Use of dual light treated albumin drug-loaded nanoparticles in the preparation of a medicine for treating cervical cancer

Abstract

This invention discloses the application of albumin-loaded drug-eluting nanoparticles for dual-phototherapy in the preparation of drugs for treating cervical cancer, belonging to the field of pharmaceutical technology. The albumin-loaded drug-eluting nanoparticles of this invention are formed by loading indocyanine green and hydroxyalizarin onto albumin; the nanoparticle system simultaneously contains ICG and purpurin; the ICG photothermal agent can not only convert light energy into heat energy through photothermal action, but also generate reactive oxygen species through photodynamic therapy to inhibit cell activity, while purpurin inhibits GDH1 activity, disrupting intracellular redox homeostasis and cutting off the nutrient supply to cancer cells. Based on the above three anti-tumor mechanisms, this invention overcomes the shortcomings of single-treatment chemotherapy, such as high toxicity and low therapeutic effect. By combining the nanosystem with photodynamic and photothermal therapy, the therapeutic effect of hydroxyalizarin on cervical cancer cells is improved, providing a new solution to the technical challenges of high recurrence and mortality rates in cervical cancer.

Description

Technical Field

[0001] This invention relates to the application of albumin-loaded drug nanoparticles for dual-light therapy in the preparation of drugs for treating cervical cancer, and belongs to the field of pharmaceutical technology. Background Technology

[0002] Currently, cancer (malignant tumors), as one of the world's deadliest diseases, is seriously threatening human health, and its incidence is showing a trend towards younger ages. When it comes to the treatment of malignant tumors, chemotherapy has always been the main treatment method, but the use of single chemotherapy drugs has limited therapeutic effects. Therefore, there is an urgent need for new methods with high safety and significant efficacy for tumor treatment. In recent years, the use of photodynamic therapy (PDT), photothermal therapy (PTT), and combination therapy for tumor treatment has attracted widespread interest from researchers. Among them, PTT and PDT have become research hotspots due to their non-invasive nature and minimal side effects. However, high levels of glutathione (GSH) in tumor cells severely affect the effectiveness of PDT, and PTT alone relies on excessive heat generation, leading to poor treatment compliance and tissue damage. Therefore, researchers are committed to using different methods to improve the effectiveness of PDT and PTT. Indocyanine green (ICG) is favored by researchers because it is approved by the FDA and can simultaneously produce photothermal and photodynamic effects; however, the short half-life and poor stability of ICG limit its efficacy.

[0003] Numerous studies have shown that albumin, as an endogenous substance in the body, is an ideal carrier material for drug delivery. Albumin-based combination therapy strategies can not only overcome the limitations of monotherapy but also improve the stability of each drug component. However, in practical applications, when albumin is used as a carrier to load indocyanine green (ICG) for use on tumor cells, there is still a problem of insufficient ICG photodynamics, which can lead to poor treatment compliance and tissue damage. Summary of the Invention

[0004] To address the shortcomings and deficiencies of existing technologies, this invention provides the application of albumin-loaded nanoparticles for dual-phototherapy in the preparation of drugs for treating cervical cancer. This invention designs and successfully optimizes the synthesis of functional albumin-loaded nanoparticles (ICG & purpurin@BSA, BIP) based on dual-phototherapy. Purpurin, a hydroxyalkaloid, can reduce GPx1 levels by inhibiting glutamate dehydrogenase (GDH1) function and limiting glutamine degradation, thereby reducing GSH content in cancer cells and achieving cell damage. Using purpurin as a nanomedicine carrier, combined with photodynamic therapy, photothermal therapy, and mechanisms such as inhibiting GDH1 to disrupt cell homeostasis and cut off nutrient supply, a drug delivery system containing this nanomedicine is constructed. Enhanced photodynamic therapy (PDT) synergistically with mild photodynamic therapy (PTT) improves the therapeutic effect of cervical cancer to a certain extent, ultimately allowing the combined therapy to fully exert its effect.

[0005] The advantages of this invention mainly include two aspects. Firstly, while improving drug stability, it avoids the local tissue hyperthermia caused by excessively high temperatures due to single PTT, thereby achieving gentle photothermal therapy and further improving patient compliance. Secondly, purpurin achieves synergistic enhancement of PDT by reducing ROS consumption. In summary, using albumin as a drug carrier to improve the stability of ICG and purpurin while achieving gentle PTT and synergistic enhancement of PDT will provide a potential new strategy for highly effective and low-toxicity tumor treatment.

[0006] The first objective of this invention is to provide the use of dual-phototherapy albumin-loaded drug nanoparticles in the preparation of drugs for treating cervical cancer, wherein the dual-phototherapy albumin-loaded drug nanoparticles are formed by loading albumin with indocyanine green and hydroxyalizarin.

[0007] In one embodiment, the albumin is any one of bovine serum albumin, human serum albumin, ovalbumin, or recombinant protein.

[0008] In one embodiment, the albumin-loaded drug nanoparticles contain an indocyanine green concentration of 30 μg / mL and a hydroxyalizarin concentration of 2.3–6.9 μg / mL.

