Binary heterojunction nanosheet photosensitizer for the photodynamic therapy of retinoblastoma
The gCN/BP heterojunction nanosheet addresses limitations in PDT by enhancing light absorption and stability, enabling deeper tissue penetration and selective cancer cell targeting, providing a non-invasive and effective treatment for retinoblastoma.
Patent Information
- Authority / Receiving Office
- WO · WO
- Patent Type
- Applications
- Current Assignee / Owner
- KOC UNIVSI
- Filing Date
- 2025-12-26
- Publication Date
- 2026-07-02
Smart Images
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Abstract
Description
[0001] 21644.92
[0002] DESCRIPTION BINARY HETEROJUNCTION NANOSHEET PHOTOSENSITIZER FOR THE PHOTODYNAMIC THERAPY OF RETINOBLASTOMA
[0003] Technical Field
[0004] The invention relates to the field of biomedical applications, specifically to photodynamic therapy for cancer treatment, involving the use of graphitic carbon nitride and black phosphorus (gCN / BP) heterojunction as photosensitizers in the form of nanosheet for the treatment of retinoblastoma, a malignant eye tumor.
[0005] Prior Art
[0006] Retinoblastoma is a type of cancer that begins with the retina, the light-sensitive tissue at the back of the eye. It is the most common type of childhood eye cancer, with prognosis depending on various factors, including whether the cancer has spread to other parts of the body. Current treatments for retinoblastoma include cryotherapy (freezing the tumor) and chemotherapy, which are the most widely used options. Additional treatments include thermotherapy, thermochemotherapy, irradiation, and brachytherapy. In more severe cases, when other treatment options have been exhausted, enucleation (removal of the eye) is the final alternative. After treatment, the focus often shifts to the long-term effects of the disease, concerns about its recurrence, and managing side effects [1].
[0007] Graphitic carbon nitride (gCN), a polymeric 2D semiconductor with a band gap of 2.7 eV (454 nm), can be activated by visible light, making it an intriguing candidate for biomedical applications. However, bulk gCN has low efficiency for photodynamic applications due to its limited surface area, low light absorption capacity and low dispersion in water. On the other hand, black phosphorus (BP), the most stable phosphorus allotrope with a three-dimensional (3D) layered structure, has attracted renewed scientific interest due to its exceptional chemical and electronic properties. However, pristine BP also has limitations, such as rapid recombination of photogenerated electron-hole pairs and low stability, which restricts its photodynamic therapy applications [2].21644.92
[0008] Photodynamic therapy (PDT) is a treatment that uses a photosensitizer to produce reactive oxygen species (ROS) upon light activation, which induces the death of cancer cells. In prior art of the retinoblastoma treatment, porphyrin-based compounds such as Photofrin and Verteporfin are commonly used photo sensitizers. These agents are used to treat small tumors in the eye, leveraging their ability to accumulate in cancerous tissues and produce cytotoxic effects when exposed to light. However, despite advancements in this field, significant limitations remain with traditional photosensitizers and other therapeutic approaches used for retinoblastoma.
[0009] One major issue with prior art photosensitizers is their limited light penetration depth. Porphyrinbased photosensitizers rely on light sources that do not penetrate deeply into tissue, limiting their use to small, superficial tumors. This becomes problematic for more advanced retinoblastoma cases, where deeper tissue penetration is necessary for effective treatment. As a result, these treatments are often ineffective for tumors located deeper within the eye or in cases where the cancer has spread. Another critical issue is the variable effectiveness of existing treatments. The efficacy of traditional photosensitizers depends on multiple factors, including the type and concentration of the agent, the light dose, and the specific characteristics of the tumor. These variables lead to inconsistent therapeutic outcomes, making it difficult to standardize treatment protocols and predict patient responses. Consequently, PDT effectiveness can vary from patient to patient, even when using similar treatment conditions [3].
