WO2022156419A1 - Preparation of ce6-loaded lipid-coated calcium carbonate carrier, preparation method therefor and application thereof - Google Patents

Abstract

The present invention relates to the technical field of pharmaceutical preparations, and in particular to a preparation of a Ce6-loaded lipid-coated calcium carbonate carrier, a preparation method therefor and an application thereof. Li/CC-Ce6 provided in the present invention has good stability, biocompatibility and tumor targeting, and can efficiently generate ROS under laser stimulation. ROS can trigger apoptosis to expose a TAA, and DCs are hopefully recruited into tumor tissues by means of an inflammation simulation mechanism. The DCs are activated in situ to release a corresponding inflammation (comprising IL-6 and TNF-α) and trigger a subsequent immune response cascade. In vitro and in vivo experiments both show that a platform has a strong capability of enhancing the vaccination of DCs, and can effectively inhibit the growth of primary and distantly growing tumors, so that a new method for effectively treating cancer may be provided.

Description

Lipid-coated calcium carbonate carrier-loaded Ce6 preparation and preparation method and application thereof

This application claims the priority of the Chinese patent application filed on January 25, 2021, with the application number of 202110097572.6 and the invention titled "Preparation of Ce6-loaded lipid-coated calcium carbonate carrier and its preparation method and application", The entire contents of which are incorporated herein by reference.

technical field

The invention relates to the technical field of pharmaceutical preparations, in particular to a preparation of lipid-coated calcium carbonate carrier-loaded Ce6 and a preparation method and application thereof.

Background technique

Immunotherapy for cancer is a novel class of therapies that includes cancer vaccines, immune checkpoint therapy, and other T-cell therapies. In particular, compared with other methods, cancer vaccines with irreplaceable advantages (such as simple preparation and low cost) are widely regarded as potential cancer treatments. Recent studies have gradually revealed the critical function of dendritic cells (DCs) in activating T cells and triggering immune responses, which are essential for immunotherapy. Multiple previous studies have focused on finding vaccines for cancer immunotherapy. However, currently available cancer vaccines often require complex processing of blood products to provide antigens/adjuvants to meet subject vaccination requirements. Therefore, an intelligent artificial vaccine that minimizes the processing procedures required to provide a robust immune response and excellent anti-cancer effects is very difficult, but also very promising.

In recent years, the use of nanotechnology has provided an efficient method to prepare DC-based cancer vaccines. In particular, calcium carbonate (CC) nanoparticles are one of the most widely used materials, which show multidimensional advantages, including high feasibility, easy fabrication, and good drug loading. Therefore, CC is recognized as a suitable carrier for drug and gene delivery, and many drug delivery systems (DDSs) have been successfully developed using CC as a backbone with satisfactory results. Furthermore, tumor-targeted drug delivery is becoming increasingly assessable in combination with tumor-associated enhanced permeability and retention (EPR) with positive targeting modifications. Nonetheless, repeated administration of high-dose DDSs was frequently required in previous reports to obtain acceptable therapeutic effects. Therefore, there is an urgent need for novel drug carriers capable of providing immune response efficacy. Previous studies have shown that photodynamic therapy (PDT) has great potential to destroy the integrity of tumor tissue, during which reactive oxygen species (ROS) can also effectively kill cancer cells to induce significant apoptosis. And exposed tumor-associated antigen (tumor-associated antigen, TAA). In addition, it has also been reported that natural inflammatory responses are also characterized by high levels of ROS. The results showed that ROS generated by artificial PDT was able to mimic the human inflammatory response to recruit immune-related cells including DCs. Unconsolidated tumor tissue after PDT favors the distribution and infiltration of dendritic cells (DCs) for immunization. Leaked TAA can activate DCs for in situ immunization, which reduces the dose and duration of effective immunotherapy more effectively than conventional routes of administration. Therefore, the preparation of lipid-coated calcium carbonate carrier-loaded photodynamic therapy drug preparations may further improve the efficacy of tumor immunotherapy, reduce the dosage and shorten the treatment period.

SUMMARY OF THE INVENTION

In view of this, the technical problem to be solved by the present invention is to provide a lipid-coated calcium carbonate carrier-loaded Ce6 preparation (Li/CC-Ce6) and its preparation method and application, which can be used as a potential for effective colon cancer immunotherapy. In situ vaccine.

The invention provides a preparation method of Li/CC-Ce6, which comprises:

Mix CTAB, n-butanol and n-hexane to prepare n-hexane emulsion;

The calcium chloride aqueous solution, the Ce6 aqueous solution and the n-hexane emulsion are mixed to make inverse microemulsion A;

The sodium carbonate aqueous solution, the DOPA aqueous solution and the n-hexane emulsion are mixed to make inverse microemulsion B;

After mixing the inverse microemulsion A and the inverse microemulsion B, and ethanol precipitation to obtain DOPA-modified CC-Ce6;

Add the DOPA-modified CC-Ce6, DOPC, FA-DSPE-PEG and cholesterol to the organic solvent, remove the solvent and disperse with PBS to obtain a solution containing Li/CC-Ce6;

The organic solvent is a mixture of ethanol and chloroform.

