Preparation and application of photoresponsive polypeptide nano-composite for removing melanoma

Abstract

According to the present invention, the R-lycosin-I / porphyrin coupling phototherapy nanometer aggregate (R-LTNPs) is designed; the multifunctional material consists of polypeptide R-lycosin-I for inhibiting tumor growth and migration and inducing apoptosis, and a porphyrin part capable of realizing fluorescence imaging, photodynamic therapy (PDT) and photothermal therapy (PTT). Through covalent coupling, the nanospheres are uniformly distributed, and excellent phototherapy performance, colloidal stability and tumor targeting specificity are shown. In-vitro experiments use B16-F10 melanoma cells show that R-LTNPs retains the intrinsic cytotoxicity and cellular uptake efficiency of peptides, and can induce strong light to activate phototoxicity under light irradiation. In in-vivo experiments, the combination of R-LTNPs and light irradiation realizes remarkable tumor growth inhibition and ablation effects which exceed the effect of single-drug treatment. According to the invention, a nano assembly material is reasonably designed, and the nano assembly material shows excellent potential in the aspect of promoting combined oncology, provides a general framework for integrating molecular targeting, imaging and multi-modal treatment, and is used for a next-generation anti-cancer strategy.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biomedical materials and tumor treatment, and particularly relates to a self-assembled photosensitive nanoparticle (R-LTNPs) based on covalent coupling of anticancer peptide R-lycosin-I and porphyrin photosensitizer (TPP), a preparation method thereof and application thereof in photodynamic therapy (PDT) of melanoma. The nanoparticle forms a stable multifunctional composite system through molecular self-assembly, has the biological activity of the targeted anticancer peptide and the phototherapy function of the porphyrin photosensitizer, and can realize efficient and low-toxicity phototherapy intervention of melanoma. BACKGROUND

[0002] Melanoma, the most deadly form of skin cancer, presents a serious clinical challenge characterized by aggressive metastasis, resistance to traditional therapies and persistently high global mortality. Despite advances in surgical resection, chemotherapy, radiotherapy and immunotherapy, these approaches are still limited by incomplete tumor eradication, systemic toxicity and acquired drug resistance, which require innovative treatment modalities.

[0003] Phototherapy methods, such as photodynamic therapy (PDT) and photothermal therapy (PTT), as an alternative to precision therapy, utilize local light activation to ablate tumors, with the advantages of spatiotemporal control and reduced off-target effects. However, traditional photosensitizers face conversion barriers, including poor water solubility, low bioavailability and skin phototoxicity, which collectively reduce treatment efficacy and hinder clinical application.

[0004] Porphyrin is a natural photosensitizer with a highly conjugated macrocyclic structure, which has attracted much attention in the biomedical field due to its excellent photophysical and chemical properties. Its unique tetrapyrrole ring structure endows it with strong photothermal conversion, reactive oxygen species generation and fluorescence imaging properties. However, single porphyrin photosensitizers have the problem of insufficient tumor targeting. The present application significantly improves the tumor selectivity of the nanoparticle by covalently coupling it with the anticancer peptide R-lycosin-I. Therefore, the development of a material that enhances stability and exhibits excellent photothermal performance solves the key defects of traditional photodynamic therapy. SUMMARY

[0005] The present application aims to solve the defects in the prior art and provides a self-assembled photosensitive nanoparticle (R-LTNPs) based on covalent coupling of anticancer peptide R-lycosin-I and porphyrin photosensitizer (TPP), a preparation method thereof and application thereof in photodynamic therapy (PDT) of melanoma. The nanoparticle overcomes the problems of poor targeting of traditional photosensitizers and single treatment effect through multi-component synergy. The present application ensures uniform formation of the nanosphere, enhances colloidal stability and exhibits excellent photothermal performance, solving the key defects of traditional carriers.