[0010] Systemic toxicity and side effects also represent significant problems in the prior art. Traditional photosensitizers can cause photosensitivity in healthy tissues, leading to prolonged adverse effects such as skin sensitivity to light. This can force patients to avoid sunlight or bright indoor lighting for extended periods after treatment, significantly impacting their quality of life. Furthermore, systemic chemotherapy, another common treatment for retinoblastoma, is associated with severe side effects like immunosuppression, nausea, and hair loss. These side effects can weaken the immune system, increase the risk of infections and causing other complications. The complex application protocols of current PDT systems present another challenge. Many existing PDT treatments require multiple light sources or different excitation wavelengths to activate the photosensitizers, adding complexity to the process. This complexity makes PDT less practical for widespread clinical use, particularly in resource-limited settings or when treating pediatric patients. The need for specialized equipment and expertise can limit the availability of these treatments, making administration more difficult [4].21644.92
[0011] Finally, there are concerns regarding the biocompatibility and stability of current photosensitizers in biological environments. Many photosensitizers in prior art lack ideal biocompatibility, leading to unwanted interactions with healthy cells and potential long-term side effects. Additionally, the stability of these compounds can be compromised in biological environments, reducing their therapeutic efficacy. Poor solubility in water and the rapid degradation of photosensitizers in vivo further limit their clinical applications.
[0012] In the prior art, the patent application CN114588279A relates to advancements in photodynamic therapy (PDT), focusing on the development of photo sensitizers that generate reactive oxygen species (ROS) to selectively destroy cancer cells upon exposure to specific light wavelengths. However, there are potential disadvantages, such as limited light penetration, which restricts the effectiveness for deeper tumors. Off-target effects can still occur, potentially harming healthy tissues. Moreover, photosensitizers like black phosphorus may suffer from stability issues, degrading quickly in biological environments. Additionally, the complex administration of PDT, requiring precise timing and coordination, and photosensitivity in patient’s post-treatment are significant challenges.
[0013] Considering these challenges, more advanced and efficient photosensitizers with better light absorption, deeper tissue penetration, and improved therapeutic effectiveness are required.
[0014] Summary of the Invention
[0015] The primary objective of the invention is to develop a binary heterojunction, comprising graphitic carbon nitride (gCN) and black phosphorus (BP), as an effective photosensitizer for photodynamic therapy (PDT) in the treatment of retinoblastoma. This heterojunction overcomes the limitations of existing photosensitizers by enhancing light absorption, provide deeper tissue penetration and promoting the generation of reactive oxygen species (ROS), which are essential for inducing cancer cell death.
[0016] Another aim of the invention is to resolve the stability issues associated with BP by combining it with gCN. The heterojunction formed with gCN stabilizes BP, enhancing the overall durability and effectiveness of the photosensitizer in clinical applications.
[0017] The invention also aims to improve the biocompatibility of PDT treatments for retinoblastoma. The gCN / BP binary heterojunction nanosheet is designed to be selective, reducing damage to healthy retinal cells while effectively targeting and destroying retinoblastoma cells. Additionally, the invention enables the photosensitizer to absorb light more efficiently at greater depths. By21644.92
[0018] improving light absorption in the NIR spectrum, the gCN / BP binary heterojunction nanosheet allows the therapy to penetrate deeper into the eye, enabling the treatment of more advanced stages of retinoblastoma.
[0019] Finally, the invention aims to simplify the application of PDT. The proposed photosensitizer can be activated by NIR light, which is non-invasive and painless, making the treatment easier to administer in clinical settings and offering a quicker, less complex solution compared to traditional therapies.
[0020] Drawings
[0021] Figure 1. Graphs of RB cell viability after BP nanosheet treatment and NIR stimulation.
[0022] Figure 1A. RB cell viability after treatment with different concentrations of BP nanosheets without light irradiation.
[0023] Figure IB. RB cell viability after BP nanosheet treatment under near-infrared (NIR) laser irradiation.
[0024] Figure 2. Graphs of BP nanosheet stability and its potential interference in RB cell viability assays at different incubation times.
[0025] Figure 2A. RB cell viability after 24 hours of treatment with different concentrations of BP nano sheets.
[0026] Figure 2B. RB cell viability after 48 hours of treatment with different concentrations of BP nano sheets.
