Carbon monoxide core-shell nanoparticles capable of intravenous administration, preparation method therefor, and use thereof

Abstract

Carbon monoxide core-shell nanoparticles capable of intravenous administration, a preparation method therefor, and use thereof, belonging to the technical field of nanomedicine. The preparation method for the carbon monoxide core-shell nanoparticles comprises: 1. preparing a polymer carrier PLGA(CO); 2. purifying the PLGA(CO); 3. weighing the PLGA(CO) and a functional material, dissolving them in THF, adding deionized water after ultrasonication, blowing off THF by using nitrogen, carrying out co-precipitation, centrifuging, washing, and carrying out co-precipitation to obtain a nanoparticle functional material @PLGA(CO); and 4. weighing DSPE-PEG, and reacting with the functional material @PLGA(CO) to obtain a functional material @PLGA(CO)@PEG. After a tail intravenous injection, the core-shell structure nanoparticles target a cancer site by means of intravenous injection under the monitoring of NIR-II fluorescence, and CO-enhanced mild temperature photothermal therapy is achieved to eliminate tumors.

Description

Intravenously administrable carbon monoxide core-shell nanoparticles and their preparation method and application Technical Field

[0001] The present invention belongs to the technical field of nanomedicine, and in particular relates to intravenously administrable carbon monoxide core-shell nanoparticles and a preparation method and application thereof. Background Art

[0002] Mild temperature photothermal therapy (MTPPT), also known as low-temperature PTT, is a localized, noninvasive approach for treating solid tumors and has attracted significant interest in the biomedical field. PTT typically utilizes a photothermal converter to convert light energy into high temperatures, thereby killing cancer cells with high specificity and efficiency. However, to achieve complete tumor elimination, PTT requires raising the temperature of cancerous tumors to above 50°C. Such high temperatures not only ablate the tumor but also damage surrounding normal tissue through thermal diffusion. To alleviate this problem, researchers have attempted to utilize MTPTT (38-43°C) to induce cancer cell death. However, the therapeutic efficacy of MTPTT is severely compromised because cancer cells can activate their self-preservation pathways, such as heat shock proteins (HSPs), to rapidly repair heat-induced cellular damage. To reduce the heat tolerance of cancer cells, several small molecule HSP inhibitors, such as gambogic acid, 17-AAG, triptolide, and STA-9090, have been developed to enhance the therapeutic efficacy of MTPTT. However, the poor solubility, acute cytotoxicity, and serum instability of these HSP inhibitors prevent them from completely silencing HSPs under complex physiological conditions. Furthermore, the complex fabrication of nanosystems using photothermal agents combined with HSP inhibitors increases pharmacokinetic uncertainty and operational difficulty, and reduces the reproducibility of clinical translation. Therefore, there is an urgent need to develop new HSP inhibitors that combine photothermal conversion capabilities with HSP silencing and perform MTPTT simultaneously.

[0003] In previous work, we developed a nanocarrier for delivering carbon monoxide (CO) that can be triggered by the tumor microenvironment to release large amounts of CO. We found that the released CO inhibited the upregulation of heat shock proteins (HSPs) in cancer cells upon thermal treatment and enhanced the performance of MTPTT (multiple photothermal transitions) based on AIE photothermal converters (PTAs). However, the application of nanocarriers in efficient tumor therapy requires the rational design of delivery nanosystems. This nanocarrier can only encapsulate AIE polymers, not small molecules; the nanoparticles formed with this carrier cannot be administered intravenously, and CO exposed on the particle surface may cause toxic effects that cannot be assessed. This core-structured nanobomb has difficulty effectively reaching tumor sites when injected intravenously, meaning it is often trapped by the liver's filtration mechanisms before reaching its intended target. This limitation not only limits its effectiveness but also hinders its further development. The liver's complex detoxification and filtration systems, designed to remove foreign substances from the blood, may recognize the nanobomb as an invader, trap it, and thus prevent it from exerting its therapeutic effect on the tumor. Therefore, the design of innovative drug delivery systems to address these issues is imperative.

