Nano-engineered microalgae for photoimmunotherapy of melanoma and preparation method of nano-engineered microalgae

Abstract

The invention discloses nano-engineered microalgae for photoimmunotherapy of melanoma and a preparation method of the nano-engineered microalgae. The preparation method comprises the following steps: S1, respectively putting tannic acid, potassium carbonate, sodium citrate and HAuCl4. 3H2O into water to prepare a tannic acid solution with the concentration of 4.3 mg / mL, a potassium carbonate solution with the concentration of 20.7 mg / mL, a sodium citrate reduction solution with the concentration of 0.7 mg / mL and a HAuCl4 solution with the concentration of 10.0 mg / mL; s2, dissolving a tannic acid solution and a potassium carbonate solution in the sodium citrate reduction solution to obtain a mixed solution; s3, the mixed solution is heated under stirring, a HAuCl4 solution is dropwise added, stirring continues, and AuNP is obtained; and S4, mixing the AuNP obtained in the step S3 with the microalgae PCC 7942, standing, incubating, and centrifugally washing, so as to obtain the nano-engineered microalgae PCC-coated AuNP. The functionalized microalgae drug delivery system (PCC-coated AuNP) prepared by the invention has the performance of respectively generating O2 and H2 through photosynthesis and photocatalytic reaction under the irradiation of 635 nm red light.

Description

Technical Field

[0001] The present invention relates to nano-engineered microalgae for melanoma photoimmunotherapy and a preparation method thereof, belonging to the technical field of gas-immunosynergistic therapy of melanoma. Background Art

[0002] Surgery, chemotherapy, and radiotherapy are traditional treatments for melanoma, but they are often accompanied by severe trauma and toxic side effects, and fail to address the problem of tumor metastasis and recurrence, resulting in high patient mortality. To address these issues, minimally invasive or non-invasive treatment options are gradually being introduced to cancer treatment, aiming to produce tumor-specific toxic substances while protecting normal tissue. Among them, gas therapy has been widely studied in cancer treatment, with hydrogen (H2) in particular attracting considerable attention due to its safety, posing no risk of poisoning even at high concentrations.

[0003] Currently, hydrogen-induced ICD to initiate systemic anti-tumor immunotherapy faces the following problems: In clinical practice, the main methods of H2 delivery include direct inhalation of hydrogen, oral administration of hydrogen-rich water or hydrogen-producing capsules, and hydrogen baths. However, the low solubility (1.6 ppm), untargeted diffusion, and uncontrolled release of H2 in these administration routes limit their therapeutic effects. In addition, the rapid proliferation of tumor cells and abnormal tumor vascular structure lead to hypoxia in tumor tissues, which in turn causes immunosuppression through multiple mechanisms, especially by recruiting tumor-associated macrophages (TAMs), myeloid-derived suppressor cells (MDSCs), and regulatory T cells (Tregs). These mechanisms often lead to the failure of immunotherapy because they limit the infiltration of T cells and deplete CD8 + T cells. Microalgae are a type of microorganism that is widely distributed in nature. They have natural photosynthetic capabilities and can effectively produce oxygen under light. Their light-controlled growth and photosynthesis properties have attracted much attention in tumor immunotherapy.

[0004] In response to the above problems, this application is filed. Summary of the Invention

[0005] This study constructed a functionalized microalgae drug delivery system (PCC@AuNP) loaded with gold nanoparticles (AuNPs) to overcome the toxic side effects of conventional cancer chemotherapy. AuNPs were prepared by a reduction method and then modified onto the surface of the microalga PCC 7942 (PCC) to create PCC@AuNPs.

[0006] The first object of the present invention is to provide a method for preparing nano-engineered microalgae for melanoma photoimmunotherapy, comprising the following steps: S1: Tannic acid, potassium carbonate, sodium citrate and HAuCl4·3H2O were respectively placed in water to prepare tannic acid solution, potassium carbonate solution, sodium citrate reduction solution and HAuCl4 solution; S2: Dissolve 40 μL of tannic acid solution and 330 μL of potassium carbonate solution in 50 mL of sodium citrate reducing solution to obtain a mixed solution; S3: The mixture was heated to 70 °C while stirring, and 325 μL of HAuCl4 solution was quickly added dropwise. The mixture was stirred at 70 °C for 10 min to obtain AuNPs. S4: The AuNPs obtained in step S3 were prepared into an AuNP solution, mixed with microalgae PCC 7942 and incubated at 25°C for 3 h, and centrifuged and washed to remove unbound AuNPs to obtain nano-engineered microalgae PCC@AuNPs.

