Lung cancer cell membrane bionic targeting precious metal-based PLGA nano drug-loaded particle as well as preparation method and application thereof

Abstract

The invention discloses a lung cancer cell membrane bionic targeting precious metal-based PLGA nano drug-loaded particle, which comprises a drug-loaded inner core and a lung cancer cell membrane coating the inner core, the drug-loaded inner core structure sequentially comprises an inner core layer and an intermediate core layer, the inner core layer is a chemotherapeutic drug cisplatin, the intermediate core layer is PLGA with good biocompatibility, and the drug-loaded inner core structure sequentially comprises an outer core layer and an inner core layer. The outer surface of the PLGA is modified with noble metal, and the noble metal is one or two of gold, ruthenium and platinum. The invention also discloses a preparation method of the lung cancer cell membrane bionic targeting noble metal-based PLGA nano drug-loaded particle. According to the drug-loaded nanoparticle and the preparation method thereof, tumor cells can be effectively targeted, heat is generated under near-infrared irradiation, the drug cis-platinum loaded in the PLGA NPs can be exposed and released from the inside, and the effect of chemotherapy-photo-thermal synergistic treatment is achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of nano-biomedicine, and in particular to a lung cancer cell membrane biomimetic targeted noble metal-based PLGA nano-drug-carrying particle and a preparation method and application thereof. Background Art

[0002] In the continuous exploration and practice of cancer treatment, although traditional chemotherapy methods can inhibit tumor growth to a certain extent, the serious toxic side effects and drug resistance caused by its systemic distribution have always been difficult problems that need to be solved urgently.

[0003] Nano-drug delivery technology achieves targeted delivery and controlled release of drugs by constructing nanoparticles with specific functions, thereby significantly improving the efficacy of drugs and reducing side effects. However, a single treatment method is often difficult to fully meet the complex and changing needs of tumor treatment. Photothermal therapy, as a non-invasive physical therapy method, uses the difference in temperature sensitivity and tolerance between tumor tissue and normal tissue to heat the tumor site to a certain temperature through laser irradiation and maintain it for a period of time, thereby killing tumor cells without damaging normal tissue. Combining nano-drug delivery technology with photothermal therapy can not only achieve precise targeted delivery of drugs, but also further enhance the treatment effect through the photothermal effect, forming a synergistic effect.

[0004] However, the existing nano-drug-assisted photothermal therapy system still has some shortcomings. For example, the long retention time of nano-drug-loaded particles (NPs) in the body may lead to their accumulation in the body and potential toxicity; the lack of specificity for the tumor site may cause thermal damage to the normal tissues around the tumor, etc. Summary of the invention

[0005] Based on the above description, the present invention provides a lung cancer cell membrane biomimetic targeted noble metal-based PLGA nano drug-carrying particle and a preparation method and application thereof.

[0006] In a first aspect, the present invention provides a lung cancer cell membrane biomimetic targeted noble metal-based PLGA nanoparticles for drug loading, wherein the nanoparticles for drug loading include a drug loading core and a lung cancer cell membrane coated on the outside of the core.

[0007] Preferably, the drug-loaded core structure includes an inner core layer and an intermediate core layer in sequence; the inner core layer is the chemotherapy drug cisplatin, the intermediate core layer is PLGA with good biocompatibility, and the outer surface of the PLGA is modified with a precious metal, wherein the precious metal is one or two of gold, ruthenium, and platinum.

[0008] In a second aspect, the present invention provides a method for preparing lung cancer cell membrane biomimetic targeted noble metal-based PLGA nano drug-loaded particles, the preparation method comprising:

[0009] (A) PLGA NPs loaded with Cis (Cis@PLGA NPs) were prepared by emulsion solvent evaporation method.

[0010] (ii) Precious metal (PM) surface modification of HS-PEG-NH2 (PM-PEG).

[0011] (iii) Couple Cis@PLGA NPs with PM-PEG by amide reaction to prepare Cis@PLGA-PEG-PM NPs.

[0012] (IV) Lung cancer cell membrane-coated precious metal-modified Cis@PLGA NPs drug-loaded nanoparticles (LCM@Cis@PLGA-PEG-PM NPs).

[0013] Furthermore, the preparation method of PLGA NPs (Cis@PLGA NPs) loaded with Cis by emulsion solvent evaporation method described in step (a) specifically includes: dissolving the drug cisplatin Cis and PLGA in 10 mL of chloroform at a mass ratio of 50:50 to form a uniform organic phase solution; then adding 20 mL of 2% polyvinyl alcohol as an aqueous phase solution of a stabilizer; after the organic phase and the aqueous phase are saturated with each other, using 250W ultrasound to emulsify the mixture for 10 minutes, evaporating the organic solvent, and centrifuging at 20000 rpm for 30 minutes to finally purify the PLGA NPs (Cis@PLGA NPs) loaded with Cis.