[0009] In one embodiment, the albumin-loaded drug nanoparticles contain a final concentration of indocyanine green of 30 μg / mL and a final concentration of hydroxyalizarin of 6.9 μg / mL.

[0010] In one embodiment, the preparation of the albumin-loaded nanoparticles for dual-light therapy specifically includes the following steps:

[0011] (1) Preparation of solution A: Dissolve indocyanine green (ICG) and purpurin in dimethyl sulfoxide, stir evenly in the dark to obtain solution A;

[0012] (2) Preparation of solution B: Dissolve albumin in ultrapure water and stir until completely dissolved to obtain solution B;

[0013] (3) Slowly add solution B dropwise to solution A and stir the reaction at room temperature in the dark. After the reaction is complete, stir the reaction solution at 2-6℃ overnight, purify it with a 100KD ultrafiltration tube, wash it with ultrapure water, repeat the ultrafiltration, and obtain albumin-loaded nanoparticles.

[0014] In one embodiment, the molar ratio of indocyanine green (ICG) to purpurin in step (1) is (0.5-2):1; preferably 1:1.

[0015] In one embodiment, the concentration of albumin in solution B in step (2) is 2-5 mg / mL; preferably 4 mg / mL.

[0016] In one embodiment, the mixing volume ratio of liquid A to liquid B in step (3) is 1:5.

[0017] In one embodiment, when the molar ratio of indocyanine green to hydroxyalizarin is 1:1, the concentration of indocyanine green in solution A is 6 mg / mL and the concentration of hydroxyalizarin is 2 mg / mL; the concentration of albumin in solution B is 4 mg / mL; and the mixing volume ratio of solution A to solution B is 1:5.

[0018] A second objective of this invention is to provide a medicament for treating cervical cancer, the medicament comprising albumin-loaded drug-eluting nanoparticles as active ingredients, wherein the albumin-loaded drug-eluting nanoparticles for dual-phototherapy are formed by loading albumin with indocyanine green and hydroxyalizarin.

[0019] The beneficial effects of this invention are:

[0020] (1) The albumin-loaded drug nanoparticles of the present invention improve the biocompatibility and therapeutic effect of the loaded drug by using bovine serum albumin as a carrier; and the nano system contains ICG (photothermal agent) and purpurin; the ICG photothermal agent can not only convert light energy into heat energy through photothermal action, but also generate reactive oxygen species (ROS) through photodynamic therapy to inhibit cell activity, while purpurin inhibits GDH1 activity, disrupts the redox homeostasis of cells and cuts off the nutrient supply to cancer cells; based on the above three anti-tumor mechanisms (photodynamic therapy, photothermal therapy, and inhibition of glutamate dehydrogenase 1 to disrupt cell homeostasis and cut off nutrient supply), the shortcomings of single chemotherapy such as high toxicity and low therapeutic effect are solved. A strategy and technical solution based on combination therapy is designed to improve the therapeutic effect of chemotherapy drug (purpurin) on cervical cancer cells by combining the nano system with photodynamic and photothermal therapy, providing a new solution to solve the technical problems of easy recurrence and high mortality of cervical cancer;

[0021] (2) The preparation of the albumin drug-loaded nanoparticles of the present invention is simple and convenient in synthesis, with a suitable particle size (about 200 nm under electron microscopy), and has good drug loading capacity as a drug carrier. It can load indocyanine green and hydroxyalizarin through hydrophobic interaction and has high biocompatibility.

[0022] In cytotoxicity assays, under 808 nm laser irradiation, compared with drug delivery systems loaded with indocyanine green alone or hydroxyalizarin alone (ICG@BSA, BI or purpurin@BSA, BP), the survival rate of HeLa cells after indocyanine green and hydroxyalizarin were co-loaded in the nanosystem (ICG&purpurin@BSA, BIP) was significantly reduced. This demonstrates that this nanosystem, through the synergistic effect of ICG and purpurin, reduces the ROS consumption of ICG while improving the therapeutic effect of cervical cancer. The antitumor effect of the drug-loaded nanosystem was determined by measuring the temperature change induced by indocyanine green photothermal therapy, the reactive oxygen species generation capacity, and the biocompatibility of the drug delivery system.

[0023] (3) The albumin-loaded nanoparticles of the present invention can reduce the dosage of chemotherapy drugs and improve the therapeutic effect of drugs on cervical cancer cells by synergistic photothermal therapy and photodynamic therapy. This has certain guiding significance for exploring combination therapies using nanomaterials as carriers to deliver anticancer drugs and provides a potential strategy for improving the effect of PDT. Attached Figure Description

[0024] Figure 1The following are schematic diagrams of electron microscopy morphology characterization of albumin-loaded nanoparticles prepared in Example 1 and Comparative Example 1 of the present invention; (A) is a BP transmission electron microscope image of albumin-loaded nanoparticles; (B) is a BI transmission electron microscope image of albumin-loaded nanoparticles; (C) is a BIP transmission electron microscope image of albumin-loaded nanoparticles.