[0006] To address the problems described in the background section, the present invention provides the following technical solutions:

[0007] A photoresponsive nanocomposite based on an anticancer peptide is obtained by covalently coupling the anticancer peptide R-lycosin-I with a porphyrin photosensitizer (TPP) to obtain self-assembled photosensitive nanoparticles R-LTNPs. The anticancer peptide R-LTNPs share 23 amino acids and covalently coupled TPP, with the following sequence: TPP-CO-NH-RGWFRAMRSIARFIARERLREHL.

[0008] The molecular weight of the self-assembled photosensitive nanomaterial is 3567.9116 Da.

[0009] By endowing porphyrins with the self-assembly and anti-tumor functions of R-lycosin-I, and then utilizing the photothermal and photodynamic therapy properties of porphyrins, a nanosystem for eradicating melanoma was obtained. This invention is the first to utilize R-lycosin-I combined with TPP to form a nanocomposite. Furthermore, when R-LTNPs are used in combination with light irradiation, superior tumor inhibition and ablation effects are achieved compared to single-therapy, attributed to enhanced targeted tumor toxicity and minimized systemic toxicity.

[0010] By coordinating the synergistic effects between the bioactivity of R-lycosin-I and the photophysical properties of porphyrins, R-LTNPs overcome key biological barriers in melanoma therapy, demonstrating the transformative potential of rationally designed supramolecular assemblies. Our findings establish a multifunctional framework for integrating combinatorial oncology paradigms, paving the way for next-generation nanomedicine to address treatment resistance and improve prognosis in aggressive cancers.

[0011] To achieve the above-mentioned technical effects, the present invention further discloses the specific preparation process of the self-assembled photosensitive nanomaterial, including:

[0012] R-lycosin-I was synthesized via a 9-fluorenemethyloxycarbonyl solid-phase synthesis method (Fmoc solid-phase synthesis), and purified and identified by high-performance liquid chromatography (HPLC) and mass spectrometry (MS / MS). Before cleavage, the R-lycosin-I peptide was linked to a porphyrin using 5-(4-carboxyphenyl)-10,15,2-triphenylporphyrin, HOBT, and HCTU to obtain crude R-lycosin-I-TPP. Further purification and identification were performed using HPLC and MS / MS. The R-lycosin-I-TPP solution was then subjected to dialysis and ultrafiltration to finally obtain R-LTNP nanoparticles. These were stored at -20°C.

[0013] Compared with existing traditional melanoma treatment techniques, the beneficial effects of this invention are as follows:

[0014] This invention significantly improves the precision, safety, and efficacy of melanoma treatment through targeted delivery, multi-mechanism synergy, and nanotechnology optimization, and has clinical translational potential.

[0015] 1. This invention improves system targeting; the introduction of the anticancer peptide R-lycosin-I endows nanoparticles with the ability to actively target tumors, reducing off-target effects and improving treatment precision.

[0016] 2. This invention employs a multi-mechanism synergistic treatment, combining photodynamic therapy (PDT) with the bioactivity of anticancer peptides. Through the dual effects of photothermal effect and biotoxicity, it synergistically kills tumor cells, overcoming the shortcomings of single therapy.

[0017] 3. This invention optimizes the nanocarrier, and the self-assembled nanostructure enhances the colloidal stability and uniformity, improves drug loading and delivery efficiency, and prolongs blood circulation time;

[0018] 4. This invention exhibits excellent photothermal properties, and can efficiently generate reactive oxygen species (ROS) at low light doses, thereby improving the efficacy of PDT and reducing side effects. This invention adopts nanoparticle technology, which may improve deep tumor penetration and enhance the killing of drug-resistant tumors by penetrating the tumor barrier (such as the EPR effect) and peptide-mediated penetration.

[0019] In summary, this invention significantly improves the precision, safety, and efficacy of melanoma treatment through targeted delivery, multi-mechanism synergy, and nanotechnology optimization, and has clinical translational potential. Attached Figure Description

[0020] To make the technical solution of the present invention more intuitive, the embodiments are described in detail below with reference to the accompanying drawings. It should be understood that the described drawings are merely illustrative and are not intended to limit the scope of protection of the present invention. Those skilled in the art can derive other embodiments based on these drawings without creative effort.