[0027] Figure 2C. RB cell viability after 72 hours of treatment with different concentrations of BP nano sheets.
[0028] Figure 2D. RB cell viability after 96 hours of treatment with different concentrations of BP nano sheets.
[0029] Figure 3. Time-dependent bright-field images of RB cells incubated with BP nanosheets, showing the morphological evolution and aggregation behavior over time. White circles indicate clumped BP nano sheets.
[0030] Figure 3A. Bright-field image of RB cells at 0 h after BP nanosheet exposure.
[0031] Figure 3B. Bright-field image of RB cells after 24 h of incubation with BP nanosheets.21644.92
[0032] Figure 3C. Bright-field image of RB cells after 48 h of incubation with BP nanosheets.
[0033] Figure 3D. Bright-field image of RB cells after 72 h of incubation with BP nanosheets.
[0034] Figure 3E. Bright-field image of RB cells after 96 h of incubation with BP nanosheets.
[0035] Figure 4. Graphs of cell viability in RB and RPE-1 cells after treatment with gCN nanosheets under dark and NIR irradiation conditions.
[0036] Figure 4A. Cell viability of RB cells after treatment with different concentrations of gCN nanosheets under dark conditions.
[0037] Figure 4B. cell viability of RPE-1 cells after treatment with different concentrations of gCN nanosheets under dark conditions.
[0038] Figure 4C. Cell viability of RB cells after combined treatment with gCN nanosheets and NIR irradiation.
[0039] Figure 4D. Cell viability of RPE-1 cells after combined treatment with gCN nanosheets and NIR irradiation.
[0040] Figure 5. Graphs of reactive oxygen species (ROS) levels in RB and RPE-1 cells following treatment with gCN nanosheets under dark and NIR irradiation conditions.
[0041] Figure 5A. ROS levels in RB cells after treatment with different concentrations of gCN nano sheets.
[0042] Figure 5B. ROS levels in RPE-1 cells after treatment with different concentrations of gCN nano sheets.
[0043] Figure 5C. ROS levels in RB cells after combined treatment with gCN nanosheets and NIR irradiation.
[0044] Figure 5D. ROS levels in RPE-1 cells after combined treatment with gCN nanosheets and NIR irradiation.
[0045] Figure 6. Graphs of the evaluation of cell toxicity in RB and RPE- 1 cells after treatment with the gCN / BP binary heterojunction nanosheet.
[0046] Figure 6A. Cell viability of RB cells after treatment with different concentrations of the gCN / BP binary heterojunction nanosheet.21644.92
[0047] Figure 6B. Cell viability of RPE-1 cells after treatment with different concentrations of the gCN / BP binary heterojunction nanosheet.
[0048] Figure 6C. Cell viability of RB cells after dual therapy with the gCN / BP binary heterojunction nanosheet and NIR irradiation.
[0049] Figure 6D. Cell viability of RPE-1 cells after dual therapy with the gCN / BP binary heterojunction nanosheet and NIR irradiation.
[0050] Figure 7. Bright-field images showing the behavior of the gCN / BP binary heterojunction nanosheet in RB cell culture medium over time.
[0051] Figure 7A. RB cells captured at the initial stage (first day) after treatment with the gCN / BP binary heterojunction nanosheet, showing early cellular interactions.
[0052] Figure 7B. RB cells after 24 hours of incubation with the gCN / BP binary heterojunction nano sheet.
[0053] Figure 7C. RB cells after 72 hours of incubation with the gCN / BP binary heterojunction nano sheet.
[0054] Figure 8. Graphs of reactive oxygen species (ROS) levels in RB and RPE-1 cells after treatment with the gCN / BP binary heterojunction nanosheet under dark and NIR irradiation conditions.
[0055] Figure 8A. ROS levels in RB cells after treatment with different concentrations of the gCN / BP binary heterojunction nanosheet under dark conditions.
[0056] Figure 8B. ROS levels in RPE- 1 cells after treatment with different concentrations of the gCN / BP binary heterojunction nanosheet under dark conditions.
[0057] Figure 8C. ROS levels in RB cells after combined treatment with the gCN / BP binary heterojunction nano sheet and NIR irradiation.
