The molecular weight cut-off of this ultrafiltration device is 30 kDa.

5. The preparation method of the near-infrared responsive lipid nanoparticle system aggregated with copper sulfide nanoparticles according to claim 1, characterized in that The specific preparation process of step S4 is as follows: The obtained Lipo@ICG film is incubated with 2 mg / mL CuS-PepQ concentrate and 2 mg / mL CuS-PepK concentrate at 60 °C for 1 h respectively for hydration to mix the copper sulfide nanoparticle solution with the green film-like mixture. After the film is completely hydrated, the mixture is transferred to a centrifuge tube and sonicated for 10 - 30 minutes, and then the mixed solution is frozen and thawed for 3 - 5 cycles to promote the loading of CuS-PepQ and CuS-PepK into the liposome. The unloaded CuS-PepQ and CuS-PepK are removed by centrifugation, and the obtained liquid is extruded through a polycarbonate membrane filter to obtain the lipid nanoparticle system Lipo@ICG@CuS-PepQ / K.

6. Application of the near-infrared responsive copper sulfide nanoparticle-aggregated lipid nanoparticle system prepared by the method according to any one of claims 1 to 5 in the preparation of a drug for photothermal and photodynamic combined therapy of tumors, wherein the lipid nanoparticle system disintegrates under near-infrared laser irradiation to promote the release of copper sulfide nanoparticles and their penetration and enzyme-responsive aggregation and retention at the tumor site, and simultaneously realizes photothermal and photodynamic therapy of tumors.

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