[0004] Core-shell nanoparticles have received considerable attention in the field of PTT. Introducing nanoparticles into PTT is considered a promising approach to address existing challenges. The appeal of these core-shell nanoparticles lies in their ability to combine the advantages of individual components into a single structure, exhibiting enhanced physical and chemical properties that are distinct from any single component. This structure further promotes active interfaces between the components, forming synergistic effects and revealing new properties. For example, certain biomolecular shells not only stabilize PTAs in biological fluids and enhance their blood circulation time, but they also confer the ability to actively target tumor sites. Importantly, by integrating functional materials or reagents, these nanoparticles can serve as multifunctional nanoplatforms, paving the way for combined therapeutic and imaging applications.

[0005] Pancreatic cancer (PC), often referred to as the "king of cancers," is a major contributor to cancer-related mortality worldwide. Compared to other types of cancer, PC possesses inherently unique characteristics. The pancreas is anatomically hidden, nestled in close proximity to vital organs such as the stomach, duodenum, liver, and gallbladder. Therefore, the diagnosis and treatment of pancreatic cancer present a dual challenge: precisely locating the elusive site of pancreatic cancer while precisely killing tumor cells without damaging adjacent normal organs. This situation underscores the enormous medical challenges posed by pancreatic cancer. Given these challenges facing PC, the use of a core-shell nanobomb with NIR-II imaging guidance has emerged as a promising strategy. This nanobomb not only facilitates precise targeting of pancreatic cancer tumors but also enables effective multi-photon-threshold-to-threshold (MTTPTT) therapy. This approach ensures precise tumor treatment while minimizing potential damage to surrounding vital organs.

[0006] Summary of the Invention

[0007] In view of the defects existing in the above-mentioned prior art, in order to solve the problem that CO carriers cannot encapsulate small molecule drugs, nanoparticles can only be injected in situ, and lack targeting, the purpose of the present invention is to design and provide a carbon monoxide core-shell nanoparticle that can be administered intravenously and its preparation method and application. The present invention can deliver carbon monoxide core-shell structured nanoparticles. After injection through the tail vein, they are injected intravenously into the cancer site under the monitoring of NIR-II fluorescence. The release of carbon monoxide is triggered by the tumor microenvironment. At the same time, the nanoparticles can carry small molecule photothermal conversion agents (such as AIE molecules), which are activated by near-infrared zone II laser to achieve CO-enhanced low-temperature photothermal therapy to eliminate tumors.

[0008] In order to achieve the above object, the present invention adopts the following technical solutions:

[0009] In one aspect, the present invention provides a method for preparing intravenously administrable carbon monoxide core-shell nanoparticles, comprising the following steps:

[0010] (1) Iron tris(II) dodecacarbonyl and methyl poly(lactic acid-glycolic acid sulfur) were weighed and dissolved in tetrahydrofuran. The mixture was stirred at high temperature and nitrogen flow to react until the color of the solution changed from dark blue to brownish yellow. The solution was cooled to room temperature, and n-hexane was added. The solution was washed with ether and dried to obtain the polymer carrier PLGA(CO).

[0011] (2) Weighing the polymer carrier PLGA(CO) and redissolving it in THF, freezing it in a low-temperature environment, and filtering the precipitated crystals to obtain purified PLGA(CO);

[0012] (3) Weighing the PLGA (CO) and functional material obtained in step (2) above, dissolving them in THF, adding deionized water after ultrasonication, blowing out the THF with nitrogen, co-precipitating, centrifuging with an ultrafiltration tube and repeatedly washing 2-5 times, co-precipitating to obtain nanoparticle functional material @PLGA (CO);

[0013] (4) Weigh DSPE-PEG and react it with the functional material @PLGA(CO) to obtain the functional material @PLGA(CO)@PEG.

[0014] In the preparation method, in step (1), the mass ratio of triiron dodecacarbonyl to methyl polylactic acid-glycolic acid sulfur is 5-100:100-600; the high temperature is 50-120° C.; and the stirring time is 1-12 hours.

[0015] In the preparation method, the methyl polylactic acid-glycolic acid sulfur in step (1) is selected from one of mPLGA5000-SH, mPLGA8000-SH, SH-mPLGA2000-SH, and SH-mPLGA8000-SH.

[0016] The preparation method is characterized in that the low temperature in step (2) is -20°C; and the freezing time is 5-24 hours.

[0017] In the preparation method, the functional material in step (3) is selected from one of small molecule drugs, polymer drugs, fluorescent probes, small molecule photothermal conversion agents or nanoparticles.