[0025] Figure 2 (A) is a physicochemical property characterization diagram of albumin-loaded drug nanoparticles; (B) is a BP hydration particle size distribution diagram of albumin-loaded drug nanoparticles. Figure 2 (A) is the particle size distribution of albumin-loaded drug nanoparticle BI; (B) is the hydrated particle size distribution of albumin-loaded drug nanoparticle BIPA; (C) is the zeta potential data of albumin-loaded drug nanoparticles BP, BI, and BIP.

[0026] Figure 3 (A) Stability test diagram of albumin-loaded drug nanoparticles (BIP); (B) Particle size variation trend diagram; (C) 7-day placement diagram of actual sample.

[0027] Figure 4 (A) is the FTIR spectrum of albumin-loaded nanoparticles BIP; (B) is the UV-vis spectrum of BIP; (C) is the fluorescence spectrum of BIP excited at 488 nm.

[0028] Figure 5 The following are the standard curves for ICG and purpurin, and the release performance of albumin-loaded drug nanoparticles (BIP); (A) is the standard curve for ICG; (B) is the standard curve for purpurin; (C) is the release performance of albumin-loaded drug nanoparticles (BIP) under different pH conditions.

[0029] Figure 6 The following diagrams illustrate the photothermal stability of albumin-loaded drug nanoparticles (BIP); (A) shows the temperature after irradiation with an 808nm laser; (B) shows the temperature change curve; (C) shows the photothermal stability data of BIP; and (D) shows the photothermal effect of different ICG concentrations in BIP.

[0030] Figure 7 This image shows the distribution of albumin-loaded drug nanoparticles (BIP) and ICG in vivo in small animals.

[0031] Figure 8 Image showing the photothermal effect of albumin-loaded drug nanoparticles (BIP) in vivo;

[0032] Figure 9 H&E staining images of tumor tissue damage after different treatments;

[0033] Figure 10The following are data graphs showing the in vivo antitumor experiment results in Example 4: (A) is a schematic diagram of the in vivo antitumor experiment; (B) is a graph showing the tumor volume inhibition effect of each drug delivery system; (C) is a visual graph showing the tumor volume inhibition effect of each experimental group after treatment; (D) is a graph showing the tumor size data of each experimental group; and (E) is a graph showing the effect of each drug delivery system on the mouse body weight during treatment.

[0034] Figure 11 This is a graph showing the inhibitory effects of albumin-loaded drug-eluting nanoparticles (BIP) on different tumor cells.

[0035] Figure 12 (A) Data graph showing the effect of nanoparticles on HeLa cell viability; (B) Data graph showing the effect of different concentrations of BSA on cancer cell activity; (C) Data graph showing the inhibitory effect of albumin-loaded nanoparticles prepared in Example 1 and Comparative Example 1 on HeLa cells with and without light exposure.

[0036] Figure 13 To observe the cellular uptake fluorescence after co-incubating albumin-loaded nanoparticles (BIP) with cells for 2, 4, and 6 hours using laser confocal microscopy (CLSM), blue represents the cell nucleus, green represents ICG, and red represents purpurin.

[0037] Figure 14 (A) A comparison of the effects of BIP and BI on ROS production; (B) A graph showing the effects of different concentrations of BIP on ROS production.

[0038] Figure 15 Fluorescence maps for ROS quantitative analysis of BIP and BI; (A) and (B) are comparison data of ROS fluorescence intensity of BIP and BI; (C) and (D) are comparison data of ROS intensity generated by different concentrations of BIP.

[0039] Figure 16 (A) and (B) are visual diagrams and data graphs of the blood compatibility of BIP prepared in Example 1 of the present invention with free drugs; (C) and (D) are visual diagrams and data graphs of the blood compatibility of different free drugs;

[0040] Figure 17 This is a schematic diagram illustrating the preparation and application of albumin-loaded drug nanoparticles (BIP) according to the present invention. Detailed Implementation

[0041] The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0042] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0043] Example 1

[0044] A method for preparing albumin-loaded drug nanoparticles, the method comprising the following steps:

[0045] (1) Preparation of solution A: Dissolve 6 mg indocyanine green (ICG) and 2 mg hydroxyalizarin (purpurin) (ICG to purpurin molar ratio 1:1) in 4 mL of dimethyl sulfoxide, stir evenly in the dark to obtain solution A;

[0046] (2) Preparation of solution B: Dissolve 80 mg of bovine serum albumin (BSA) in 20 mL of ultrapure water and stir until completely dissolved to obtain solution B with a concentration of 4 mg / mL.

[0047] (3) Add solution B slowly dropwise to solution A, one drop every 2 seconds. This process is a stirring reaction at room temperature, which requires protection from light and slow reaction. Afterward, stir the resulting reaction solution overnight at 4°C in the dark. Purify with a 100KD ultrafiltration tube, centrifuge at 3000 RCF and take the supernatant. Wash with ultrapure water multiple times, repeat ultrafiltration, and collect the combined solution to obtain albumin-loaded nanoparticles, namely ICG&purpurin@BSA, abbreviated as BIP-loaded nanoparticles.