[0007] Preferably, the concentration of the tannic acid solution in step S1 is 4.3 mg / mL, the concentration of the potassium carbonate solution is 20.7 mg / mL, the concentration of the sodium citrate reducing solution is 0.7 mg / mL, and the concentration of the HAuCl4 solution is 10.0 mg / mL.

[0008] Preferably, the concentration of the AuNP solution in step S4 is 50 μg / mL.

[0009] Preferably, the density of microalgae PCC 7942 in step S4 is 3.0 × 10 8 cell / mL.

[0010] The second object of the present invention is to provide a nano-engineered microalgae loaded with gold nanoparticles prepared by the above method, wherein the PCC@AuNP produces O2 and H2 through photosynthesis and photocatalytic reaction respectively under 635 nm red light irradiation.

[0011] The third purpose of the present invention is to provide the application of the nano-engineered microalgae loaded with gold nanoparticles in the gas-immunotherapy of melanoma, using H2 to induce ICD of tumor cells to promote DCs maturation, and using O2 to reverse the immunosuppressive microenvironment, and the two synergistically promote CD8 + T infiltration to inhibit proximal and distal tumor growth.

[0012] Beneficial effects of the present invention: The functionalized microalgae drug delivery system (PCC@AuNP) prepared by the present invention can produce O2 and H2 through photosynthesis and photocatalytic reaction respectively under 635 nm red light irradiation. In vitro experiments have shown that H2 produced under light irradiation can specifically destroy the redox dynamic balance in B16F10 cells, leading to B16F10 cell apoptosis, causing strong ICD and accompanied by the release of DAMPs in the cells. In vivo results have verified that PCC@AuNP can alleviate tumor hypoxia through photosynthetic oxygen production under 635 nm red light irradiation, thereby reducing the recruitment of MDSCs and Tregs, and re-inducing M2 macrophages to M1 phenotype, ultimately reversing the tumor immunosuppressive microenvironment. In addition, H2-induced ICD synergistically promotes DCs maturation with ARS-2, thereby increasing CD8 + T cell infiltration activates anti-tumor immune responses and significantly inhibits the growth of proximal and distal tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 Figure a in the middle shows the TEM image of PCC 7942, and Figure b shows the TEM and HRTEM images of PCC@AuNP.

[0014] Figure 2 Zeta potential (Figure a) and UV-vis graph (Figure b) of PCC, AuNP and PCC@AuNP.

[0015] Figure 3 Figure a is the dissolved oxygen release curve of PCC 7942 and PCC@AuNP, and figure b is a schematic diagram of the hydrogen production of PCC@AuNP.

[0016] Figure 4 is the relative cell survival rate of B16F10 cells after co-incubation with different materials of the present invention.

[0017] Figure 5 Figure a in the middle shows the corresponding mean fluorescence intensity statistics of CRT expression in B16F10 cells after different treatments; Figure b shows the release amount of HMGB1 in the supernatant of B16F10 cells after different treatments; Figure c shows the ATP level in B16F10 cells after different treatments.

[0018] Figure 6 Graph showing the volume changes of the proximal tumor (Fig. a) and distal tumor (Fig. b) in mice treated with the present invention for 15 days.

[0019] Figure 7 Statistical graphs of mature DCs in proximal TDLNs (Figure a) and distal TDLNs (Figure b) of the present invention.

[0020] Figure 8 The CD8+ T cell statistics. DETAILED DESCRIPTION

[0021] In order to make the technical means, creative features, objectives and effects achieved by the present invention easier to understand, the present invention is further described below in conjunction with specific implementation methods.

[0022] Example 1 A method for preparing nano-engineered microalgae for melanoma photoimmunotherapy comprises the following steps: S1: Tannic acid, potassium carbonate, sodium citrate and HAuCl4·3H2O were respectively placed in water to prepare tannic acid solution, potassium carbonate solution, sodium citrate reduction solution and HAuCl4 solution; S2: Dissolve 40 μL of tannic acid solution and 330 μL of potassium carbonate solution in 50 mL of sodium citrate reducing solution to obtain a mixed solution; S3: The mixture was heated to 70 °C while stirring, and 325 μL of HAuCl4 solution was quickly added dropwise. The mixture was stirred at 70 °C for 10 min to obtain AuNPs. S4: The AuNPs obtained in step S3 were prepared into an AuNP solution, mixed with microalgae PCC 7942, and incubated at 25°C for 3 h, followed by centrifugation and washing to obtain nano-engineered microalgae PCC@AuNPs.