[0014] Furthermore, the PLGA in step (i) is carboxyl functionalized PLGA, and its molecular weight is 80,000.

[0015] Furthermore, the method for surface modification of precious metals with HS-PEG-NH2 (PM-PEG) in step (ii) specifically includes: preparing a 100 mM precious metal salt solution and a 100 mM HS-PEG-NH2 solution, and adjusting the pH of the reaction solution to a weakly alkaline condition; then, slowly adding a reducing agent, sodium borohydride (NaBH4, 10 mM), to the precious metal salt solution under stirring, centrifuging at 16000 g for 10 min at 4°C, and washing twice to obtain PM-PEG.

[0016] Furthermore, the noble metal salt ion in step (ii) is Au 3+ 、Ru 3+ , Pt 4+ One or two of the above; the reaction molar ratio of the noble metal salt, HS-PEG-NH2 and NaBH4 is 1:(3-4):(3-4).

[0017] Further, the amide reaction coupling method described in step (iii) specifically includes: dispersing Cis@PLGA NPs in a 30% ethanol solution, adding EDC-NHS acylation catalyst (5mM, EDC and NHS are mixed in a molar ratio of 1:1.2), stirring and reacting for 30 minutes to activate the carboxyl groups on the surface of Cis@PLGA NPs. Subsequently, PM-PEG prepared in step (ii) is added in a molar ratio of 1:1, and stirred for 20 minutes to make the activated carboxyl groups on the surface of PLGA NPs react with the amino groups on PM-PEG to form an amide bond, thereby obtaining Cis@PLGA-PEG-PM NPs.

[0018] Furthermore, the method for preparing lung cancer cell membrane-coated precious metal-modified Cis@PLGA NPs nano-drug-carrying particles in step (iv) specifically includes: taking A549 lung cancer cells growing in the logarithmic phase, scraping the cells with a cell scraper, collecting the cells by centrifugation at 500 g, washing with isotonic pre-cooled PBS, centrifuging, discarding the supernatant, adding a hypotonic solution and ice bathing for 20 minutes, repeatedly freezing and thawing the cell solution after the ice bath at room temperature and in liquid nitrogen until the cells are completely broken, then centrifuging at 500 g to obtain the supernatant, centrifuging at 15,000 g to obtain the precipitate, which is the cell membrane fragments, and then ultrasonically treating the cell disruptor in an ice bath to obtain uniformly dispersed nanoscale cell membrane fragments. The precious metal-modified Cis@PLGA NPs nano-drug carrier particles and lung cancer cell membrane fragments were then vortex-mixed evenly in a mass ratio of 1:3, allowed to stand for 30 minutes, and then filtered 3 to 5 times through 0.75 μm, 0.45 μm and 0.22 μm filter membranes according to the pore size from large to small to obtain lung cancer cell membrane bionic targeting precious metal-based PLGA nano-drug carrier particles.

[0019] In a third aspect, the present invention provides a use of the lung cancer cell membrane biomimetic targeted noble metal-based PLGA nano-drug-loaded particles as described in the first aspect in the treatment of lung cancer.

[0020] The mechanism of action of the nano drug-carrying particles of the present invention is that the drug cisplatin is encapsulated in PLGA with biocompatibility and biodegradability to form nanoparticles (NPs), and noble metal atoms are modified on these NPs. Since these NPs are near-infrared resonant and PLGA is biodegradable, heat is generated during near-infrared irradiation, which accelerates the hydrolysis of PLGA NPs, so that the drug cisplatin loaded in the PLGA NPs is exposed and released from the inside, and the chemical-photothermal therapy shows a significant synergistic effect.

[0021] Beneficial effects of the present invention:

[0022] First, nanoparticles disguised with cancer cell membranes can actively sense and target tumor cells, reducing the clearance effect of the immune system. Secondly, by encapsulating the drug in PLGA with good biocompatibility, the premature release of the drug in the blood circulation is reduced, and the nonspecific toxicity to normal tissues is reduced. In addition, since precious metal materials (gold, ruthenium, and platinum) have superior photothermal conversion properties and can efficiently convert light energy into thermal energy, local temperature rise not only promotes the degradation of the PLGA matrix, but also accelerates the release of cisplatin drugs. Combining the dual mechanisms of chemotherapy (the cytotoxic effect of cisplatin) and photothermal therapy (NIR-induced local high temperature killing of cancer cells), a significant synergistic effect is shown. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 a is a TEM morphology of LCM@Cis@PLGA-PEG-Au / Ru NPs nano-drug-carrying particles prepared in Example 4 of the present invention; Figure 1 b is a TEM morphology of LCM@Cis@PLGA-PEG-Au / Pt NPs nano-drug-carrying particles prepared in Example 5 of the present invention; Figure 1 c is a TEM morphology image of LCM@Cis@PLGA-PEG-Ru / Pt NPs nanodrug-carrying particles prepared in Example 6 of the present invention, with a scale of 100 nm.