After the solution containing Li/CC-Ce6 is centrifuged, washed with straight-chain alcohol, and dried to obtain a white powder product, which is Li/CC-Ce6. Specifically, the straight chain alcohol is n-butanol.

In some embodiments, in the n-hexane emulsion, the mass ratio of CTAB, n-butanol and n-hexane is (4-5):(8-10):(18-36).

In some specific embodiments, in the n-hexane emulsion, the mass ratio of CTAB, n-butanol and n-hexane is 3:1:25.

In some specific embodiments, the ratio of the calcium chloride aqueous solution, the Ce6 aqueous solution and the n-hexane emulsion is (2.5mL-12.5mL): (2.5mL-12.5mL): (90g-110g);

In some embodiments, the ratio of the calcium chloride aqueous solution, the Ce6 aqueous solution and the n-hexane emulsion is (2.5mL-12.5mL): (2.5mL-12.5mL): (108g-109g);

In a specific embodiment, the ratio of the calcium chloride aqueous solution, the Ce6 aqueous solution and the n-hexane emulsion is 12.5mL: 12.5mL: 108.5g;

The invention has carried out experimental verification on the dosage of calcium chloride aqueous solution of 2.5mL, 7.5mL and 12.5mL, and carried out experimental verification on the dosage of DOPA solution of 2.5mL, 7.5mL and 12.5mL. The results show that the prepared nanometer Neither the particle size nor the encapsulation efficiency of the particles is as good as the effect when the ratio of the calcium chloride aqueous solution, the Ce6 aqueous solution and the n-hexane emulsion is 12.5 mL: 12.5 mL: 108.5 g.

In some embodiments, the ratio of the sodium carbonate aqueous solution, the DOPA aqueous solution and the n-hexane emulsion is (2.5 mL-12.5 mL): (2.5 mL-12.5 mL): (90 g-110 g).

In some specific embodiments, the ratio of the sodium carbonate aqueous solution, the DOPA aqueous solution and the n-hexane emulsion is (2.5 mL-12.5 mL): (2.5 mL-12.5 mL): (108 g-109 g).

In a specific embodiment, the ratio of the sodium carbonate aqueous solution, the DOPA aqueous solution and the n-hexane emulsion is 12.5 mL: 12.5 mL: 108.5 g.

The invention has carried out experimental verification on the dosage of sodium carbonate aqueous solution of 2.5mL, 7.5mL and 12.5mL, and carried out experimental verification on the dosage of DOPA solution of 2.5mL, 7.5mL and 12.5mL. The results show that the prepared nanoparticles Neither the particle size nor the encapsulation efficiency of the sodium carbonate solution is as good as the effect when the ratio of sodium carbonate aqueous solution, DOPA solution and the n-hexane emulsion is 12.5mL:12.5mL:108.5g.

In the calcium chloride aqueous solution, the concentration of calcium chloride is 2mol/L;

In the Ce6 aqueous solution, the concentration of Ce6 is 2mol/L;

In the sodium carbonate aqueous solution, the concentration of sodium carbonate is 2mol/L;

In the DOPA aqueous solution, the concentration of DOPA is 2 mol/L.

The preparation of the inverse microemulsion A includes mixing the calcium chloride aqueous solution, the Ce6 aqueous solution and the n-hexane emulsion, and then aging for 1 hour to 24 hours;

The preparation of the inverse microemulsion B includes mixing the sodium carbonate aqueous solution, the DOPA aqueous solution and the n-hexane emulsion, and then aging for 1 h to 24 h.

The volume ratio of the inverse microemulsion A and the inverse microemulsion B is 1:1; the conditions for mixing the inverse microemulsion A and the inverse microemulsion B include stirring at room temperature for 20 minutes.

In the ethanol precipitation step, the volume of absolute ethanol is 1/2 of the sum of the volumes of inverse microemulsion A and inverse microemulsion B.

The added amount of CC-Ce6, DOPC, FA-DSPE-PEG and cholesterol will affect the particle size of the obtained nanoparticles. It has been repeatedly verified in the present invention that under the conditions described in the examples, the obtained particle size is most suitable for entering the organism as a drug for treating tumors. The mass ratio of the DOPA-modified CC-Ce6, DOPC, FA-DSPE-PEG and cholesterol was 9.79:7.86:50.01:3.87.

Li/CC-Ce6 prepared by the preparation method of the present invention.