[0048] Example 2

[0049] The only difference from Example 1 is that the molar ratio of indocyanine green and hydroxyalizarin in step (1) is adjusted to 2:1 and 1:2 respectively; other parameters and conditions are the same as in Example 1.

[0050] Comparative Example 1

[0051] The only difference from Example 1 is that step (1) omits the addition of indocyanine green or hydroxyalizarin; other parameters and conditions are the same as in Example 1, and albumin-loaded drug nanoparticles BI and BP are prepared respectively.

[0052] Performance characterization of albumin-loaded nanoparticles

[0053] 1. The particle size of the albumin-loaded drug nanoparticles prepared in Examples 1 and 2 were measured, and the results are shown in Table 1:

[0054] Table 1. Changes in BIP particle size (nm) and PDI (mean ± SD, n = 3) under different feed ratios for ICG and purpurin

[0055]

[0056] 2. The albumin-loaded drug nanoparticles prepared in Example 1 and Comparative Example 1 were characterized.

[0057] The average particle size of the prepared albumin-loaded nanoparticles was measured using transmission electron microscopy (TEM) and dynamic light scattering (DLS).

[0058] Figure 1 The average particle size of the prepared albumin-loaded drug nanoparticles was measured by transmission electron microscopy (TEM). The results showed that the particle sizes of BP and BI were less than 200 nm, and the particle size of BIP was around 200 nm. The drug-loaded nanoparticles had relatively uniform particle size and good dispersibility.

[0059] Figure 2 The average particle size of the prepared albumin-loaded nanoparticles was measured by dynamic light scattering (DLS). The results showed that the particle sizes of the albumin-loaded nanoparticles BP, BI, and BIP were 190 nm, 220 nm, and 359 nm, respectively. Figure 2 The zeta potentials of different drug-loaded nanoparticles indicate successful drug loading.

[0060] Stability tests were performed on albumin nanoparticles (BIP), and the results are as follows: Figure 3 As shown in Figure A, the particle size of BIP nanoparticles changes little with time, and their dispersibility remains good. Figure 3 B shows that no precipitation was found at the bottom of the sample solution, and the 7-day stability test indicates that the BIP solution has good physical stability.

[0061] Successful loading of purpurin was verified using FTIR and 488nm characteristic fluorescence, as shown in the results. Figure 4 As shown in Figure A, the FTIR results show that BIP and purpurin are at 1049 and 880 cm⁻¹. -1 The same absorption peak is observed at 488 nm, and the BIP lyophilized powder exhibits the characteristic fluorescence of purpurin at 488 nm. Figure 4 C).

[0062] Validating the successful payload of ICG using UV-vis Figure 4 Characterization results showed that BIP and ICG had the same characteristic peak at 780 nm, and FTIR results showed that at 667 cm⁻¹... -1 The characteristic peak of ICG is present at this location. Figure 4 A) The above results all indicate that BIP can be successfully prepared.

[0063] like Figure 5 As shown, ICG (internal chromatogram) was plotted at 778 nm and 486 nm using an external spectrophotometer. Figure 5 A) and purpurin( Figure 5The standard curves for B) were then used to measure the UV absorption of purified free ICG and purpurin, and their contents in the nanoparticles were calculated. Ultimately, the encapsulation efficiency and drug loading level of purpurin in BIP were 61.2% and 3.4%, respectively, while those for ICG were 88.7% and 14.7%.

[0064] To investigate the drug release characteristics of BIP nanoparticles, BIP was placed in PBS under different conditions for release studies. The drug release from BIP under different pH conditions was observed, and the results are as follows: Figure 5 As shown in C, a weakly acidic environment is more conducive to the release of drugs in BIP.

[0065] 3. Study on the photothermal properties of albumin-loaded drug nanoparticles (BIP)

[0066] The BIP prepared according to Example 1 and the BI prepared according to Comparative Example 1 were centrifuged in five plastic tubes containing 1 mL of different samples (BSA, purpurin, ICG, BI, and BIP) at 808 nm (where the concentration of BSA was 400 μg / mL, the concentration of ICG was 30 μg / mL, the concentration of purpurin was 6.9 μg / mL, and the concentration of ICG in both BI and BIP was 30 μg / mL) at a power intensity of 1 W / cm². -2 The samples were irradiated with a laser for 4 minutes; then, the temperature of each group was recorded every 30 seconds using a near-infrared thermal imager (real-time temperature recorder) for 4 minutes.

[0067] like Figure 6 As shown in (A), BSA does not generate heat with free purpurin, while the heat mainly comes from the photothermal agent ICG and the drug delivery systems BI and BIP containing ICG.

[0068] according to Figure 6 (B) The temperature curve shows that the more light exposure time, the more heat the drug delivery system generates, which is the main reason why the drug delivery system produces a good cell inhibition effect.

[0069] Figure 6 (C) This shows that, compared with free ICG, BIP still exhibits good photothermal stability after four consecutive light-cooling cycles. Figure 6 As shown in (D), the photothermal effect of ICG increases with increasing concentration and is concentration-dependent. After 4 minutes of light irradiation, the temperature can reach a maximum of 48.5°C. More importantly, this temperature can exert photothermal therapy without damaging other surrounding tissues, which is very beneficial for cervical cancer patients. The above results indicate that the BIP prepared in this invention can treat cancer through mild photothermal therapy.