[0024] Figure 2 These are the results of biocompatibility experiments on precious metal-based PLGA nano-drug-carrying particles with and without lung cancer cell membrane coating.

[0025] Figure 3 These are the results of targeting analysis of precious metal-based PLGA nanodrug-loaded particles targeting lung cancer cell membranes.

[0026] Figure 4 To simulate near-infrared irradiation conditions, the PLGA on the surface of the nano-drug-carrying particles is heated and ruptured, releasing the internal drugs.

[0027] Figure 5 The tumor killing ability of precious metal-based PLGA nano-drug-loaded particles is biomimetic and targeted to lung cancer cell membranes.

[0028] Figure 6 This is the curve of tumor volume changes in tumor-bearing mice after receiving different treatments.

[0029] Figure 7H&E staining images of tumors in tumor-bearing mice after receiving different treatments, where a: PBS; b: Cis; c: Cis@PLGA-PEG-Au NPs; d: Cis@PLGA-PEG-Au NPs+NIR; e: LCM@Cis@PLGA-PEG-Au NPs+NIR; f: LCM@Cis@PLGA-PEG-Au / Ru NPs+NIR. DETAILED DESCRIPTION

[0030] The present invention will be further described below in conjunction with embodiments.

[0031] Example 1: A method for preparing lung cancer cell membrane biomimetic targeting precious metal (gold) PLGA nanoparticles containing drugs, comprising the following steps:

[0032] (1) The drug cisplatin Cis and carboxyl-functionalized PLGA were dissolved in 10 mL of chloroform at a mass ratio of 50:50 to form a uniform organic phase solution; 20 mL of 2% polyvinyl alcohol was then added to the organic solution as an aqueous phase solution of a stabilizer; after the organic phase and the aqueous phase were saturated with each other, the mixture was emulsified using 250 W ultrasound for 10 minutes, the organic solvent was evaporated, and the mixture was centrifuged at 20,000 rpm for 30 minutes to finally purify the Cis-loaded PLGA NPs (Cis@PLGA NPs).

[0033] (2) Prepare 100 mM HAuCl4 solution and 100 mM HS-PEG-NH2 solution, adjust the pH of the reaction solution to 7.4, slowly add reducing agent sodium borohydride (NaBH4, 10 mM) to the HAuCl4 solution under stirring, the reaction molar ratio of HAuCl4, HS-PEG-NH2 and NaBH4 is 1:3:3. Centrifuge at 16000 g for 10 min at 4°C and wash twice to obtain PM-PEG.

[0034] (3) EDC and NHS were mixed at a molar ratio of 1:1.2 to prepare a 5 mM EDC-NHS acylation catalyst, and then Cis@PLGA NPs were dispersed in a 30% ethanol solution, and the EDC-NHS acylation catalyst was added and stirred for 30 minutes to activate the carboxyl groups on the surface of Cis@PLGA NPs. Subsequently, PM-PEG prepared in step (2) was added at a molar ratio of 1:1 and stirred for 20 minutes to allow the activated carboxyl groups on the surface of PLGA NPs to undergo an amidation reaction with the amino groups on PM-PEG to form an amide bond, thereby obtaining Cis@PLGA-PEG-PM NPs.

[0035] (4) Take A549 lung cancer cells in logarithmic phase, scrape the cells with a cell scraper, collect the cells by centrifugation at 500g, wash with isotonic pre-cooled PBS, discard the supernatant after centrifugation, add hypotonic solution and ice bath for 20 minutes, freeze and thaw the cell solution after ice bath repeatedly at room temperature and in liquid nitrogen until the cells are completely broken, then centrifuge at 500g to obtain the supernatant, centrifuge at 15000g to obtain the precipitate, which is the cell membrane fragment, and then ultrasonic cell disruptor ice bath ultrasonic treatment to obtain uniformly dispersed nanoscale cell membrane fragments. Then, Cis@PLGA-PEG@Au NPs nano drug-carrying particles and lung cancer cell membrane fragments were vortexed and mixed evenly at a mass ratio of 1:3, and allowed to stand for 30 minutes, and then filtered through 0.75μm, 0.45μm and 0.22μm filter membranes from large to small pore size for 3 to 5 times to obtain lung cancer cell membrane biomimetic targeted precious metal (gold) PLGA nano drug-carrying particles LCM@Cis@PLGA-PEG-Au NPs.