[0021] Figure 1 The structural formula, purification, identification, and characterization of R-lycosin-I-TPP were determined.

[0022] Figure 2 This relates to cellular uptake and intracellular ROS production.

[0023] Figure 3 This indicates cell viability, cell migration, and staining results for live and dead cells.

[0024] Figure 4 This provides in vivo imaging in mice, in vitro fluorescence images of tumors and organs, fluorescence intensity at tumor sites, and information on tumor treatment.

[0025] Figure 5 The H&E staining and TUNEL staining of the tumor tissue.

[0026] Figure 6 The H&E staining status of the major organs.

[0027] Figure 7 The attached figure is for the abstract. Detailed Implementation

[0028] To facilitate understanding of the present invention, the present invention will be described more fully and in detail below with reference to the accompanying drawings and preferred embodiments, but the scope of protection of the present invention is not limited to the following specific embodiments.

[0029] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by those skilled in the art. The technical terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the scope of the invention.

[0030] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be purchased from the market or prepared by existing methods.

[0031] Example 1: Preparation of R-LTNP nanoparticles

[0032] This invention uses the self-assembled anticancer peptide R-lycosin-I as a nanocarrier, and obtains R-lycosin-I-TPP through coupling with porphyrin, ultimately yielding R-LTNP nanoparticles. The R-LTNP sequence is as follows: TPP-CO-NH-RGWFRAMRSIARFIARERLREHL. The specific preparation steps are as follows:

[0033] (1) Synthesis of R-lycosin-I

[0034] The Fmoc solid-phase synthesis method was used to synthesize the peptide R-lycosin-I from the C-terminus to the N-terminus. The specific steps are as follows: Resin swelling: Weigh 0.159 g of resin into the synthesis column, dissolve it with 2-3 mL of DMF, then swell it in a mixer for 40 min. Dry the swelled resin using a circulating water vacuum pump, wash it 8 times with DMF, and then dry it again. Deprotection: Perform two deprotection reactions on the resin. Add 4-6 mL of 20% piperidine to the synthesis column, react it in a mixer for 7 min, and then dry it. Add another 4-6 mL of 20% piperidine, react it in a mixer for 8 min, and then dry it again. Wash it 8 times with DMF. Amino acid activation: Weigh 0.8 mmol of HOBT, HCTU, and amino acids into 1.5 mL centrifuge tubes, and use a total of 3 mL of 5% N-methylmorpholine solution. Dissolve the amino acids, then mix them in the same centrifuge tube and activate them in a mixer for 15 min. For amino acid coupling: pour the activated amino acids into a synthesis column containing resin, cap the column, invert to mix, and react in a mixer for 1 h. Dry the column, then wash 8 times with DMF. Repeat the above steps for the second amino acid coupling until the last amino acid is coupled. For peptide lysis: after the last amino acid coupling is complete, dry the column, wash 8 times with DMF, remove protection with 20% piperidine for 15 min, and then wash 8 times each with DMF and anhydrous methanol. Add 6 mL of freshly prepared lysis buffer (prepared at a ratio of 5.4 mL) to the synthesis column. TFA, 120 μL anisole, 120 μL anisole sulfide and 300 μL β-mercaptoethanol), capped and inverted to mix, reacted in a mixer for 2.5 h; Peptide precipitation: Prepare a new 50 mL centrifuge tube, blow the liquid in the synthesis column into the centrifuge tube below the synthesis column with a rubber bulb, add 30-45 mL of ice-cold ether, mix well, centrifuge at 4000 g for 10 min, remove the supernatant, and air dry to obtain crude peptide. The crude product was purified and identified by high performance liquid chromatography and mass spectrometry.