The application of Li/CC-Ce6 prepared by the preparation method of the present invention in preparing a medicine for treating colorectal cancer.

The present invention provides a Li-coated CC nanoparticle (Li/CC) as a carrier for loading Ce6 (Li/CC-Ce6), and ultimately as a potential in situ vaccine for effective colorectal cancer immunotherapy. It has been verified many times that compared with other preparation steps or parameters, the Li/CC-Ce6 prepared under the parameters provided by the present invention has higher stability, biocompatibility and tumor targeting, and can be effectively produced under laser stimulation. ROS. ROS can trigger apoptosis to expose TAA and are expected to recruit DCs into tumor tissues through an inflammation-mimicking mechanism. DCs are activated in situ to release corresponding inflammation (including IL-6 and TNF-α) and trigger subsequent immune response cascades. Both in vitro and in vivo experiments demonstrate the powerful ability of this platform to enhance DC vaccination, which can effectively inhibit the growth of primary and distantly growing tumors, which may be a new approach to effectively treat cancer.

Description of drawings

Figure 1 shows size distribution and water stability, (A) size distribution of Li/CC-Ce6, (B) Li/CC-Ce6 in PBS (pH 7.4) and mouse plasma at 37°C for up to 48 hours Colloidal stability, data are shown as mean ± standard deviation (n=3);

Figure 2 shows the biocompatibility of Li/CC-Ce6, (A) hemolysis of 2% RCB incubated with various concentrations of Li/CC-Ce6, (B) Li/CC-Ce6 to various concentrations of BSA of protein adsorption, data are shown as mean ± standard deviation (n=3);

Figure 3 shows the ROS generation capacity of Li/CC-Ce6, (A) the absorbance changes of DPBF at 418 nm after Li/CC-Ce6 (Ce6 concentration of 0.1 mg/mL) irradiation (1W/cm ² ) at different time intervals, (B) Absorbance change of DPBF in the presence of different concentrations of Li/CC-Ce6 (Ce6 concentration: 0.1-0.5 mg/mL) under 60s irradiation, data are shown as mean ± SD (n=3);

Figure 4 shows in vitro ROS generation analysis of Li/CC-Ce6, (A) representative CLSM images of cells treated with different formulations with/without laser irradiation, scale bar: 20 μm, (B) main image shows the Flow cytometric analysis of MFI in cells treated with different formulations after 5 minutes of irradiation (1 W/cm ² ), data are shown as mean ± standard deviation (n=3), *P<0.05;

Figure 5. In vitro and in vivo targeting of Li/CC-Ce6, (A) Quantitative analysis of intracellular time-dependent uptake of different formulations in CT-26 cells (pretreated with FA/no FA), (B) Mean fluorescence intensity of dissected tumors and major organs of mice treated with CC-Ce6 and Li/CC-Ce6 at 48 hours after injection, data are presented as mean ± SD (n=3), *P<0.05;

Figure 6 shows the cytotoxicity of Li/CC-Ce6 assessed by MTT assay, (A) cytotoxicity of Li/CC-Ce6 (without laser irradiation) after 48 hours of incubation with CT-26 cells, (B) after 48 hours of incubation Cytotoxicity of free Ce6, CC-Ce6 and Li/CC-Ce6 (laser stimulated) with different Ce6 concentrations on CT-26 cells after 1 hour, data are shown as mean ± SD (n=3);

Figure 7 shows the cytotoxicity of Li/CC-Ce6 assessed by MCTS, the volume change of MCTS after different treatments (A) and the corresponding optical images (B), data are shown as mean ± SD (n=3) , *P＜0.05;

Figure 8 shows the DC simulation ability of Li/CC-Ce6, the levels of IL-6 (A) and TNF-α (B) in the serum of mice isolated on days 0, 2 and 4 after different treatments, data are shown as mean Value±standard deviation (n=3), *P<0.05;

Figure 9 shows the in vivo anticancer efficacy of different nanoformulations in CT-26 tumor-bearing C57BL/6 mice, (A) PDT-based Li/CC-Ce6 inhibits CT-26 tumors by dead tumor cells and orthotopic DC vaccination Schematic representation of TAA dissemination for growth and progeny effects, recording tumor volume over time for (B) primary and (C) distant tumors, *P<0.05.

Detailed ways

The invention provides a preparation of lipid-coated calcium carbonate carrier-loaded Ce6, a preparation method and application thereof, and those skilled in the art can learn from the content of this article and appropriately improve process parameters to achieve. It should be particularly pointed out that all similar substitutions and modifications are obvious to those skilled in the art, and they are deemed to be included in the present invention. The method and application of the present invention have been described through the preferred embodiments, and it is obvious that relevant persons can make changes or appropriate changes and combinations of the methods and applications herein without departing from the content, spirit and scope of the present invention, so as to realize and apply the present invention. Invention technology.