[0058] Figure 8D. ROS levels in RPE-1 cells after combined treatment with the gCN / BP binary heterojunction nanosheet and NIR irradiation.
[0059] Figure 9. Graphs of Western blot analysis of apoptotic pathway markers in RB cells after treatment with the gCN / BP binary heterojunction nanosheet and NIR irradiation.21644.92
[0060] Figure 9A. Bcl-2 protein expression levels in RB cells in the control group, after treatment with the gCN / BP binary heterojunction nanosheet alone, after NIR irradiation alone, and after combined gCN / BP binary heterojunction nanosheet and NIR treatment.
[0061] Figure 9B. Caspase-9 protein expression levels in RB cells in the control group, after treatment with the gCN / BP binary heterojunction nanosheet alone, after NIR irradiation alone, and after combined gCN / BP binary heterojunction nanosheet and NIR treatment.
[0062] Figure 9C. Bax protein expression levels in RB cells in the control group, after treatment with the gCN / BP binary heterojunction nanosheet alone, after NIR irradiation alone, and after combined gCN / BP binary heterojunction nanosheet and NIR treatment.
[0063] Figure 10. Graphs of Western blot analysis of apoptotic pathway markers in RPE-1 cells after treatment with the gCN / BP binary heterojunction nanosheet and NIR irradiation.
[0064] Figure 10A. BCL-2 protein expression levels in RPE-1 cells in the control group, after treatment with the gCN / BP binary heterojunction nanosheet alone, after NIR irradiation alone, and after combined gCN / BP binary heterojunction nanosheet and NIR treatment.
[0065] Figure 10B. BAX protein expression levels in RPE-1 cells in the control group, after treatment with the gCN / BP binary heterojunction nanosheet alone, after NIR irradiation alone, and after combined gCN / BP binary heterojunction nanosheet and NIR treatment.
[0066] Figure 11. Immunofluorescence images showing cell proliferation and structural integrity of RB cells after treatment with the gCN / BP binary heterojunction nanosheet, NIR irradiation, and combined therapy.
[0067] Figure 11A. DAPLstained nuclei of untreated RB control cells, showing clustered nuclear morphology.
[0068] Figure 11B. Ki-67 immunofluorescence image indicating proliferating RB control cells.
[0069] Figure 11C. Merged DAPI and Ki-67 image of RB control cells, demonstrating colocalization of Ki-67 within nuclei and active cell proliferation.
[0070] Figure 11D. DAPLstained nuclei of RB cells after treatment with the gCN / BP binary heterojunction nanosheet showing impairment of cellular structural integrity.
[0071] Figure HE. Ki-67 immunofluorescence image of RB cells after treatment with the gCN / BP binary heterojunction nanosheet.21644.92
[0072] Figure 11F. Merged DAPI and Ki-67 image of RB cells after treatment with the gCN / BP binary heterojunction nanosheet, showing nuclear localization of Ki-67.
[0073] Figure 11G. DAPI-stained nuclei of RB cells after NIR irradiation, showing deterioration of cellular structure.
[0074] Figure 11H. Ki-67 immunofluorescence image of RB cells after NIR irradiation.
[0075] Figure 111. Merged DAPI and Ki-67 image of RB cells after NIR irradiation, showing nuclear Ki-67 localization.
[0076] Figure 11J. DAPI-stained nuclei of RB cells after combined treatment with the gCN / BP binary heterojunction nanosheet and NIR irradiation, showing severe disruption of cellular structure.
[0077] Figure 11K. Ki-67 immunofluorescence image of RB cells after combined gCN / BP binary heterojunction nano sheet and NIR treatment.
[0078] Figure 11L. Merged DAPI and Ki-67 image of RB cells after combined gCN / BP binary heterojunction nanosheet and NIR treatment, showing altered proliferation behavior at the nuclear level.
[0079] Figure 12. Immunofluorescence images showing cell proliferation behavior of RPE- 1 cells after treatment with the gCN / BP binary heterojunction nanosheet, NIR irradiation, and combined therapy.