[0070] 4. Study on cellular uptake of albumin-loaded drug delivery systems

[0071] The distribution of intracellular drugs at different emission wavelengths was imaged using a laser confocal microscope. 1 mL of a 1×10⁻⁶ m³ drug was added to a laser confocal microscopy-specific culture dish. 5 HeLa cells were incubated at a concentration of 10 cells / mL in an incubator until they fully adhered to the culture dish. The ICG&purpurin@BSA (BIP) drug delivery system dispersion prepared in Example 1 was incubated with HeLa cells for 2, 4, and 6 hours, with the concentration of ICG in the BIP being 30 μg / mL. After washing the confocal culture dishes three times with PBS to remove any residual drug, 1 mL of 4% paraformaldehyde solution was added to fix the cells. Then, 500 μL of DAPI staining solution was added, and the cell nuclei were stained after 10 min. The cells were then washed five times with 1 mL of PBS on a shaker for 10 min each time to remove any residual DAPI staining solution. After washing, 1 mL of PBS was added to each dish and stored at 4°C in the dark. The phagocytic effect of the drug delivery system on HeLa cells and the distribution of intracellular drugs at different emission wavelengths were observed using a laser scanning confocal microscope (CLSM).

[0072] Figure 13 The results showed that when the incubation time was 2 hours, only a small amount of fluorescence signal (green fluorescence of ICG and red fluorescence of purpurin) was observed. However, when the incubation time was extended to 6 hours, the fluorescence signal could be clearly observed. This demonstrates that the ICG&purpurin@BSA (BIP) drug delivery system can be rapidly taken up by tumor cells and successfully enter the cells, thus confirming the feasibility of using the ICG&purpurin@BSA (BIP) drug delivery system of the present invention for the treatment of cervical cancer.

[0073] 5. Detection of intracellular ROS production under specific wavelength light stimulation

[0074] First, add 1 mL of a 1×10⁻⁶ solution to the 24-well plate. 4 HeLa cells were incubated at 100 cells / mL in an incubator for 48 hours. Then, serum-free DMEM-dispersed ICG & purpurin@BSA (BIP)(C ICG(0, 10, 20, 30 μg / mL), and incubated for another 6 h. Then, the ROS production capacity was detected using a ROS detection kit. Detailed procedures were as follows: The wells were washed with PBS to remove residual drug and culture medium, and then washed three times (10 min each time) on a shaker at 200 rpm, avoiding light during washing. Subsequently, DCFH-DA was diluted with serum-free DMEM to a concentration of 10 μM, and incubated with the cells in an incubator for 30 min. Afterward, the cells were washed with serum-free culture medium in the dark, and then washed three times (10 min each time) on a shaker at 200 rpm. Finally, the 24-well plate was irradiated with light of a specific wavelength at 808 nm for 4 min at 1 W / cm². 2 The generation of ROS was then observed using a fluorescence microscope with a FITC channel.

[0075] like Figure 14 As shown in Figure A, the albumin-based drug delivery system prepared in Example 1 successfully generated a large amount of ROS under irradiation at a specific wavelength. The DCF generated in the ROS kit bound to the ROS and exhibited strong green fluorescence. Moreover, compared with the BI group, the BIP group was found to generate more ROS after light irradiation. Figure 14 B indicates that the ROS generated by BIP after illumination is concentration-dependent. Figure 15 Yes Figure 14 Data obtained from flow cytometry analysis of the generated ROS. Figure 15 A shows that, under the same ICG concentration and illumination, BIP produces more ROS than BI. Figure 15 B indicates that ROS increases in a concentration-dependent manner with increasing BIP concentration. In summary, Figure 14 , Figure 15 This study demonstrates that the ICG&purpurin@BSA (BIP) drug delivery system has the ability to generate ROS, and that the amount of ROS generated increases with the increase of ICG concentration. At an ICG concentration of 30 μg / mL, BIP generates more ROS than BI after light irradiation, indicating that BIP can be used for cancer treatment through enhanced photodynamic therapy.

[0076] Example 3: Stability and therapeutic efficacy study of albumin-loaded drug-eluting nanoparticles (BIP) in mice.

[0077] HeLa cells in good growth condition were selected for the construction of the tumor model. Healthy female nude mice (4-6 weeks old) weighing 18-22g were used and purchased from Spiford (Beijing) Biotechnology Co., Ltd. All animal experiments were conducted in accordance with the requirements of the Ethics Committee of Jiangnan University.

[0078] First, HeLa cells in good growth condition were digested with trypsin, resuspended in PBS, and the cell concentration was adjusted to 1×10⁶. 6100 μL of resuspended HeLa cells were subcutaneously injected into the right axilla of nude mice to establish a tumor model. The size of the tumor was measured using digital calipers. The tumor was allowed to reach a certain volume (100 mm²) before being monitored. 3 At this point, the tumor model is established.