[0023] In this embodiment, the concentration of the tannic acid solution in step S1 is 4.3 mg / mL, the concentration of the potassium carbonate solution is 20.7 mg / mL, the concentration of the sodium citrate reducing solution is 0.7 mg / mL, and the concentration of the HAuCl4 solution is 10.0 mg / mL.

[0024] In this embodiment, the concentration of the AuNP solution in step S4 is 50 μg / mL.

[0025] In this embodiment, the density of microalgae PCC 7942 in step S4 is 3.0 × 10 8 cell / mL.

[0026] The PCC@AuNPs prepared in this example produce O2 and H2 through photosynthesis and photocatalytic reaction, respectively, under 635 nm red light irradiation.

[0027] Experimental Example 1 Morphological Characterization of PCC@AuNP The PCC@AuNPs were observed by transmission electron microscopy (TEM) to characterize the morphology of PCC@AuNPs. Figure 1 The AuNP nanoparticles were successfully attached to the microalgae PCC 7942.

[0028] Experimental Example 2 Potential and UV Characterization of PCC@AuNP Figure 2 The Zeta potential of PCC@AuNP in a is −20.1 mV, while the Zeta potentials of PCC and AuNP are −3.1 mV and −30.8 mV, respectively. Figure 2 b UV spectrum results show that PCC@AuNP has the characteristic absorption peak of AuNP at 510 nm, and the characteristic absorption peaks of PCC 7942 are at 442 nm, 630 nm, and 680 nm. These results indicate that AuNP nanoparticles were successfully modified onto microalgae.

[0029] Experimental Example 3: PCC@AuNP oxygen and hydrogen production performance verification A dissolved oxygen meter was used to study the oxygen production performance of PCC@AuNP under light. Figure 3 As shown in Figure a, oxygen can be continuously produced under 635 nm red light irradiation, and the oxygen production of PCC@AuNP is basically the same as that of PCC 7942. Gas chromatography (GC) was used to measure the accumulation of H2 in the PCC@AuNP solution under continuous light irradiation. Figure 3 As shown in (b), the H2 concentration of the PCC@AuNP solution gradually increased within 40 min under 635 nm red light irradiation. These results indicate that PCC@AuNP has the ability to produce oxygen and hydrogen under light irradiation.

[0030] Experimental Example 4: In vitro verification of the cancer cell killing performance of PCC@AuNP materials Different treatment groups were set up, and the CCK-8 method was used to evaluate the killing effect of PCC@AuNP materials on cancer cells. Figure 4 As shown in the figure, the PBS group did not produce significant cytotoxicity to B16F10 cells, while the PCC@AuNP+Laser group showed up to 77.4% cytotoxicity to B16F10 cells. This indicates that the H2 generated by the PCC@AuNP material under red light irradiation can effectively induce apoptosis of B16F10 cells.

[0031] Experimental Example 5 Study on Hydrogen-Induced Immunogenic Cell Death To test the potential of H2 generated by PCC@AuNPs to induce apoptosis and activate ICD-mediated immunotherapy, flow cytometry was used to evaluate the surface exposure of CRT in B16F10 cells after treatment with each treatment group. Figure 5 As shown in a, the PBS group had a negligible effect on the induction of CRT expression. In contrast, the PCC@AuNP+Laser-treated cells showed the highest CRT expression level. An ELISA kit was used to detect H2-induced HMGB1 secretion in B16F10 cells. Figure 5As shown in b, the extracellular secretion of HMGB1 by B16F10 cells after treatment with PCC@AuNP+Laser group was more than 2 times that of PBS group. In addition, the intracellular ATP of B16F10 cells treated with PCC@AuNP+Laser group decreased, while the extracellular secretion of ATP increased, which was due to H2-induced mitochondrial dysfunction ( Figure 5 c). The above results verify that H2 generated by PCC@AuNP under light irradiation can effectively induce ICD of cancer cells.