The test materials used in the present invention are all common commercial products and can be purchased in the market.

Among them, calcium chloride (calcium chloride), sodium carbonate (sodium carbonate), 1,3-diphenylisobenzofuran (1,3-diphenylisobenzofuran, DPBF), methylthiazolyl tetrazolium (MTT), 2 ',7'-Dichlorodihydrofluorescein diacetate (DCFH-DA), chlorin e6 (Ce6), folic acid (FA), TritonX-100, paraformaldehyde and paraformaldehyde cholesterol, by Sigma -Courtesy of Aldrich (St. Louis, MO, USA). The inner leaflet lipid (the inner leaflet, DOPA), dioleoylphosphatidylcholine (dioleoylphosphatidylcholine, DOPC), folic acid-modified distearoylphosphatidylethanolamine-polyethylene glycol (The folate modified distearoylphosphatidyl ethanolamine-polyethylene glycol, FA-DSPE-PEG), purchased from Ponsure Biotechnology (Shanghai, China).

CT-26 (mouse colorectal cancer cell line) and NIH3T3 (mouse embryonic fibroblast) cell lines were obtained from Shang Model Cell Center (Shanghai, China). All cell lines were cultured using the protocol in the previous report. Multicellular tumor spheroids (MCTS) were established based on previous studies. Sixty male C57BL/6J mice (6-8 weeks old) were purchased from Wuhan Institute of Model Animals (Wuhan, China) and housed under standard conditions. The establishment of the CT26 tumor xenograft model followed a previously reported protocol. All animal procedures used were approved by the IEC (Institutional Ethics Committee) of Jeonbuk National University.

Below in conjunction with embodiment, the present invention is further elaborated:

Example Preparation of Li/CC-Ce6 Nanoparticles

_CaCl2 and Ce6 were dispersed in a water-in-oil microemulsion. _The same microemulsion containing _Na2CO3 and DOPA was also prepared. The two solutions were mixed for 20 minutes to prepare hydrophobic DOPA-modified CC-Ce6, and the product was precipitated with absolute ethanol and centrifuged (CR22, Hitachi, Japan). After thorough washing with ethanol, DOPA-modified CC-Ce6 was mixed with appropriate amount of DOPC, FA-DSPE-PEG and cholesterol in a mixed solution of chloroform and ethanol (1:1, v/v). After evaporating the solvent by steam, the formed film was dispersed in medium PBS to obtain Li/CC-Ce6 solution. Specific steps include:

1) Preparation of calcium chloride solution: weigh 55.49 grams of calcium chloride, add distilled water to dissolve, and configure it into a 2mol/L calcium chloride solution in a 250ml volumetric flask;

2) Preparation of chlorin e6 solution: weigh 156.18 grams of chlorin e6, add distilled water to dissolve, and configure into a 2mol/L chlorin e6 solution in a 250ml volumetric flask;

3) preparation of sodium carbonate aqueous solution: weigh 53 grams of sodium carbonate, add distilled water to dissolve, and configure into a 2mol/L sodium carbonate solution in a 250ml volumetric flask;

4) Preparation of DOPA solution: Weigh 98.59 grams of DOPA, add distilled water to dissolve, and prepare a 2 mol/L DOPA solution in a 250ml volumetric flask;

5) Preparation of oil emulsion: Weigh 20.46g CTAB (cetyltrimethylammonium bromide, also known as cetyltrimethylammonium bromide, hexadecyltrimethylammonium bromide, cetyltrimethylammonium bromide,), 40.68g n-butanol, 180.00g n-hexane was added to the conical flask in turn, heated to 50°C, magnetically stirred for 15min, 3000rpm, and prepared into n-hexane emulsion;

6) Preparation of calcium-containing salt and Ce6 inverse microemulsion: take 12.5ml of calcium chloride aqueous solution obtained in step 1), and 12.5ml of Ce6 solution obtained in step 2) and add it to 108.5g of n-hexane emulsion obtained in step 5), After fully stirring to make it react completely, it is aged for 1 to 24 hours; it is formulated into calcium chloride and Ce6 inverse microemulsion;

7) Preparation of sodium salt and DOPA inverse microemulsion: take 12.5 ml of the sodium carbonate aqueous solution obtained in step 3), and 12.5 ml of DOPA solution obtained in step 4) into the 108.5 g n-hexane emulsion obtained in step 5), fully After stirring to make it react completely, it is aged for 1 to 24 hours; it is formulated into sodium carbonate and DOPA inverse microemulsion;

8) The two solutions of 6) and 7) were mixed for 20 minutes to prepare the hydrophobic DOPA-modified CC-Ce6. The product was precipitated with absolute ethanol and centrifuged. Centrifugation speed 3000rpm, 40min.