[0079] Two experimental groups (free ICG group and BIP group) were set up, with one mouse in each group. 100 μL of the drug containing the same concentration of ICG (70 μg) was injected into the tail vein of each mouse. Eight hours after the injection, the distribution of the drugs (ICG and BIP) in the mice was detected using a small animal in vivo imaging system (excitation 745 nm / emission 840 nm).

[0080] The results are as follows Figure 7 As shown, in vivo imaging and tissue imaging analysis revealed that after free ICG entered the mice, it was mainly distributed in the liver and kidneys, with only a small amount being taken up by the tumor site. This indicates that free ICG may have a relatively short half-life in vivo and is easily metabolized by the body, which is not conducive to treatment. While the BIP group showed strong ICG fluorescence in the liver and kidneys, it also accumulated to some extent in the tumor site, indicating that BIP can be taken up by tumor tissue. This may be attributed to albumin's targeting ability, making it easily taken up by starved tumor cells. Furthermore, the organ distribution of BIP shows that it is mainly metabolized by the liver. Compared to free ICG, drugs delivered via BSA have lower renal clearance and better stability. Therefore, these results demonstrate that BIP can be taken up by tumor tissue and exhibits good in vivo stability.

[0081] To further analyze the photothermal conversion effect of albumin-loaded nanoparticles in vivo, two experimental groups (PBS-NIR group and BIP-NIR group) were set up two weeks after modeling. Mice were injected intravenously with 100 μL of PBS and 100 μL of BIP (containing 70 μg ICG), respectively. Eight hours later, mice were anesthetized and subjected to laser irradiation (808 nm, 1 W cm⁻¹) at the tumor site. -2 (4 min), then the temperature change of the tumor site after irradiation was recorded using a near-infrared imager, and finally the temperature change curve was plotted.

[0082] The results are as follows Figure 8 As shown, Figure 8 A indicates that BIP can achieve good photothermal conversion in vivo. Figure 8The temperature change results showed that after 4 minutes of light exposure, the highest temperature of BIP reached 45.1℃, indicating that BIP could be successfully taken up by tumor tissue and exert a mild photothermal effect. In contrast, the temperature of the PBS group after light exposure was close to normal body temperature and did not produce a PTT effect. The comparison of the photothermal effects of the two groups in vivo revealed that BIP can achieve mild photothermal therapy in vivo and has two main characteristics: firstly, the mild photothermal effect kills tumor cells without affecting normal tissues, which greatly improves the compliance of photothermal therapy; secondly, it verifies the successful delivery of BIP in vivo, providing a foundation for an integrated albumin drug delivery system based on interference with redox homeostasis.

[0083] To further investigate the effects of the BIP drug delivery system on tumor tissue, hematoxylin and eosin (H&E) staining was used to examine tumor tissue sections. Hematoxylin is a natural basic dye that stains cell nuclei blue, while eosin stains the cytoplasm pink. Figure 9 It can be seen that the PBS group showed almost no tissue damage. As the light exposure continued, each drug-treated group showed varying degrees of tissue damage, with the ICG-NIR, BI-NIR, and BIP-NIR phenomena being the most obvious. It is speculated that this may be related to the corresponding nuclear lysis, tissue necrosis, fibrosis, inflammatory infiltration, and vacuolation phenomena in the cells. The vacuolation phenomenon was more severe in the BIP-NIR group, further demonstrating the effectiveness of BIP treatment.

[0084] Example 4

[0085] Application of BIP drug-loaded nanoparticles in the treatment of cervical cancer

[0086] HeLa cells in good growth condition were selected for the construction of the tumor model. Healthy female nude mice (4-6 weeks old) weighing 18-22g were used and purchased from Spiford (Beijing) Biotechnology Co., Ltd. All animal experiments were conducted in accordance with the requirements of the Ethics Committee of Jiangnan University.

[0087] First, HeLa cells in good growth condition were digested with trypsin, resuspended in PBS, and the cell concentration was adjusted to 1×10⁶. 6 100 μL of resuspended HeLa cells were subcutaneously injected into the right axilla of nude mice to establish a tumor model. The size of the tumor was measured using digital calipers. The tumor was allowed to reach a certain volume (100 mm²) before being monitored. 3 At this point, the tumor model is established.

[0088] Seven experimental groups were set up (PBS-NIR, Pur-NIR, ICG-NIR, BI, BIP, BI-NIR, and BIP-NIR groups), with four mice in each group. NIR stands for Near-infrared irradiation, indicating the light treatment group. Drug administration was scheduled every two days, with 100 μL of either PBS or the drug group (containing 70 μg ICG and 161 μg purpurin) injected via the tail vein for a total of six administrations (on days 1, 3, 5, 7, 9, and 11). The light-treated group (NIR group) was exposed to light 8 hours after each administration, while the non-light-treated group was not exposed to light. (808 nm, 1 W cm⁻¹) -2 (4 min), during the administration process, the body weight and tumor size of the mice were recorded every two days for subsequent experimental analysis;

[0089] After treatment, all mice were euthanized and their tumors and tissues were removed for experimental research. The tumor size of each experimental group was compared to determine the anti-tumor effect.