[0036] Example 2: A method for preparing lung cancer cell membrane biomimetic targeted noble metal (ruthenium) PLGA nanoparticles containing drugs, comprising the following steps:

[0037] The difference between this embodiment and embodiment 1 is that the noble metal modified on the surface of Cis@PLGA is Ru, the noble metal salt solution used is RuCl3, and the reaction molar ratio of RuCl3, HS-PEG-NH2 and NaBH4 is 1:3:3. The other steps are the same as those in embodiment 1, and finally LCM@Cis@PLGA-PEG-Ru NPs are obtained.

[0038] Example 3: A method for preparing lung cancer cell membrane biomimetic targeted noble metal (platinum) PLGA nanoparticles containing drugs, comprising the following steps:

[0039] The difference between this embodiment and embodiment 1 is that the noble metal modified on the surface of Cis@PLGA is Pt, and the noble metal salt solution used is H2PtCl6, Pt 4+ The reaction molar ratio of HS-PEG-NH2 and NaBH4 was 1:4:4, and the other steps were the same as in Example 1, and finally LCM@Cis@PLGA-PEG-Pt NPs were obtained.

[0040] Example 4: A method for preparing lung cancer cell membrane biomimetic targeting noble metal (gold, ruthenium) PLGA nanoparticles containing drugs, comprising the following steps:

[0041] The difference between this embodiment and embodiment 1 is that the noble metals modified on the surface of Cis@PLGA are Au and Ru, and the noble metal salt solution used is a mixture of HAuCl4 and RuCl3. 3+ 、Ru 3+The reaction molar ratio of HS-PEG-NH2 and NaBH4 was 0.5:0.5:3:3, and the other steps were the same as in Example 1, and finally LCM@Cis@PLGA-PEG-Au / Ru NPs were obtained.

[0042] Example 5: A method for preparing lung cancer cell membrane biomimetic targeting noble metal (gold, platinum) PLGA nanoparticles loaded with drugs, comprising the following steps:

[0043] The difference between this embodiment and embodiment 1 is that the noble metals modified on the surface of Cis@PLGA are Au and Pt, and the noble metal salt solution used is a mixture of HAuCl4 and H2PtCl6. 3+ , Pt 4+ The reaction molar ratio of HS-PEG-NH2 and NaBH4 was 0.5:0.5:4:4, and the other steps were the same as in Example 1, and finally LCM@Cis@PLGA-PEG-Au / Pt NPs were obtained.

[0044] Example 6: A method for preparing lung cancer cell membrane biomimetic targeting noble metal (ruthenium, platinum) PLGA nanoparticles containing drugs, comprising the following steps:

[0045] The difference between this embodiment and embodiment 1 is that the precious metals modified on the surface of Cis@PLGA are Ru and Pt, and the precious metal salt solution used is a mixture of RuCl3 and H2PtCl6. 3+ , Pt 4+ The reaction molar ratio of HS-PEG-NH2 and NaBH4 was 0.5:0.5:4:4, and the other steps were the same as in Example 1, and finally LCM@Cis@PLGA-PEG-Au / Pt NPs were obtained.

[0046] Example 7: Morphological characterization of the nanoparticles prepared in Examples 4-6 of the present invention

[0047] Take an appropriate amount of the prepared sample to be tested and dissolve it in deionized water. After ultradispersion, take 5 μL and drop it on the carbon-coated copper mesh. Naturally adsorb and dry it at room temperature. Observe the morphology of the nanoparticles under a transmission electron microscope at 200 KV.

[0048] TEM characterization images of LCM@Cis@PLGA-PEG-Au / Ru NPs, LCM@Cis@PLGA-PEG-Au / Pt NPs, and LCM@Cis@PLGA-PEG-Ru / Pt NPs nanoparticles prepared in Examples 4, 5, and 6 of the present invention are shown in FIG. Figure 1 As shown in ac, it has a regular spherical structure, uniform size, and obvious membrane coating characteristics, which indicates that lung cancer cell membrane-coated nano-drug-loaded particles have been successfully synthesized.