9) After thorough washing with ethanol, the DOPA-modified CC-Ce6 was mixed with an appropriate amount of DOPC, FA-DSPE-PEG and cholesterol in a mixed solution of chloroform and ethanol (1:1, v/v). After evaporating the solvent by steam, the formed film was dispersed in medium PBS to obtain Li/CC-Ce6 solution.

10) Select nanoparticles with a particle size of 60-120 nm and an encapsulation efficiency of 82.13% into the experimental group.

Preparation of Comparative Example CC-Ce6 Nanoparticles

CC-Ce6 alone was used as a control and prepared using a method similar to Example 1, but without the addition of DOPA and lipids. The encapsulation efficiency of Ce6 in the formulation was determined to be 8.1%.

1) Preparation of calcium chloride solution: same as Example 1.

2) Preparation of chlorin e6 solution: same as Example 1.

3) Preparation of sodium carbonate aqueous solution: the same as in Example 1.

4) Preparation of oil emulsion: same as Example 1.

5) Preparation of calcium salt and Ce6 inverse microemulsion: take 2.5mL (3.05g), 7.5ml, 12.5ml of calcium chloride aqueous solution obtained in step 1), and 2.5ml (4.06g) of Ce6 solution obtained in step 2) , 7.5ml and 12.5ml were added to the 108.5g n-hexane emulsion obtained in step 4), fully stirred to make it fully react, and then aged for 1 to 24 hours; formulated into calcium chloride, Ce6 inverse microemulsion;

6) Preparation of sodium salt-containing inverse microemulsion: take 12.5 ml of the sodium carbonate aqueous solution obtained in step 3) and 108.5 g of n-hexane emulsion obtained in step 4) to prepare a sodium carbonate inverse microemulsion;

7) The two solutions of 5) and 6) were mixed for 20 minutes to prepare CC-Ce6. The product was precipitated with absolute ethanol and centrifuged. Centrifugation speed 3000rpm, 40min. After thorough washing with ethanol, CC-Ce6 was mixed with an appropriate amount of DOPC, FA-DSPE-PEG and cholesterol in a mixed solution of chloroform and ethanol (1:1, v/v). After evaporating the solvent by steam, the formed film was dispersed in medium PBS to obtain a CC-Ce6 solution.

8) Select nanoparticles with a particle size of 60-120 nm to enter the experimental group.

Properties and efficacy verification

1. Characterization of physical and chemical properties

The size of Li/CC-Ce6 was measured using ZS90 (Malvern, UK) and its morphology was observed by transmission electron microscopy (TEM, JEM-2100, JEOL, Japan). The size change of Li/CC-Ce6 in PBS/mouse plasma was observed for 48 hours to assess colloidal stability. Hemolysis assays were performed according to previous reports. The adsorption capacity of BSA was determined as reported previously. Western blot analysis was performed using standard protocols with the corresponding antibodies from Abcam (UK). IL-6 and TNF-α levels in serum were determined by ELISA kits (Abeam) according to the manufacturer's instructions.

2. In vitro release and ROS production

The drug release profiles of Li/CC-Ce6 were evaluated as previously reported. To generate ROS, 20 μL of LDPF (10 mM) was incubated with Li/CC-Ce6 aqueous solution. The solution was then irradiated with laser light (680 nm, 1 W/cm ² ), and the UV absorption peak of DPBF at 418 nm was monitored to reflect the production of ROS. The UV absorption of the solution at 418 nm was normalized with the untreated solution.

3. Anticancer detection in vivo

Li/CC-Ce6 (5-100 μg/mL) or Li/CC-Ce6 (Ce6 concentration, 0.25-5 μg/mL, 680 nm, 400 mW/cm for ¹⁰ min) were incubated with CT26 cells without laser irradiation. Forty-eight hours after incubation, standard MTT analysis was performed as previously reported. Furthermore, MCTS were incubated with different formulations at a Ce6 concentration of 5 μg/mL for 24 h before laser irradiation. The MCTS volume was monitored for 5 days.

4. Intracellular Ce6 accumulation and ROS production

CT26 cells were pretreated with/without FA (1 mM) for 2 h and then incubated with free Ce6, CC-Ce6 and Li/CC-Ce6. At predetermined time points, cells were harvested and assessed for intracellular accumulation of Ce6 by flow cytometry (FCM, DxFLEX, Beckman Coulter, Miami, USA).

Intracellular ROS levels were measured with a fluorescent probe (DCFH-DA). Briefly, cells (1×10 ⁶ ) were treated differently (Ce6 concentration: 5 μg/mL) for 4 h and then loaded with DCFH-DA (25 mM, 30 min). Afterwards, cells were treated with/without laser irradiation (1 W/cm ² , 5 min), and intracellular ROS levels were controlled by a confocal laser scanner (CLSM, FV3000, Olympus, Tokyo, Japan).