[0035] (2) Synthesis of R-lycosin-I-TPP

[0036] After the synthesis of peptide R-lycosin-I, before lysis, 0.1 mmol of HOBT, HCTU, and 0.04 mmol of 5-(4-carboxyphenyl)-10,15,2-triphenylporphyrin (TPP-COOH) were weighed into a centrifuge tube and dissolved in 3 mL of 5% N-methylmorpholine solution. The mixture was then reacted in the same centrifuge tube for 15 min. The mixture was then poured into a synthesis column, shaken until the resin was dissolved, and reacted overnight on a mixer. The solution was then dried and washed 8 times with DMF. Lysis and precipitation were performed as described above to obtain crude R-lycosin-I-TPP. The crude product was purified and identified by high-performance liquid chromatography and mass spectrometry.

[0037] (3) Preparation of R-LTNPs

[0038] The specific steps for preparing R-LTNP nanoparticles are as follows: First, 100 μL of R-lycosin-I-TPP DMSO solution (10, 20 mg / mL) is added. -1 R-lycosin-I-TPP was mixed with 900 μL of ddH2O to obtain a dispersed product. After standing at room temperature for 24 h, dialysis was performed. During dialysis, 1 mL of R-lycosin-I-TPP was sealed in a dialysis bag (molecular weight cutoff: 5 kDa) and soaked in 1 L of ddH2O for 48 h, with the water changed 3 times. Finally, the R-lycosin-I-TPP was transferred to an ultrafiltration tube (molecular weight cutoff: 3 kDa) and centrifuged at 4500 rpm for 40 min. Then, the liquid retained in the upper part of the ultrafiltration tube was collected and redispersed with ddH2O to a concentration of 1 mg·mL⁻¹. -1 2 mg·mL -1 R-LTNPs should be stored at -20°C.

[0039] (4) Physicochemical characterization methods of R-LTNPs

[0040] Transmission electron microscopy (TEM): Observation of concentrations of 2 mg / mL -1 The diameter of the R-LTNPs nanoparticles is approximately 40 nm.

[0041] Dynamic light scattering (DLS) assay: The average particle sizes of freshly prepared R-LTNPs (1 mg / mL and 2 mg / mL) were 107.5 nm and 153.8 nm, respectively. After aging for 42 days, the particle size decreased slightly (1 mg / mL: 77.08 nm; 2 mg / mL: 112.9 nm). Diluting 2 mg / mL R-LTNPs (fresh and aged samples) to different concentrations did not result in significant changes in particle size.

[0042] Zeta potential test: 2 mg·mL -1 The Zeta potential of R-LTNPs is approximately 58.87 mV, and optical images show that the prepared R-LTNPs are a clear, transparent brown liquid.

[0043] Hydrated particle size and Zeta potential of R-LTNPs: Detected using a dynamic light scattering analyzer (DLS) at room temperature, with each sample measured in triplicate.

[0044] Photothermal conversion performance of R-LTNPs: R-LTNPs exhibit excellent photothermal conversion performance and stability. At a concentration of 400 μg·mL⁻¹, as the laser power increases from 0.3 W·cm⁻¹, the photothermal conversion performance decreases. -2 Increased to 1.2 W·cm -2The solution temperature can rise by 20°C within 10 minutes; while at 1.2 W·cm -2 At a constant power, the temperature increased significantly with increasing nanoparticle concentration. Notably, a 400 μg·mL⁻¹ R-LTNPs solution exhibited a rapid temperature increase under laser irradiation, returned to room temperature after irradiation was stopped, and maintained stable photothermal performance even after five laser-on-off cycles, fully demonstrating its excellent photothermal stability and reusability. Figure 1 ).

[0045] Example 2: Investigation of the in vitro anticancer activity and mechanism of action of R-LTNPs

[0046] (1) Cytotoxicity assay: Melanoma cells (B16-F10) were treated with different concentrations of R-lycosin-I, R-LTNPs, and TPP-COOH for 12 hours. Afterward, the culture medium was replaced with serum-free medium. Then, 96-well plates were subjected to a wavelength of 638 nm and an intensity of 0.3 W / cm². 2 Cells were irradiated with a laser for 90 seconds (the control group was not irradiated with a laser) and incubated overnight in the dark. Cells were then incubated with culture medium containing 10% CCK-8 solution for 1 to 5 hours. After incubation, absorbance was measured at 450 nm using an enzyme-linked immunosorbent assay (ELISA) plate reader.