[0049] Example 8: Comparison of biocompatibility of precious metal-based PLGA nanoparticles with and without lung cancer cell membrane coating

[0050] In order to further verify the biosafety performance of the material of the present invention, Cis@PLGA-PEG-Au NPs nanoparticles, Cis@PLGA-PEG-Ru NPs nanoparticles, Cis@PLGA-PEG-Pt NPs nanoparticles, LCM@Cis@PLGA-PEG-AuNPs nanoparticles, LCM@Cis@PLGA-PEG-Ru NPs nanoparticles, and LCM@Cis@PLGA-PEG-Pt NPs nanoparticles were prepared according to the preparation method of lung cancer cell membrane biomimetic targeting noble metal-based PLGA nanoparticles of the present invention. Normal human lung epithelial cells BEAS-2B were selected as cell models. BEAS-2B cells (8×10 3 ) were inoculated into a 96-well plate, with 100 μL of culture medium added to each well. After 24 h of cell culture, the original culture medium was aspirated, and the culture medium containing the above six nano-drug carrier particles was added. The cells were placed in a 37°C, 5% CO2 incubator and continued to be cultured for 24 h. 10 μL of CCK-8 solution was added to each well of the plate. After incubation for 1-3 hours, the absorbance at a wavelength of 450 nm was measured by an enzyme reader to calculate the cell viability.

[0051] like Figure 2 As shown in the figure, the average cell viability of the single precious metal-based PLGA nanoparticles without cell membrane coating (Cis@PLGA-PEG-AuNPs, Cis@PLGA-PEG-Ru NPs, Cis@PLGA-PEG-Pt NPs) is about 80%, indicating that the single precious metal-based PLGA nanoparticles without cell membrane coating have certain biocompatibility; the viability of BEAS-2B cells treated with the single precious metal-based PLGA nanoparticles coated with cancer cells (LCM@Cis@PLGA-PEG-Au NPs, LCM@Cis@PLGA-PEG-Ru NPs, LCM@Cis@PLGA-PEG-Pt NPs) increased to more than 90%, further improving the biocompatibility of the nanoparticles.

[0052] Example 9: Targeting analysis of precious metal-based PLGA nanoparticles targeting lung cancer cell membrane

[0053] Under the condition of laser irradiation, the specific evaluation of lung cancer cell membrane biomimetic targeting precious metal-based PLGA nanoparticles was evaluated by flow cytometry. After pancreatic cancer cells Panc02, breast cancer cells MDA-MB-231, and lung cancer cells A549 were inoculated in a 60mm dish and cultured for 24 hours, the nanoparticles prepared in Example 1 were used as an example, and the culture medium containing LCM@Cis@PLGA-PEG-Au NPs nanoparticles was added. After continuing to culture for 24 hours, the Annexin V-FITC apoptosis detection kit and flow cytometry were used to determine the cell apoptosis and necrosis analysis.

[0054] The cytotoxicity of LCM@Cis@PLGA-PEG-Au NPs to homologous cells was further investigated by flow cytometric analysis of cell apoptosis / necrosis. To evaluate the percentage of viable cells (Q4), early apoptotic cells (Q3), late apoptotic cells (Q2), and necrotic cells (Q1), the in vitro cells after different treatments were stained with Annexin V / PI. Figure 3 The results showed that compared with the PBS control group, the percentages of apoptosis and necrosis of pancreatic cancer cells Panc02 and breast cancer cells MDA-MB-231 treated with laser irradiation + LCM@Cis@PLGA-PEG-Au NPs nanodrug carrier particles were low, indicating that LCM@Cis@PLGA-PEG-Au NPs nanodrug carrier particles had no tumor killing ability on heterologous cells and could not specifically identify and kill them; A549 cells treated with laser irradiation LCM@Cis@PLGA-PEG-Au NPs nanodrug carrier particles showed obvious cell apoptosis and necrosis, about 92%, confirming the high specific killing effect of LCM@Cis@PLGA-PEG-Au NPs nanodrug carrier particles on homologous cells.

[0055] Example 10: Under simulated near-infrared irradiation conditions, the PLGA on the surface of the nanoparticles is ruptured by heat to release the internal drugs

[0056] A549 cells were plated at 1×10 5 / mL of cells were inoculated in a 6-well cell plate and cultured in a cell culture incubator at 37°C and 5% CO2 for 24h. The original culture medium was removed, and the cells were treated with culture medium containing LCM@Cis@PLGA-PEG-Au NPs nanoparticles and LCM@Cis@PLGA-PEG-Au / Ru NPs nanoparticles, respectively, and the cells were cultured in a 37°C, 5% CO2 incubator for 24h. At the same time, A549 cells were cultured normally as a negative control. After 24 hours, the two groups containing LCM@Cis@PLGA-PEG-Au NPs nanoparticles and LCM@Cis@PLGA-PEG-Au / Ru NPs nanoparticles were irradiated with 808nm laser for 10 minutes, and the other two groups were not treated with laser irradiation. 10μL CCK-8 solution was added to each well of the plate. After incubation for 1-3 hours, the absorbance at a wavelength of 450nm was measured by an enzyme reader to calculate the cell viability.