5. In vivo tumor targeting

CT26 tumor-bearing mice with only primary tumors were injected intravenously with CC-Ce6 and Li/CC-Ce6 (5 mg/kg Ce6). 48 hours after distribution, mice were sacrificed for major organs and tumors. The levels of Ce6 in these tissues were assessed by in vivo imaging (In-Vivo Xtreme, Bruker, German).

6. Anticancer research in vivo

24 mice were randomly divided into 4 groups (n=6) and injected intravenously with normal saline (as control), free Ce6, CC-Ce6 and Li/CC-Ce6 (5 mg/kg Ce6). Primary tumors were irradiated with a 680 nm laser at 100 mW/cm2 for 20 minutes. Dosing was performed twice every two days at a Ce6 concentration of 5 mg/kg, while irradiation was performed 24 hours after administration. Tumor volumes were measured in all animals for 15 days every 3 days.

Results and discussion

The preparation of Li/CC-Ce6 involves two steps. First, the CC core is formed in a size-controlled microemulsion, during which Ce6 is loaded into the CC matrix through the complexation between the Ca2 ⁺ of CC and the carboxyl group of Ce6. To facilitate the surface modification of Li, CC-Ce6 was surface-modified using DOPA to generate a hydrophobic layer to further immobilize Li. Combining these two components (Li and CC-Ce6) into a system finally constructs Li/CC-Ce6. As shown in Figure 1A, the obtained Li/CC-Ce6 are nanoparticles with a narrow size distribution of 60-120 nm and the highest peak at 100 nm. Previous reports reported that nanoparticles with a size of about 100 nm were best suited to exploit tumor-based EPR effects relative to other size ranges for targeted drug delivery.

To test the long-term stability of Li/CC-Ce6, we cultured Li/CC-Ce6 in PBS and plasma for 48 h and monitored the change in particle size during this period to determine its stability. As shown in Fig. 1B, the diameter of Li/CC-Ce6 exhibits only small fluctuations (less than 10%) throughout the process, which indicates that the stability of Li/CC-Ce6 can be well obtained under physiological conditions save. Applicable to be a promising DDS in cancer treatment.

To further verify the safety of Li/CC-Ce6, we also performed a hemolysis test. As shown in Figure 2A, a positive correlation between the hemolysis rate of Li/CC-Ce6 and DDS concentration was observed after incubation in 2% RCB for 1 h. However, hemolysis was less than 1% at all concentrations tested. Considering that the plasma concentrations of nanoparticles in practical applications will be much lower than those used in experiments, we speculate that there is no risk of hemolysis for Li/CC-Ce6. In addition, we also performed a comparative study on protein adsorption properties between CC-Ce6 (without Li modification) and Li/CC-Ce6. Previous studies have shown that excess calcium ions on the CC surface will positively charge the surface of the nanoparticles, and the positively charged nanoparticles preferentially react with the large amount of negatively charged proteins in the blood to form larger aggregates. Not only did these aggregates alter the behavior of the nanoparticles in the body, but they also increased the risk of embolism, which could lead to death. Therefore, the protein adsorption properties of nanoparticles have an important impact on their safety. The results are shown in Fig. 2B, in the CC-Ce6 group without Li protection, the adsorption of the model protein (BSA) increased significantly with the concentration and was more severe than that in the Li/CC-Ce6 group, which was consistent . with previous reports. In contrast, Li/CC-Ce6 hardly adsorbs onto BSA, while the change in OD278 is small. These results indicate that Li/CC-Ce6 can well protect the loaded drugs from the external environment, thus exerting good biosafety, which is a promising DDS.

Although Li/CC-Ce6 can provide satisfactory protection for loaded Ce6, whether the packaged Ce6 can generate ROS in response to laser stimulation remains to be explored. Therefore, we used DPBF as a detector of ROS in our experiments to evaluate the ability of Li/CC-Ce6 to generate ROS under laser stimulation. According to previous reports, DPBF can react with ROS to quench its UV absorption peak at 418 nm, and the degree of quenching is positively correlated with the ROS concentration in the environment. This is a convenient method to determine the ROS-producing capacity of a formulation. As shown in Figure 3A, the ROS production of Li/CC-Ce6 (Ce6 concentration: 0.1 mg/mL) was significantly increased under laser stimulation, which was reflected as a sharp quenching (89%) of the DPBF absorption peak at 418 nm. under 3 minutes of stimulation). In addition, the results in Fig. 3B also show that the ROS generation of Li/CC-Ce6 by ROS is positively correlated with the encapsulated Ce6 concentration. These results suggest that Ce6 in Li/CC-Ce6 retains the ability to generate ROS, which is beneficial for cancer therapy utilizing PDT.