[0080] Figure 12A. DAPI-stained nuclei of untreated RPE-1 control cells.
[0081] Figure 12B. Ki-67 immunofluorescence image indicating proliferating RPE-1 control cells.
[0082] Figure 12C. Merged DAPI and Ki-67 image of RPE-1 control cells, showing nuclear colocalization of Ki-67 and active cell proliferation.
[0083] Figure 12D. DAPI-stained nuclei of RPE-1 cells after treatment with the gCN / BP binary heterojunction nano sheet.
[0084] Figure 12E. Ki-67 immunofluorescence image of RPE-1 cells after treatment with the gCN / BP binary heterojunction nanosheet.21644.92
[0085] Figure 12F. Merged DAPI and Ki-67 image of RPE-1 cells after treatment with the gCN / BP binary heterojunction nanosheet, showing no significant change in proliferation compared to the control group.
[0086] Figure 12G. DAPI-stained nuclei of RPE-1 cells after NIR irradiation.
[0087] Figure 12H. Ki-67 immunofluorescence image of RPE-1 cells after NIR irradiation. Figure 121. Merged DAPI and Ki-67 image of RPE-1 cells after NIR irradiation, indicating maintained proliferative activity.
[0088] Figure 12J. DAPI-stained nuclei of RPE-1 cells after combined treatment with the gCN / BP binary heterojunction nanosheet and NIR irradiation.
[0089] Figure 12K. Ki-67 immunofluorescence image of RPE-1 cells after combined treatment with the gCN / BP binary heterojunction nanosheet and NIR irradiation.
[0090] Figure 12L. Merged DAPI and Ki-67 image of RPE-1 cells after combined gCN / BP binary heterojunction nanosheet and NIR treatment, showing no significant difference in cell proliferation compared to the control group.
[0091] Figure 13. Immunofluorescence images showing cell integrity and morphological characteristics of RPE-1 cells after treatment with the gCN / BP binary heterojunction nanosheet, NIR irradiation, and combined therapy.
[0092] Figure 13A. DAPI-stained nuclei of untreated RPE-1 control cells.
[0093] Figure 13B. ZO-1 immunofluorescence image showing the tight junction morphology of RPE-1 control cells.
[0094] Figure 13C. Merged DAPI and ZO-1 image of RPE-1 control cells, demonstrating preserved cellular morphology.
[0095] Figure 13D. DAPI-stained nuclei of RPE-1 cells after treatment with the gCN / BP binary heterojunction nano sheet.
[0096] Figure 13E. ZO-1 immunofluorescence image of RPE-1 cells after treatment with the gCN / BP binary heterojunction nanosheet.
[0097] Figure 13F. Merged DAPI and ZO- 1 image of RPE- 1 cells after treatment with the gCN / BP binary heterojunction nanosheet, showing preserved cellular morphology.21644.92
[0098] Figure 13G. DAPI-stained nuclei of RPE-1 cells after NIR irradiation.
[0099] Figure 13H. ZO-1 immunofluorescence image of RPE-1 cells after NIR irradiation. Figure 131. Merged DAPI and ZO-1 image of RPE-1 cells after NIR irradiation, indicating maintained cell morphology.
[0100] Figure 13 J. DAPI-stained nuclei of RPE-1 cells after combined treatment with the gCN / BP binary heterojunction nanosheet and NIR irradiation.
[0101] Figure 13K. ZO-1 immunofluorescence image of RPE-1 cells after combined treatment with the gCN / BP binary heterojunction nanosheet and NIR irradiation.
[0102] Figure 13L. Merged DAPI and ZO-1 image of RPE-1 cells after combined treatment with the gCN / BP binary heterojunction nanosheet and NIR irradiation, showing largely preserved epithelial morphology.
[0103] Figure 14. TEM, HAADF-STEM, and elemental mapping images of the gCN / BP binary heterojunction nanosheet.
[0104] Figure 14A. Low-magnification TEM image of the gCN / BP binary heterojunction nano sheet.
[0105] Figure 14B. TEM image of the gCN / BP binary heterojunction nanosheet at higher magnification.