[0047] (2) Cell uptake assay: After culturing B16-F10 cells for 24 h, the culture medium was replaced with serum-free medium containing 10 μM R-LTNPs or TPP-COOH, and the cells were cultured in the dark for 3 h, 6 h, 12 h and 24 h respectively. The cell nuclei were stained with DAPI, and finally, fluorescence images were obtained using a laser confocal microscope.

[0048] (3) Generation of intracellular reactive oxygen species (ROS): B16-F10 cells were seeded in 12-well plates and cultured for 24 h, then treated with 0.2 μM MR-LTNPs or TPP-COOH for 12 h, and incubated with 10 μM DCFH-DA for 30 min. The cells were then exposed to a 638 nm laser at a laser density of 0.3 W / cm². -2 Irradiate the cells for 90 seconds. After irradiation, stain the cell nuclei with DAPI, then observe and photograph them under a fluorescence microscope.

[0049] (4) Calcein-AM / PI double staining of live and dead cells: B16-F10 cells were seeded in 12-well plates and cultured for 24 h, then treated with 0.2 μM R-LTNPs or TPP-COOH for 12 h, and a 638 nm laser with a laser density of 0.3 W·cm² was used. -2Cells were irradiated for 90 seconds, with the un-illuminated group serving as a negative control. After culturing for another 12 hours, the cells were stained and then observed under a fluorescence microscope.

[0050] In summary, the prepared R-LTNPs exhibit anticancer and selectivity of the peptide R-lycosin-I, as well as strong phototoxicity under laser irradiation. The analysis of their in vitro anticancer activity and mechanism of action lays the foundation for further investigation into the in vivo PDT effect and biosafety of R-LTNPs. Figure 2 , Figure 3 ).

[0051] Example 3: Investigation of the in vivo anticancer activity of R-LTNPs

[0052] (1) Establishment of a melanoma mouse model: BALB / c mice (5-6 weeks old) were used in this experiment by subcutaneous injection of BALB / c mice (5-6 weeks old). First, B16-F10 cells in good growth condition were digested, centrifuged, the supernatant was discarded, washed with 1×PBS, resuspended, and counted. Then, 100 μL (6×10⁶ cells) was taken. 7 Cell suspension ( / mL) was subcutaneously inoculated into mice, and the tumor volume was increased to 100 mm². 3 Then proceed with the next experiment.

[0053] (2) Intratumoral accumulation of drugs: Six tumors with a volume of 100 mm were collected. 3 B16-F10 tumor-bearing mice were randomly divided into two groups: the R-LTNPs group and the TPP-COOH group. 50 μL of R-LTNPs or TPP-COOH was injected intratumorally. Mice were anesthetized with sodium pentobarbital (50 mg / kg) at 1, 4, 8, 12, 24, 36, and 48 hours after administration. Real-time fluorescence images of the mice were observed using a small animal in vivo imaging system at an excitation wavelength of 605 nm and an emission wavelength of 430 nm.

[0054] (3) In vivo anti-tumor experiment: The tumor volume reached 100 mm. 3 B16-F10 tumor-bearing mice were randomly divided into 7 groups: (a) R-LTNPs + laser (R-LTNPs concentration in 5% glucose aqueous solution was 10.71 mg·mL⁻¹). -1 (b) TPP-COOH + laser, (the concentration of TPP-COOH in 5% glucose aqueous solution is 1.98 mg·mL) -1 (c) 5% Glu+laser, (d) R-LTNPs, (e) TPP-COOH, (f) 5% Glu, (g) R-lycosin-I, (the concentration of R-lycosin-I in 5% glucose aqueous solution is 8.78 mg·mL) -1Mice were injected intratumorally with 50 μL of LR-LTNPs, TPP-COOH, R-lycosin-I, and 5% glucose solution on days 1 and 5, respectively. At 48 h and 72 h post-injection, mice in groups (a), (b), and (c) were treated with 638 nm (0.2 W·cm⁻¹) -2 The mice were irradiated with laser for 7 minutes. The body weight and tumor volume of the mice were recorded every 2 days, and the tumor site was photographed for a total of 9 days. After 9 days, the mice were sacrificed, the tumor and major organs were removed, and the tissue was washed with 1×PBS.