DCFH-DA can easily penetrate into the cell interior and is not fluorescent. DCF is produced after intracellular lipase degradation. After ROS oxidation, the fluorescence of the probe was greatly increased compared with the unreacted probe, which provided the possibility to evaluate the intracellular ROS generation profile of Li/CC-Ce6. As shown in Figure 4A, in the absence of stimulation, the fluorescence generated in the free Ce6 group was weak. The increase in fluorescence upon exposure to light indicated that laser stimulation was an important factor in the generation of ROS. In contrast, the intracellular fluorescence of the Li/CC-Ce6 group was higher than that of the free Ce6 group without laser irradiation, indicating that more Ce6 was absorbed into the cells with the help of DDS. This is consistent with previous reports that DDS can better deliver drugs to the interior of cells via endocytosis. Similarly, after laser irradiation, the fluorescence intensity of Li/CC-Ce6-treated cells was much stronger than the other groups, indicating the best ROS generation capacity. Using flow cytometry in Figure 4B to further quantify the results, it was found that the ROS production of the Li/CC-Ce6 group was 3.86 times higher than that of the free Ce6 group after laser irradiation, indicating the effective delivery efficacy of Li/CC-Ce6 for cancer-related PDT.

Tumor homing of different nanoparticles in CT-26 cells was explored. As shown in Figure 5A, the intracellular Ce6 signal was positively correlated with incubation time in all groups. Free Ce6 showed only weak cell retention compared to both preparations. DDS has been shown to play a beneficial role in increasing drug entry into cells in experiments with intracellular ROS, which is consistent with our observations here. Furthermore, in the absence of FA pretreatment, the Li/CC-Ce6 group consistently showed higher intracellular Ce6 signal than CC-Ce6 at all tested time points. In addition, after folic acid pretreatment, the intracellular Ce6 signal decreased sharply in the Li/CC-Ce6 group, while the changes in the free Ce6 and CC-Ce6 groups were small. All results suggest that FA modification may increase the intrinsic entry of Li/CC-Ce6 into CT-26 cells through FA-mediated endocytosis.

To validate the in vivo tumor-homing ability of Li/CC-Ce6 on the same cell model, the distribution of Ce6 in tumors/major organs was assessed by in vitro imaging. As shown in Figure 5B, CC-Ce6 exhibited poor tumor accumulation while being mainly trapped in major organs, especially in the reticuloendothelial system (liver and spleen). In contrast, the Ce6 signal in the tumor tissues of the Li/CC-Ce6 group was much higher than that of the main organs, suggesting that the non-targeted distribution of CC-Ce6 was improved by surface modification of Li and FA.

Next, we investigated the in vitro anticancer efficacy of different formulations on CT-26 cells using the MTT method. First, non-laser-irradiated Li/CC-Ce6 was incubated with cells to study its biocompatibility. As shown in Figure 6A, cells treated with a vehicle concentration of 100 μg/mL still had high viability after 48 hours of incubation. These results suggest that Li/CC-Ce6 is highly biocompatible and suitable for use as a drug vehicle in drug delivery.

Thereafter, the anticancer effects of drug-loaded Li/CC-Ce6 were evaluated using free Ce6 and CC-Ce6 as negative controls. According to Figure 6B, it was observed that the PDT effect of all Ce6-containing formulations was positively correlated with Ce6 concentration under a given laser condition. Additionally, unlike DDS, free Ce6 showed lower anticancer effects, suggesting that DDS could have a positive role in drug delivery. Furthermore, the benefits of Li/CC-Ce6 are greatly enhanced at all Ce6 concentrations compared to CC-Ce6. In particular, at the highest drug dose, the cell viability after PDT was less than 10%, indicating a strong anticancer effect.

MCTS, composed of fibroblasts and tumor cells, are widely used to mimic solid tumors and evaluate the therapeutic effect of DDS. As shown in Figure 7A, the saline group without any treatment showed a sustained increase in the volume of MCTS, which was almost 3 times its original size at the end of the experiment and was representative of the development of solid tumors. However, the amount of MCTS was significantly reduced in all PDT groups. Furthermore, consistent with the MTT analysis, Li/CC-Ce6 exhibits the most robust anticancer properties, supported by the MCTS volume results, which are only 72% of the original size. The optical observations in Figure 7B also show similar conclusions. MCTS after PDT treatment were observed to have structural damage and apoptosis, and MCTS in the Li/CC-Ce6 group had the smallest volume and the most significant structural damage.