[0018] In the preparation method, the nanoparticles are 2TT-OC46B, PBPTV or Bodipy, the fluorescent probe is TPB, and the small molecule photothermal conversion agent is an AIE molecule.

[0019] In the preparation method, the mass-to-volume ratio of the PLGA (CO), functional material and deionized water in step (3) is 1-50 mg:5-10 mg:10 mL.

[0020] In the preparation method, the ultrasonic time in step (3) is 5-10 minutes; the centrifugation time is 5 minutes.

[0021] A carbon monoxide core-shell nanoparticle capable of intravenous administration, wherein the carbon monoxide core-shell nanoparticle is prepared by any one of the preparation methods described above.

[0022] The application of the carbon monoxide core-shell nanoparticles in intravenous administration.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] 1. The preparation method of the present invention is simple and easy to implement, and can be successfully prepared in only two steps, which is convenient for operation and promotion.

[0025] 2. The nanoparticles of the present invention have very low toxicity to normal tissues while killing tumor tissues and can target tumors.

[0026] 3. The nanoparticles prepared by the present invention can exist stably in a normal environment and no sedimentation or flocculation occurs after 7 days.

[0027] 4. The present invention proposes a method of using carbon monoxide gas molecules to inhibit heat shock proteins, thereby improving the effect of low-temperature photothermal therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 is a Nanobomb of the present invention 2G Preparation flow chart of

[0029] Figure 2 is a Nanobomb of the present invention 2G UV spectrum of

[0030] Figure 3 is a Nanobomb under the hydrated particle size analyzer of the present invention 2G Particle size;

[0031] Figure 4 is a Nanobomb of the present invention 2G Photothermal conversion efficiency;

[0032] Figure 5 shows the CCK 8 experiment of the present invention to prove Nanobomb 2G cytotoxicity to cancer cells;

[0033] Figure 6 is a Nanobomb of the present invention 2G Targeting of tumors in vivo;

[0034] Figure 7 is a Nanobomb in the present invention 2G Therapeutic effect on in situ pancreatic cancer;

[0035] Figure 8 is a Nanobomb of the present invention 2G Figure 3. Suppression of HSP expression in tumors. DETAILED DESCRIPTION

[0036] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0037] Example 1: Preparation of nanoparticles 2TT-OC46B@PLGA(CO)@PEG

[0038] (1) The substitution reaction of iron dodecacarbonyl with thiol was used to modify one end of methyl polylactic acid-glycolic acid sulfide (mPLGA SH) to serve as a reactive oxygen species (ROS) responsive amphiphilic polymer carrier PLGA (CO). The specific preparation process is as follows: iron dodecacarbonyl (5-100 mg) and mPLGA-SH (MW≈2000) (100-600 mg) were dissolved in tetrahydrofuran (THF) and stirred at 50-120 ° C under nitrogen flow for 1 12 h. At the end of the reaction, the solution changed from dark blue to brownish yellow. After cooling to room temperature, n-hexane was added to obtain a brown precipitate, which was washed with ether and dried to obtain (PLGA-CO).

[0039] (2) PLGA(CO) was redissolved in THF and frozen at -20°C for 5-24 hours. The precipitated crystals were filtered to obtain a novel intelligent carbonyl iron carrier PLGA(CO).

[0040] (3) 1-50 mg of PLGA(CO) and 5-10 mg of 2TT-OC46B dye were dissolved in THF. After sonication for 5-10 min, 10 mL of deionized water was added, and the THF was blown out with nitrogen gas for coprecipitation. The mixture was centrifuged in an ultrafiltration tube for 5 min and washed three times. Coprecipitation was used to form stable and uniform nanoparticles of 2TT-OC46B@PLGA(CO).

[0041] (4) A layer of DSPE-PEG was wrapped around the outer layer of the nanoparticles to form 2TT-OC46B@PLGA(CO)@PEG, named Nanobomb 2G Nanobomb 2G The preparation flow chart is shown in Figure 1.

[0042] Example 2: UV spectrum and particle size of nanoparticles

[0043] 2TT-OC46B, 2TT-OC46B@PLGA(CO) and Nanobomb 2G Dissolve in pure water to a final concentration of 5-10 mM, take 0.5-1 mL into a cuvette, and measure its UV absorption in the range of 200-1000 nm using a UV spectrophotometer. The results are shown in Figure 2.