[0032] Experimental Example 6 Investigation of anti-tumor effects in vivo A bilateral melanoma model on the back of mice was established to explore the therapeutic effect of PCC@AuNP on solid tumors. When the right tumor (proximal tumor) of C57BL / 6 tumor-bearing mice grew to about 60 mm 3 At 14 days (defined as day 0), all C57BL / 6 tumor-bearing mice were randomly divided into two groups: PBS group and PCC@AuNP+Laser group. The proximal tumors of the mice were injected intratumorally on the designated days 0 and 3. Thirty minutes after the injection, the PCC@AuNP+Laser group was irradiated with 635 nm red light. Figure 6 As shown, the PCC@AuNP+Laser group exhibited superior tumor growth inhibition compared to the PBS group. This was attributed to the synergistic enhancement of H2-induced ICD release of DAMPs and O2-based TME reversal. In addition, tumor growth in the left tumor (distal tumor) of mice in the PCC@AuNP+Laser group was significantly inhibited even without drug administration, indicating that PCC@AuNP+Laser can induce systemic anti-tumor effects through immune response.

[0033] Experimental Example 7 Investigation of Induced Dendritic Cell Maturation in Vivo In order to explore the anti-tumor mechanism of PCC@AuNP materials under 635 nm red light irradiation, flow cytometry was used to detect the immune cells in the tumor-draining lymph nodes (TDLNs) of mice after treatment. Figure 7 Flow cytometry results showed that compared with the PBS group, the PCC@AuNP+Laser group had the highest proportion of mature DCs in the proximal and distal TDLNs of mice, 4.5 times and 4.3 times that of the PBS group, respectively. These results indicate that PCC@AuNP material induces the release of DAMPs and ARS-2 under 635 nm red light irradiation, synergistically promoting DC maturation.

[0034] Experimental Example 8 Investigation of the promotion of cytotoxic T lymphocyte infiltration in vivo Since PCC@AuNP material can alleviate hypoxia and induce ICD under red light irradiation, flow cytometry was used to study CD8 +T cell infiltration. Figure 8 The results showed that compared with the PBS group, the CD8 + The number of T cells in proximal and distal tumors increased by 1.4 and 1.1 times, respectively. This indicates that PCC@AuNP combined with 635 nm red light irradiation significantly increased CD8 + The infiltration of T cells is beneficial for killing tumor cells.

[0035] It should be noted that the PCC@AuNPs involved in Experimental Examples 1 to 8 are all products prepared in Example 1, the PBS group involved is the Control group, and the PCC@AuNP+Laser group is the final material group.

[0036] The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention. It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the present invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the present invention. Therefore, the embodiments should be regarded as illustrative and non-restrictive in all respects. The scope of the present invention is defined by the appended claims rather than the foregoing description, and all changes that come within the meaning and range of equivalents of the claims are intended to be included therein.

[0037] In addition, it should be understood that although this specification is described in terms of implementation methods, not every implementation method contains only one independent technical solution. This narrative method of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment can also be appropriately combined to form other implementation methods that can be understood by those skilled in the art.

Claims

1. A method for preparing nano-engineered microalgae for melanoma photoimmunotherapy, characterized in that: The steps include: S1: Tannic acid, potassium carbonate, sodium citrate and HAuCl4·3H2O were respectively placed in water to prepare tannic acid solution, potassium carbonate solution, sodium citrate reduction solution and HAuCl4 solution; S2: Dissolve 40 μL of tannic acid solution and 330 μL of potassium carbonate solution in 50 mL of sodium citrate reducing solution to obtain a mixed solution; S3: The mixture was heated to 70 °C while stirring, and 325 μL of HAuCl4 solution was quickly added dropwise. The mixture was stirred at 70 °C for 10 min to obtain AuNPs. S4: The AuNPs obtained in step S3 are prepared into an AuNP solution, mixed with microalgae PCC 7942, and incubated at 25° C. for 3 h, followed by centrifugation and washing to obtain nano-engineered microalgae PCC@AuNPs.

2. The method for preparing nano-engineered microalgae for melanoma photoimmunotherapy according to claim 1, characterized in that: The concentration of the tannic acid solution in step S1 is 4.3 mg / mL, the concentration of the potassium carbonate solution is 20.7 mg / mL, the concentration of the sodium citrate reducing solution is 0.7 mg / mL, and the concentration of the HAuCl4 solution is 10.0 mg / mL.

3. The method for preparing nano-engineered microalgae for melanoma photoimmunotherapy according to claim 1, characterized in that: The concentration of the AuNP solution in step S4 was 50 μg / mL.

4. The method for preparing nano-engineered microalgae for melanoma photoimmunotherapy according to claim 1, wherein: The density of microalgae PCC 7942 in step S4 was 3.0 × 10 8 cell / mL.

5. A nano-engineered microalgae prepared by the method according to any one of claims 1 to 4, characterized in that: The PCC@AuNPs produced O2 and H2 through photosynthesis and photocatalytic reactions, respectively, under 635 nm red light irradiation.

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