Results from previous studies have shown that PDT disrupts the structural integrity of tumors and mimics inflammatory processes in vivo. Thus, after PDT, ROS generated in tumor tissue triggers the recruitment of DCs, while TAA exposed in tumor tissue due to apoptosis provides in situ vaccination of recruited DCs, leading to a subsequent immune response. Additionally, the disrupted structural integrity of tumor tissue further facilitated the spread of DCs for adequate vaccination. Therefore, plasma concentrations of two representative cytokines (IL-6 and TNF-α) over time after different treatments were assessed using ELISA kits. Since IL-6 and TNF-α are two characteristic proteins secreted by DCs upon activation, the levels of these two proteins were positively correlated with the activation curves of DCs. As shown in Figure 8, among the three groups, the Li/CC-Ce6 group had the highest cytokine levels, suggesting that Li/CC-Ce6 could more effectively activate the in situ activation of DCs to provide strong immunity.

The anticancer effect of Li/CC-Ce6 as a DC vaccine was evaluated using mouse models with primary and distant tumors. The experimental protocol is shown in Figure 9A, and the results are summarized in Figures 9B and C. It was observed that the Li/CC-Ce6 group (206 mm ³ of the primary tumor) significantly inhibited the growth of the primary tumor and distant tumors. The treated distance was 81 mm ³ ). However, tumor volume in the CC-Ce6 group continued to grow, with final volumes of 432 mm ³ (primary) and 238 mm ³ (distant), respectively, indicating the importance of increased stability and tumor homing after Li and FA modification. Altogether, the results strongly suggest that in situ DC vaccination of Li/CC-Ce6 is important for the effective treatment of cancer.

The above are only the preferred embodiments of the present invention. It should be pointed out that for those skilled in the art, some improvements and modifications can be made without departing from the principles of the present invention, and these improvements and modifications should also be regarded as It is the protection scope of the present invention.

Claims (10)

The preparation method of Li/CC-Ce6, it comprises: Mix CTAB, n-butanol and n-hexane to prepare n-hexane emulsion; The calcium chloride aqueous solution, the Ce6 aqueous solution and the n-hexane emulsion are mixed to make inverse microemulsion A; The sodium carbonate aqueous solution, the DOPA aqueous solution and the n-hexane emulsion are mixed to make inverse microemulsion B; After mixing the inverse microemulsion A and the inverse microemulsion B, and ethanol precipitation to obtain DOPA-modified CC-Ce6; Add the DOPA-modified CC-Ce6, DOPC, FA-DSPE-PEG and cholesterol to the organic solvent, remove the solvent and disperse with PBS to obtain a solution containing Li/CC-Ce6; The organic solvent is a mixture of ethanol and chloroform. The preparation method according to claim 1, wherein, In the n-hexane emulsion, the mass ratio of CTAB, n-butanol and n-hexane is (4～5):(8～10):(18～36); The ratio of the calcium chloride aqueous solution, the Ce6 aqueous solution and the n-hexane emulsion is (2.5mL-12.5mL): (2.5mL-12.5mL): (90g-110g); The ratio of the sodium carbonate aqueous solution, the DOPA aqueous solution and the n-hexane emulsion is (2.5 mL-12.5 mL): (2.5 mL-12.5 mL): (90 g-110 g). The preparation method according to claim 1 or 2, characterized in that, In the calcium chloride aqueous solution, the concentration of calcium chloride is 2mol/L; In the Ce6 aqueous solution, the concentration of Ce6 is 2mol/L; In the sodium carbonate aqueous solution, the concentration of sodium carbonate is 2mol/L; In the DOPA aqueous solution, the concentration of DOPA is 2 mol/L. The preparation method according to claim 1, wherein, The preparation of the inverse microemulsion A includes mixing the calcium chloride aqueous solution, the Ce6 aqueous solution and the n-hexane emulsion, and then aging for 1 hour to 24 hours; The preparation of the inverse microemulsion B includes mixing the sodium carbonate aqueous solution, the DOPA aqueous solution and the n-hexane emulsion, and then aging for 1 h to 24 h. The preparation method according to claim 1 or 4, wherein the volume ratio of the inverse microemulsion A and the inverse microemulsion B is 1:1; the inverse microemulsion A and the inverse microemulsion B are The mixing conditions include stirring at room temperature for 20 min. The preparation method according to claim 1, wherein, in the ethanol precipitation step, the volume of absolute ethanol is 1/2 of the sum of the volumes of inverse microemulsion A and inverse microemulsion B. The preparation method according to claim 1, wherein the volume ratio of ethanol and chloroform in the organic solvent is 1:1. The preparation method according to claim 1, wherein the mass ratio of the DOPA-modified CC-Ce6, DOPC, FA-DSPE-PEG and cholesterol is 9.79:6.86:50.01:3.87. Li/CC-Ce6 obtained by the preparation method according to any one of claims 1 to 8. Application of the Li/CC-Ce6 prepared by the preparation method of any one of claims 1 to 8 in preparing a medicine for treating colorectal cancer.

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