[0044] The nanoparticles were dissolved in pure water to a final concentration of 5-10 mM, 0.5-1 mL was taken into a cuvette, and the average particle size was measured using a DLS particle size analyzer. The results are shown in Figure 3. The average particle size of the 2TT-OC46B@PLGA(CO) nanoparticles is about 166 nm.

[0045] The average particle size of 2TT-OC46B@PLGA(CO)@PEG nanoparticles is about 190 nm.

[0046] Example 3: Photothermal conversion efficiency of nanoparticles

[0047] The nanoparticles were dissolved in pure water to a final concentration of 50-100 mM. 0.5-1 mL was placed in a centrifuge tube and illuminated with an 808 nm laser probe for 5 minutes. Simultaneously, an infrared camera was used to monitor the temperature of the nanoparticle solution. After 5 minutes, the laser probe was turned off, and the solution was allowed to cool naturally while the temperature change was recorded. The results, shown in Figure 4, show that the solution heated up rapidly over the first 3 minutes, with the heating time roughly equal to the cooling time.

[0048] Example 4: Cytotoxicity of Nanoparticles to Cancer Cells

[0049] Panc02 pancreatic cancer cells were cultured in a 96-well plate with PBS in the outer circle. Each column of six wells in the middle of the plate was grouped together. On the second day, 10-20 μL of 2TT-OC46B, PLGA(CO), and 2TT-OC46B@PLGA(CO)@PEG solutions at varying concentrations (1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 mg / mL) were added to the wells. The cells were then incubated in an incubator for 24 hours. CCK-8 dye was then added, and cell viability in the plate was measured using a microplate reader. This serves as the first set of data.

[0050] In the above three experiments, after adding 2TT-OC46B, PLGA (CO) and Nanobomb 2G After the solution was added, the plate was irradiated with 808nm laser for 1-2 minutes and the cell viability was measured after 24 hours of culture. The results are shown in Figure 5. The horizontal axis is the concentration. It can be seen from the figure that the dye 2TT-OC46B has no inhibitory effect on the growth of cancer cells regardless of its concentration, while PLGA (CO) and Nanobomb 2G The inhibitory effect of nanoparticles on cancer cells increases with the increase of concentration. At the same concentration, the inhibitory effect on cancer cells is from large to small. 2G Nanoparticles plus light, PLGA(CO) plus light, PLGA, Nanobomb 2G Nanoparticles.

[0051] Example 5: Targeted imaging of pancreatic cancer tumors using nanoparticles

[0052] Two groups of Panc02 tumor-bearing BALB / c mice were selected, with 5 mice in each group. One group of mice was injected with 0.5-1 mL of 20-30 mM nanoparticle solution. 2G ), and the other group was injected with 0.5-1mL of 20-30mM nanoparticle solution (2TT-OC46B@PLGA(CO)). After 12 hours, the tumor site of each mouse was irradiated with 808nm NIR for 1-2 minutes. The fluorescence signal of the mouse tumor site was monitored using a near-infrared two-zone fluorescence camera. The results are shown in Figure 6. 2G The tumor site of the 2TT-OC46B@PLGA(CO) group emitted obvious fluorescence signals, while the tumor site of the 2TT-OC46B@PLGA(CO) group emitted almost no fluorescence, indicating that Nanobom 2G Tumor targeting.

[0053] Example 6: Therapeutic Effects of Nanoparticles on In Situ Pancreatic Cancer Tumors

[0054] Two groups of Panc02 tumor-bearing BALB / c mice were selected, with 5 mice in each group. One group of mice was injected with 0.5-1 mL of 20-30 mM Nanobomb.2G Nanoparticle solution, another group was injected with 0.5-1mL of 20-30mM Nanobomb 2G After 12 hours of exposure to the nanoparticle solution, each mouse's tumor site was irradiated with 808nm NIR for 1-2 minutes. Tumor volume was recorded every two days until the mice were sacrificed on the 14th day. Figure 7 shows tumor volume changes. While tumor volume was suppressed in mice injected with PBCO alone, it continued to grow. However, tumor volume in mice injected with PBCO followed by photothermal therapy remained virtually unchanged, demonstrating a significant inhibitory effect on tumor growth.