[0090] The results are as follows Figure 10 As shown: Figure 10 (A) is a schematic diagram of an in vivo anti-tumor experiment; Figure 10 Figures (B) and (C) show statistical and visual representations of tumor volume in each group after treatment. As can be seen, the BIP-NIR group exhibited the most significant tumor inhibition effect. The tumor volume in the control group (PBS-NIR group) was approximately 10 times that of the BIP-NIR group, demonstrating BIP's effective ability to inhibit tumor growth. Compared to the free drug groups (purpurin-NIR, ICG-NIR), BIP-NIR also showed enhanced anti-tumor effects. Compared to free ICG-NIR, the tumor growth inhibition rate of BIP-NIR increased by 32.6%, and compared to free purpurin-NIR, the tumor growth inhibition rate increased by approximately 47.6%.

[0091] In addition, compared with the BI-NIR group, although the BI-NIR group also had a certain therapeutic effect, the tumor volume in the BIP-NIR group was smaller after 11 days of treatment, and the effect was more obvious compared with the blank group (tumor growth inhibition rate of 87%). This indicates that the overall therapeutic effect of BIP-NIR is greater than that of the BI-NIR group. This is because, compared with BI-NIR, the BIP-NIR group is loaded with purpurin. The purpurin loaded in BIP, as a redox homeostasis regulator, can reduce the GSH content in tumor cells, thereby reducing the consumption of ROS generated during ICG photodynamic therapy and enhancing the ICG photodynamic effect. Compared with the BI-NIR group alone, it ultimately achieves a better anti-tumor effect than BI-NIR.

[0092] Figure 10(D) shows the tumor weight of each experimental group. The results show that the BIP-NIR group had the lowest tumor weight, which corresponds to the tumor size, further indicating that the BIP-NIR group had the most ideal treatment effect. Figure 10 (E) shows that the mice’s weight did not change significantly during the treatment, which indirectly reflects the safety of the treatment strategy.

[0093] Comparative Example 2

[0094] Referring to the application method in Example 4, the inhibitory effect of BIP on A431 (human skin squamous cell carcinoma cells), PATU-8988 (human pancreatic cancer cells), and MDA-MB-231 (human breast cancer cells) was investigated using the MTT assay (thiazolyl blue). The results are as follows: Figure 11 As shown, when the ICG concentration in BIP was 30 μg / mL and the purpurin concentration was 6.9 μg / mL, the survival rate of HeLa cells was the lowest (19.4%) under light conditions (808 nm, 1 W cm⁻², 4 min), and the therapeutic effect was the most obvious.

[0095] Example 5: Investigation into the dosage of albumin drug delivery system

[0096] The in vitro cytotoxicity of the drug-loaded albumin delivery system in Example 1 to HeLa cells was studied using the MTT (thiazolyl blue) method.

[0097] First, BIP nanoparticles were prepared according to Example 1, and BI and BP nanoparticles were prepared according to Comparative Example 1 for later use. Then, HeLa cells were cultured at 1×10⁻⁶. 4 Cells were seeded at a density of 1 / well into 96-well plates and incubated for 36 h (37 °C, 5% CO2).

[0098] Because BIP is synthesized as a single unit, to better represent concentration relationships and to investigate the inhibitory effect of the drug actually loaded in BIP on cells, the concentration relationship is expressed as the concentration of the loaded drug. The culture medium in the 96-well plate was removed using a pipette, and the albumin-loaded drug system dispersion diluted with culture medium (so that the ICG concentration in BIP was 0 μg / mL, 10 μg / mL, 20 μg / mL, 30 μg / mL, and 40 μg / mL, and the corresponding purpurin concentrations were 0 μg / mL, 2.3 μg / mL, 4.6 μg / mL, 6.9 μg / mL, and 9.2 μg / mL) was added to the wells, and incubation continued for 6 hours. After incubation, the drug-loaded drug system dispersion was removed, fresh culture medium was added, and the plate was irradiated with an 808 nm laser at a power intensity of 1 W / cm² for 4 minutes. -2After illumination, cells were cultured for 24 hours, then the culture medium was removed, and 100 μL of fresh culture medium and 10 μL of MTT solution (5 mg / mL) were added. Incubation continued for 4 hours. Subsequently, 100 μL of MTT buffer (triple dissolution solution) was added, and the plate was read overnight. The optical density (OD) value at 570 nm for each well was recorded using a microplate reader. Each experiment was repeated three times. Cell viability was calculated using the following formula: Cellviability = OD 实验 / OD 对照 .

[0099] like Figure 12 (A) shows that high doses of BSA do not inhibit HeLa cells. The survival rate of HeLa cells is above 90% in the range of BSA concentration from 0 to 800 μg / mL, indicating that BSA has good safety as a carrier.

[0100] like Figure 12 (B) The inhibitory effect of different concentrations of BIP on HeLa cells was investigated, and the inhibition rate of the albumin-loaded drug system at different concentrations was detected by the MTT assay. It was observed that when there was no light and the concentration of ICG in the drug-loaded system was 30 μg / mL, the survival rate of HeLa cells could still reach 80%, indicating that BIP has good safety at this concentration.