[0055] (4) Pathological and histological examination: After treatment, mice were euthanized and tumor tissues and major organs (heart, liver, spleen, lung, and kidney) were removed. After cleaning and drying, the weight of each group of tumors was measured. Then, the tumor tissues and major organs (heart, liver, spleen, lung, and kidney) of the mice were fixed with 4% paraformaldehyde solution, embedded in paraffin, sectioned, and subsequently analyzed by H&E and TUNEL staining.

[0056] Compared to free TPP-COOH, R-LTNPs exhibit stronger fluorescence intensity at tumor sites and enhanced tumor-specific accumulation. Figure 4 During in vivo treatment, compared to the other five groups, the R-LTNPs+laser group showed significant crusting or ablation of tumor sites in mice. H&E staining and TUNEL assays of the tumor sites further confirmed the in vivo antitumor efficacy of R-LTNPs. Figure 5 Histopathological examination using H&E staining further confirmed that R-LTNPs are highly efficient and safe photodynamic anticancer nanoparticles. Figure 6 ).

Claims

1. A light-responsive polypeptide nanocomposite (R-LTNPs) for clearing melanoma, comprising R-lycosin-I and a porphyrin photosensitizer (TPP), wherein the R-LTNPs sequence is as follows: TPP-CO-NH-RGWFRAMRSIARFIARERLREHL.

2. The method for preparing R-LTNPs according to claim 1 includes the following steps. (1) Synthesis of R-lycosin-I: The peptide R-lycosin-I was synthesized from the C-terminus to the N-terminus using the Fmoc solid-phase synthesis method. (2) Synthesis of R-lycosin-I-TPP: After the synthesis of peptide R-lycosin-I, before cleavage, HOBT, HCTU, and 5-(4-carboxyphenyl)-10,15,2-triphenylporphyrin (TPP-COOH) were placed in a centrifuge tube and dissolved in N-methylmorpholine solution. The solutions were then mixed and reacted in the same centrifuge tube. The mixture was then poured into a synthesis column, shaken until the resin dissolved, and reacted overnight on a mixer. The solution was then dried and washed 8 times with DMF. Cleavage and precipitation were performed according to the above method to obtain crude R-lycosin-I-TPP. The crude product was purified and identified by high-performance liquid chromatography and mass spectrometry. (3) Preparation of R-LTNPs: First, R-lycosin-I-TPP DMSO solution was mixed with ddH2O to obtain dispersed R-lycosin-I-TPP, which was then dialyzed after standing. Finally, the R-lycosin-I-TPP was transferred to an ultrafiltration tube. Then, the liquid retained in the upper part of the ultrafiltration tube was collected, redispersed with ddH2O, and stored at -20℃. (4) Physicochemical characterization methods of R-LTNPs: The photothermal conversion performance of R-LTNPs was analyzed by transmission electron microscopy (TEM), dynamic light scattering (DLS) test, Zeta potential test, and dynamic light scattering analyzer (DLS).