[0057] like Figure 4 As shown, under the conditions of near-infrared irradiation in vivo, the precious metals act as photothermal converters to efficiently convert the absorbed NIR light energy into heat energy, resulting in an increase in the local temperature around the nanoparticles, which in turn triggers the thermal responsive degradation of the PLGA polymer and accelerates the release of the anticancer drug cisplatin (Cis) encapsulated therein. The cell viability of the LCM@Cis@PLGA-PEG-Au NPs+NIR group is about 33%, and the cell viability of the LCM@Cis@PLGA-PEG-Au / Ru NPs+NIR group is about 22%. This not only reflects the killing effect of photothermal synergistic drugs on lung cancer cells, but also reflects the performance advantages of dual precious metal materials. For the group without NIR irradiation treatment, due to the lack of photothermal conversion effect, PLGA has no thermal response degradation, and the rate of natural degradation remains relatively slow, resulting in only a weak release of the encapsulated drug Cis. The material of the nano-drug-loaded particles did not show any additional effects on cell activity, indicating that its photothermal effect was in an inactive state under this condition and did not directly participate in or affect the survival state of the cells. The cell viability of the two groups of cells was maintained at a high level, with the cell viability of the LCM@Cis@PLGA-PEG-Au NPs group being approximately 73% and the cell viability of the LCM@Cis@PLGA-PEG-Au / Ru NPs group being approximately 66%.

[0058] Example 11: Tumor killing ability of precious metal-based PLGA nanoparticles with biomimetic targeting of lung cancer cell membranes

[0059] According to the preparation method of lung cancer cell membrane biomimetic targeted noble metal-based PLGA nanoparticles, LCM@Cis@PLGA-PEG-Au NPs, LCM@Cis@PLGA-PEG-Ru NPs, LCM@Cis@PLGA-PEG-Pt NPs, LCM@Cis@PLGA-PEG-Au / Ru NPs, LCM@Cis@PLGA-PEG-Au / Pt NPs, LCM@Cis@PLGA-PEG-Ru / Pt NPs and Cis@PLGA-PEG-Ru NPs were prepared. A549 cells (8×10 3 ) were inoculated into 96-well plates, with 100 μL of culture medium added to each well. After 24 h of cell culture, the original culture medium was aspirated, and the culture medium containing the above six nano-drug carrier particles was added. The cells were placed in a 37°C, 5% CO2 incubator and continued to be cultured for 24 h. They were irradiated with 808 nm laser for 10 min, and 10 μL of CCK-8 solution was added to each well of the plate. After incubation for 1-3 hours, the absorbance at a wavelength of 450 nm was measured by an enzyme reader to calculate the cell viability.

[0060] The cell proliferation experiment was used to study the cancer cell killing efficiency of lung cancer cell membrane biomimetic targeting noble metal-based PLGA nanoparticles. Figure 5As shown, the average viability of A549 cells after treatment with single precious metal-based PLGA nanoparticles coated with cancer cells (LCM@Cis@PLGA-PEG-Au NPs, LCM@Cis@PLGA-PEG-Ru NPs, LCM@Cis@PLGA-PEG-Pt NPs) combined with near-infrared irradiation was approximately 33%, indicating that single precious metal-based PLGA nanoparticles coated with cancer cells can specifically recognize target cells and effectively kill tumor cells. It is worth noting that the cell viability of the dual noble metal-based PLGA nanoparticles (LCM@Cis@PLGA-PEG-Au / Ru NPs, LCM@Cis@PLGA-PEG-Au / Pt NPs and LCM@Cis@PLGA-PEG-Ru / Pt NPs) coated with cancer cells further decreased after treatment with near-infrared irradiation. Among them, the LCM@Cis@PLGA-PEG-Au / Ru NPs nanoparticle treatment group combined with near-infrared irradiation had a more significant tumor killing effect, which may be related to the synergistic enhancement of the photothermal effect between the bimetallic nanostructures. Under NIR light irradiation, the bimetallic triggered a stronger localized surface plasmon resonance (LSPR) effect, which promoted the efficient conversion of light energy to thermal energy. Compared with single noble metal-based nanoparticles, the bimetallic nanostructure provides more hot spots, making the heat more concentrated, thereby generating a higher temperature gradient in the target cells, effectively improving the local hyperthermia effect.