[0101] Therefore, an ICG concentration below 30 μg / mL is considered reasonable for use in albumin-loaded drug delivery systems. Taking into account the drug loading capacity in subsequent experiments, an application experiment was conducted using an ICG concentration of 30 μg / mL in the drug delivery system as an example. At an ICG concentration of 30 μg / mL, the cell inhibition rate of BIP after light irradiation was 19.4%. Furthermore, under 808 nm laser irradiation, the BIP drug delivery system, simultaneously loaded with ICG and purpurin, exhibited superior cell inhibition compared to BP or BI loaded with only one drug, and this cell inhibition effect was concentration-dependent.

[0102] Example 6: Blood compatibility study of albumin drug delivery system

[0103] The blood compatibility of nanoparticles was studied by co-incubating erythrocytes from nude mouse blood with free drugs and BIP. Whole mouse blood was placed in anticoagulant tubes to prevent clotting. Erythrocytes were obtained by centrifugation. Two hemolysis experimental groups were set up (free ICG group and BIP group). The positive control group was PBS containing Triton X-100 (10%), and the blank control was PBS. Then, 800 μL of different concentrations of drugs (containing the same ICG concentration of 0, 10, 20, 30, and 40 μg / mL) were added to each tube. 200 μL of freshly prepared PBS suspension containing 2% erythrocytes was then added to the EP tube. Finally, each sample was incubated in a constant temperature incubator at 37℃ for 3 hours. After incubation, the sample was centrifuged (3000 rpm, 5 min), and 200 μL of the supernatant was measured at 541 nm using a microplate reader. The hemolysis rate of the sample was determined using the following formula. Figure 9 As shown, BIP exhibits good biocompatibility compared to free drugs.

[0104]

[0105] Figure 16 A and B are the results of the blood compatibility test for the free drug. Figure 16 A shows that as the concentration of free ICG increases, the hemolysis in the fluid layer becomes more pronounced. Figure 16 B represents the change in hemolysis rate corresponding to the free drug. The results show that the hemolysis rate increases with increasing drug concentration, indicating that the higher the concentration of the free drug, the worse the safety. When the free ICG concentration is 30 μg / mL (at which point the purpurin concentration is 6.9 μg / mL), the hemolysis rate is 48.33%. Figure 16 The blood compatibility test results of CD BIP showed that when the ICG concentration in the BIP was 30 μg / mL (purpurin concentration was 6.9 μg / mL), the hemolysis rate was only 4.8%. Comparatively, at this concentration, BSA improved the safety of ICG and purpurin by approximately 10 times. When the ICG concentration in the BIP was 40 μg / mL, the hemolysis rate increased from 4.8% to 6.3%. Excessively high hazard ratios (HR) are not conducive to treatment (generally below 5%). These findings indicate that the BIP drug delivery system has good blood compatibility, and that an ICG concentration of 30 μg / mL in the particles can meet both safety requirements and achieve good therapeutic effects.

[0106] The embodiments provided above are not intended to limit the scope of the invention, nor are the described steps intended to limit the order of execution. Any obvious modifications made to the invention by those skilled in the art based on existing common knowledge also fall within the scope of protection defined by the claims.

Claims

1. The application of albumin-loaded drug-eluting nanoparticles for dual-light therapy in the preparation of drugs for treating cervical cancer, characterized in that, The albumin-loaded drug-eluting nanoparticles for dual-phototherapy are formed by loading indocyanine green and hydroxyalizarin onto albumin. The albumin is bovine serum albumin; The albumin-loaded drug nanoparticles contained an indocyanine green concentration of 30 μg / mL and a hydroxyalizarin concentration of 6.9 μg / mL.

2. The application according to claim 1, characterized in that, The preparation of the albumin-loaded nanoparticles for dual-light therapy specifically includes the following steps: (1) Preparation of solution A: Dissolve indocyanine green (ICG) and hydroxyalizarin purpurin in dimethyl sulfoxide, stir evenly in the dark to obtain solution A; (2) Preparation of solution B: Dissolve albumin in ultrapure water and stir until completely dissolved to obtain solution B; (3) Add solution B slowly dropwise to solution A and stir at room temperature in the dark. After the reaction is complete, stir the reaction solution at 2~6℃ overnight, purify with a 100KD ultrafiltration tube, wash with ultrapure water, repeat ultrafiltration to obtain albumin-loaded nanoparticles.

3. The application according to claim 2, characterized in that, Step (1) The molar ratio of indocyanine green (ICG) to hydroxyalizarin purpurin is (0.5~2):

1.

4. The application according to claim 2, characterized in that, The concentration of albumin in solution B in step (2) is 2~5 mg / mL.

5. The application according to claim 2, characterized in that, In step (3), the volume ratio of liquid A to liquid B is 1:

5.

6. The application according to claim 2, characterized in that, When the molar ratio of indocyanine green to hydroxyalizarin is 1:1, the concentration of indocyanine green in solution A is 6 mg / mL and the concentration of hydroxyalizarin is 2 mg / mL; the concentration of albumin in solution B is 4 mg / mL; and the mixing volume ratio of solution A to solution B is 1:5.

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