[0055] Example 7: Nanobomb 2G Inhibition of heat shock proteins in tumors

[0056] Panc02 tumor-bearing BALB / c mice were divided into six groups, with five mice in each group. After 12 hours, the tumor area was irradiated with 808nm NIR. The tumor volume and weight of the mice were recorded every 2 days. The mice were killed on the 14th day, and the main organs and tumors were obtained for histological examination. The tumors of five mice implanted with tumors at the same time were treated differently: injection of 0.5-1mL PBS; injection of 0.5-1mL PBS and irradiation with 4-5W 808nm laser for 3-5min; injection of 0.5-1mL 20-30mM 2TTOC46B solution; injection of 0.5-1mL 20-30mM 2TTOC46B solution and irradiation with 4-5W 808nm laser for 3-5min; injection of 0.5-1mL 20-30mM Nanobomb 2G Solution: Inject 0.5-1mL of 20-30mM Nanobomb 2G Solution and irradiate with 808nm laser with power of 4-5W for 3-5min. The mouse tumor tissue was removed and immersed in formalin solution for 40-50 hours and embedded in wax blocks. After making paraffin sections, dewax to water as usual and perform antigen heat repair. Add primary antibody, incubate in 37℃ constant temperature box for 1 hour, wash with PBS, add secondary antibody, incubate in 37℃ constant temperature box for 1 hour, wash with PBS, add DAPI dye, incubate in 37℃ constant temperature box for 10 minutes, wash with PBS, develop color with DBA, and seal after conventional counterstaining. Confocal microscopy observation, the results are shown in Figure 8, indicating that Nanobomb 2G It can inhibit the expression of HSP in tumors.

Claims

1. A preparation method of carbon monoxide core-shell nanoparticles for intravenous administration, characterized in that, it comprises the following steps: (1) Weigh ferric dodecacarbonyl and methyl poly(lactic-co-glycolic acid) sulfide, dissolve them in tetrahydrofuran, stir and react under high temperature and nitrogen flow until the color of the solution changes from dark blue to yellowish brown, cool to room temperature, add n-hexane, wash with ether and dry to obtain the polymer carrier PLGA(CO); (2) Weigh the polymer carrier PLGA(CO), redissolve it in THF, freeze it in a low-temperature environment, filter the precipitated crystals to obtain purified PLGA(CO); (3) Weigh the PLGA(CO) obtained in the above step (2) and the functional material, dissolve them in THF, add deionized water after ultrasonic treatment, blow out THF with nitrogen, perform co-precipitation, centrifuge with an ultrafiltration tube and repeat washing 2-5 times, perform co-precipitation to obtain nanoparticle functional material @PLGA(CO); (4) Weigh DSPE-PEG and react it with the functional material @PLGA(CO) to obtain the functional material @PLGA(CO)@PEG.

2. The preparation method according to claim 1, characterized in that, in step (1), the mass ratio of the ferric dodecacarbonyl to the methyl poly(lactic-co-glycolic acid) sulfide is 5-100:100-600; the high temperature is 50-120 °C; the stirring time is 1-12 h.

3. The preparation method according to claim 1, characterized in that, in step (1), the methyl poly(lactic-co-glycolic acid) sulfide is selected from one of mPLGA5000-SH, mPLGA8000-SH, SH-mPLGA2000-SH, SH-mPLGA8000-SH.

4. The preparation method according to claim 1, characterized in that, in step (2), the low temperature is -20 °C; the freezing time is 5-24 h.

5. The preparation method according to claim 1, characterized in that, in step (3), the functional material is selected from one of small molecule drugs, polymer drugs, fluorescent probes, small molecule photothermal conversion agents or nanoparticles.

6. The preparation method according to claim 5, characterized in that, the nanoparticles are 2TT-OC46B, PBPTV or Bodipy, the fluorescent probe is TPB, and the small molecule photothermal conversion agent is an AIE molecule.

7. The preparation method according to claim 1, characterized in that, in step (3), the mass-to-volume ratio of the PLGA(CO), the functional material and the deionized water is 1-50 mg:5-10 mg:10 mL.

8. The preparation method according to claim 1, characterized in that, in step (3), the ultrasonic time is 5-10 min; the centrifugation time is 5 min.

9. A carbon monoxide core-shell nanoparticle for intravenous administration, characterized in that, the carbon monoxide core-shell nanoparticle is prepared by the preparation method according to any one of claims 1-6.

10. Use of the carbon monoxide core-shell nanoparticle according to claim 9 in intravenous administration and delivering carbon monoxide to the tumor environment.

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