[0061] Example 12: Tumor growth inhibition effect of noble metal-based PLGA nanoparticles coupled with near-infrared irradiation and biomimetic targeting of lung cancer cell membranes

[0062] A549 cells (3×10 6 cells, 100 μL Ham's F-12K medium) were subcutaneously inoculated into male Balb / c nude mice (8-10 weeks old, 20 g). The tumor volume was measured with a digital caliper and calculated according to the following formula: Tumor volume = (tumor length) × (tumor width) 2 / 2. When the tumor volume reaches 50mm 3 The mice were treated with Cis, Cis@PLGA-PEG@Au NPs, Cis@PLGA-PEG@Au NPs+NIR, LCM@Cis@PLGA-PEG@Au NPs+NIR and LCM@Cis@PLGA-PEG@Au / Ru NPs+NIR (40 μL, Cis concentration equivalent to 5 μg / mL) respectively. All preparations were intravenously injected into the mice through the tail vein every 2 days, and were given / not given near-infrared irradiation according to the group. Three hours after injection, the mice were irradiated with 808 nm laser (2.83 W / cm 2) for 10 minutes, and the control group mice were injected with PBS. The tumor volume (V) of the mice was measured every 2 days for 16 days. The change in tumor volume during the entire treatment process was expressed as the relative tumor volume V / V0 (V0 is the tumor volume at the beginning of treatment). After 16 days, the mice were killed, and the tumors were fixed in 10% neutral buffered formalin for 24 hours, washed with PBS, and stored in 70% ethanol (4°C). After OCT embedding, it was cut into ~5μm, stained with hematoxylin and eosin (H&E), and analyzed by optical microscopy.

[0063] In order to maximize the therapeutic effect, the cells were divided into the following five groups: Cis, Cis@PLGA-PEG-Au NPs, Cis@PLGA-PEG-Au NPs+NIR, LCM@Cis@PLGA-PEG-Au NPs+NIR, and LCM@Cis@PLGA-PEG-Au / Ru NPs+NIR, and a control group without treatment was set up. Figure 6 As shown, at the end of the experiment, the untreated tumor grew to 5.3 times its initial size. Free Cis treatment initially only slightly reduced the tumor size compared to the original size. Comparing the tumor growth rates of the Cis@PLGA-PEG-Au NPs and Cis@PLGA-PEG-Au NPs+NIR treatment groups, it was found that near-infrared light irradiation did enhance the efficacy of the nanoparticles; this indicates that the precious metal gold on the nanoparticles played a photothermal effect and achieved a good lung cancer treatment effect. At the same time, comparing the tumor growth rates of the Cis@PLGA-PEG-Au NPs+NIR and LCM@Cis@PLGA-PEG-Au NPs+NIR treatment groups, it was found that LCM@Cis@PLGA-PEG-Au NPs+NIR coated with cancer cell membranes had better therapeutic efficacy. This is due to the clever wrapping of the lung cancer cell membrane, and the nano-drug-carrying particles have acquired the ability to target specific cancer cells, promoting the accumulation and release of drugs at the tumor site. Similar to the in vivo experimental results, the dual-noble metal-based PLGA nanoparticles coated with cancer cells showed superior anti-tumor effects under NIR irradiation than single-noble metal-based PLGA nanoparticles, and the dual-metal photothermal effect was synergistically enhanced.

[0064] Figure 7af are representative H&E-stained tumor sections after different treatments. The tumors of mice injected with PBS showed typical pathological features of lung cancer, such as close arrangement of tumor cells. The tumor tissues of Cis, Cis@PLGA-PEG-Au NPs, Cis@PLGA-PEG-Au NPs+NIR, LCM@Cis@PLGA-PEG-Au NPs+NIR, and LCM@Cis@PLGA-PEG-Au / Ru NPs+NIR groups all showed punctate necrosis and intercellular spaces. In addition, LCM@Cis@PLGA-PEG-Au / RuNPs irradiated with 808nm laser had the most obvious tumor cell necrosis and shrinkage. The lung cancer cell membrane specifically targets the tumor tissue. Under 808nm infrared irradiation, the heat generated by the outermost noble metal base and PLGA nanoshell and the released drugs can kill cancer cells together, thereby enhancing the anticancer effect. Therefore, the combination of noble metal-based near-infrared photothermal therapy with cisplatin chemotherapy can achieve better anticancer efficacy than the current single therapy.

[0065] 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 lung cancer cell membrane biomimetic targeting noble metal-based PLGA nanoparticles, characterized by: It includes a drug-loaded core and a lung cancer cell membrane coated on the outside of the core; wherein the drug-loaded core structure includes an inner core layer and an intermediate core layer in sequence; the inner core layer is the chemotherapy drug cisplatin, the intermediate core layer is PLGA, and the outer surface of the PLGA is modified with a precious metal, wherein the precious metal is one or two of gold, ruthenium, and platinum.