3. The method for preparing R-LTNPs according to claim 2, characterized in that, In step (1), 0.159g of resin was weighed into the synthesis column, dissolved with 2-3ml of DMF, and then placed in a mixer to expand for 40min. The expanded resin was then dried using a circulating water vacuum pump, washed with DMF 8 times, and dried again. The resin underwent two deprotection reactions. 4-6 mL of 20% piperidine was added to the synthesis column, and the column was reacted in a mixer for 7 min. The mixture was then dried, and another 4-6 mL of 20% piperidine was added, reacted in a mixer for 8 min, dried, and washed 8 times with DMF. 0.8 mmol of HOBT, HCTU, and amino acids were weighed into 1.5 mL centrifuge tubes and dissolved in a total of 3 mL of 5% N-methylmorpholine solution. The solutions were then mixed in the same centrifuge tube and activated in a mixer for 15 min. The activated amino acids were poured into the synthesis column containing the resin, the top cap was closed, and the column was inverted to mix. The mixture was reacted in a mixer for 1 h, dried, and washed 8 times with DMF. This process was repeated for the second amino acid coupling until the last amino acid was coupled. After the last amino acid coupling was completed, the column was dried, washed 8 times with DMF, deprotected with 20% piperidine for 15 min, and then washed 8 times each with DMF and anhydrous methanol. 6 mL of freshly prepared lysis buffer (prepared at a ratio of 5.4 mL) was added to the synthesis column. The mixture of TFA, 120 μL anisole, 120 μL anisole sulfide, and 300 μL β-mercaptoethanol was inverted and mixed after capping, and then reacted in a mixer for 2.5 h. A new 50 mL centrifuge tube was prepared, and the liquid in the synthesis column was blown into the centrifuge tube below the synthesis column using a bulb syringe. 30-45 mL of ice-cold diethyl ether was added, and the mixture was mixed. The mixture was centrifuged at 4000 g for 10 min, the supernatant was removed, and the mixture was air-dried to obtain the crude peptide. The crude peptide was purified and identified by high performance liquid chromatography and mass spectrometry.

4. The method for preparing R-LTNPs according to claim 2, characterized in that, In step (2), 0.1 mmol of HOBT, HCTU and 0.04 mmol of TPP-COOH were weighed and dissolved in 3 mL of 5% N-methylmorpholine solution for 15 min.

5. The method for preparing R-LTNPs according to claim 2, characterized in that, In step (3), R-lycosin-I-TPP DMSO solution (10, 20 mg·mL) -1 The amounts of R-lycosin-I-TPP and ddH2O were 100 μL and 900 μL respectively, and the mixture was left to stand at room temperature for 24 h before dialysis. During dialysis, 1 mL of R-lycosin-I-TPP (molecular weight cutoff: 5 kDa) was sealed in a dialysis bag and soaked in 1 L of ddH2O for 48 h, changing the water three times during this period. Finally, the R-lycosin-I-TPP was transferred to an ultrafiltration tube (molecular weight cutoff: 3 kDa) and centrifuged at 4500 rpm for 40 min. The liquid retained in the upper part of the ultrafiltration tube was then collected and redispersed with ddH2O to a concentration of 1 mg / mL. -1 2 mg·mL -1 R-LTNPs should be stored at -20°C.

6. The method for preparing R-LTNPs according to claim 2, characterized in that, In step (4), the concentration of 2 mg·mL was observed using a transmission electron microscope (TEM). -1 R-LTNPs nanoparticles. Freshly prepared R-LTNPs (1 mg / mL and 2 mg / mL) were used for dynamic light scattering (DLS) testing. The 2 mg / mL R-LTNPs (fresh and aged samples) were then diluted to different concentrations, and particle size changes were observed. (2 mg / mL) -1 Zeta potential of R-LTNPs was measured. Hydrated particle size and Zeta potential of R-LTNPs: The hydration particle size and Zeta potential of R-LTNPs were measured at room temperature using a dynamic light scattering analyzer (DLS).

7. The application of the photoresponsive polypeptide nanocomposite according to claim 1 in the treatment of melanoma.

8. The application of the photoresponsive polypeptide nanocomposite according to claim 1 in preparation and photodynamic therapy.

9. The application of R-LTNPs in integrated diagnosis and treatment according to claim 1, characterized in that: By utilizing the fluorescence imaging capabilities of TPP, the accumulation of drugs at the tumor site can be monitored in real time.

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