2. A method for preparing lung cancer cell membrane biomimetic targeting noble metal-based PLGA nanoparticles, characterized in that: The steps include: S1: The drug cisplatin Cis and PLGA were dissolved in 10 mL of chloroform at a mass ratio of 1:1 to form a uniform organic phase solution; then 20 mL of 2% polyvinyl alcohol was added to mix with the aqueous phase as a stabilizer; after the organic phase and the aqueous phase were saturated with each other, the mixture was emulsified using 250W ultrasound for 10 minutes; the organic solvent was evaporated and centrifuged at 20,000 rpm for 30 minutes to finally purify Cis-loaded Cis@PLGA NPs; S2: prepare 100 mM noble metal salt solution and 100 mM HS-PEG-NH2 solution, adjust the pH value of the reaction solution to a weakly alkaline condition, slowly add 10 mM reducing agent sodium borohydride NaBH4 to the noble metal salt solution under stirring; the noble metal ions, HS-PEG-NH2 and NaBH4 are reacted in a certain proportion; centrifuge at 16000 g for 10 min at 4°C, and wash twice to obtain PM-PEG; S3: coupling Cis@PLGA NPs with PM-PEG by amide reaction to prepare Cis@PLGA-PEG-PM NPs; S4: Lung cancer cell membrane-coated precious metal-modified Cis@PLGA NPs nanodrug-loaded particles.

3. The method for preparing drug-loaded nanoparticles according to claim 2, characterized in that: The PLGA described in step S1 is carboxyl functionalized PLGA, and its molecular weight is 80,000.

4. The method for preparing drug-loaded nanoparticles according to claim 2, characterized in that: The pH of the reaction solution in step S2 is 7.

4.

5. The method for preparing drug-loaded nanoparticles according to claim 2, characterized in that: The noble metal salt ion in step S2 is Au 3+ 、Ru 3+ , Pt 4+ One or two of the above; the reaction molar ratio of the noble metal salt, HS-PEG-NH2 and NaBH4 is 1:(3-4):(3-4).

6. The method for preparing drug-loaded nanoparticles according to claim 2, characterized in that: The amide reaction coupling method described in step S3 specifically includes: mixing EDC and NHS in a molar ratio of 1:1.2 to prepare a 5mM EDC-NHS acylation catalyst, then dispersing Cis@PLGA NPs in a 30% ethanol solution, adding the EDC-NHS acylation catalyst, stirring the reaction for 30 minutes to activate the carboxyl groups on the surface of Cis@PLGANPs; then, adding the PM-PEG prepared in step S2 in a certain proportion, stirring for 20 minutes, so that the activated carboxyl groups on the surface of PLGANPs react with the amino groups on the PM-PEG to form an amide bond, and obtain Cis@PLGA-PEG-PM NPs.

7. The method for preparing drug-loaded nanoparticles according to claim 6, characterized in that: The reaction molar ratio of Cis@PLGANPs to PM-PEG is 1:

1.

8. The method for preparing drug-loaded nanoparticles according to claim 2, characterized in that: The method for preparing lung cancer cell membrane-coated precious metal-modified Cis@PLGA NPs nano-drug-carrying particles in step S4 specifically includes: taking A549 lung cancer cells growing in the logarithmic phase, scraping the cells with a cell scraper, collecting the cells by centrifugation at 500 g, washing with isotonic pre-cooled PBS, centrifuging, discarding the supernatant, adding a hypotonic solution and ice bathing for 20 minutes, repeatedly freezing and thawing the cell solution after the ice bath at room temperature and in liquid nitrogen until the cells are completely broken, then centrifuging at 500 g to obtain the supernatant, centrifuging at 15,000 g to obtain the precipitate, which is the cell membrane fragments, and then ultrasonically treating the cell disruptor in an ice bath to obtain evenly dispersed nanoscale cell membrane fragments. The precious metal-modified Cis@PLGA NPs nano-drug carrier particles and lung cancer cell membrane fragments were then vortex-mixed evenly in a mass ratio of 1:3, allowed to stand for 30 minutes, and then filtered 3 to 5 times through 0.75 μm, 0.45 μm and 0.22 μm filter membranes according to the pore size from large to small to obtain lung cancer cell membrane bionic targeting precious metal-based PLGA nano-drug carrier particles.

9. Use of the lung cancer cell membrane biomimetic targeted noble metal-based PLGA nano drug-carrying particles as claimed in any one of claims 1 to 8 in the preparation of drugs for treating lung cancer.

10. The use according to claim 9, characterized in that: The precious metal-based near-infrared photothermal therapy was combined with cisplatin chemotherapy, and the chemo-photothermal therapy showed obvious synergistic effect.

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