[0106] Figure 14C. High-resolution TEM image showing the layered morphology of the gCN / BP binary heterojunction nano sheet.
[0107] Figure 14D. HAADF-STEM image of the gCN / BP binary heterojunction nanosheet, confirming the hybrid structure.
[0108] Figure 14E. Elemental mapping image showing the spatial distribution of carbon (C), nitrogen (N), and phosphorus (P) in the gCN / BP binary heterojunction nanosheet.
[0109] Figure 14F. Elemental mapping image showing the distribution of carbon (C) in the gCN / BP binary heterojunction nanosheet.
[0110] Figure 14G. Elemental mapping image showing the distribution of nitrogen (N) in the gCN / BP binary heterojunction nanosheet.21644.92
[0111] Figure 14H. Elemental mapping image showing the distribution of phosphorus (P) in the gCN / BP binary heterojunction nanosheet.
[0112] Figure 15. X-ray diffraction (XRD) patterns of the gCN / BP binary heterojunction nanosheet, pristine gCN, and crystalline BP.
[0113] Figure 16. Graphs of optical absorption properties and band gap analysis of the gCN / BP binary heterojunction nanosheet and pristine gCN.
[0114] Figure 16A. UV-vis-NIR diffuse reflectance spectra of pristine gCN and the gCN / BP binary heterojunction nano sheet.
[0115] Figure 16B. Tauc plots of pristine gCN and the gCN / BP binary heterojunction nanosheet for estimation of the optical band gap energies.
[0116] Figure 17. Graphs of photoluminescence and time-resolved photoluminescence characteristics of the gCN / BP binary heterojunction nanosheet and pristine gCN.
[0117] Figure 17A. Steady-state photoluminescence spectra of pristine gCN and the gCN / BP binary heterojunction nano sheet.
[0118] Figure 17B. Time-resolved photoluminescence (TRPL) decay curves of pristine gCN and the gCN / BP binary heterojunction nanosheet.
[0119] Detailed Description
[0120] The invention pertains to binary heterojunction as photosensitizer for use in photodynamic therapy (PDT) in the treatment of retinoblastoma in form of nanosheet.
[0121] Hereinafter, the binary heterojunction nanosheet photosensitizer according to the present invention may also be referred to as a gCN / BP binary heterojunction nanosheet without departing from the scope of the invention.
[0122] A binary heterojunction nanosheet photosensitizer for use in photodynamic therapy (PDT) for treating retinoblastoma, comprising:
[0123] Graphitic carbon nitride (gCN), a two-dimensional (2D) polymeric semiconductor with a bandgap of 2.7 eV, and21644.92
[0124] Few-layer black phosphorus (BP) with a bandgap of 1.4 eV, wherein the heterojunction formed between gCN and BP enhances light absorption and improves charge carrier separation, minimizing electron-hole recombination. gCN is a polymeric semiconductor with a bandgap of approximately 2.7 eV, which allows for being activated by visible light. It is known for its stability owing to its polymeric structure, making it suitable for biomedical applications like PDT. However, bulk gCN is not highly effective in PDT applications due to its large particle size, limited surface area and poor dispersion in water. By combining it with few-layer black phosphorus (BP), the invention enhances the photosensitizer's dispersion and functionality. Few-layer BP, another 2D semiconductor, is highly stable and has a bandgap of 1.4 eV, making it responsive to near- infrared (NIR) light, which is crucial for deeptissue treatments. The wavelength of NIR light used is 810 nm. However, BP suffers from rapid degradation and recombination of electron-hole pairs, limiting its effectiveness in photodynamic therapy applications. The heterojunction formed with gCN stabilizes BP, improving its stability and effectiveness.
[0125] The binary heterojunction photosensitizer is prepared through the liquid-phase assembly method involving ultrasonication-assisted dispersion of gCN and BP in N-methyl-2-pyrrolidone (NMP) solvent.
[0126] A method of synthesizing the binary heterojunction nanosheet photo sensitizer for use in photodynamic therapy (PDT) comprises the following steps:
[0127] i. synthesizing mesostructured graphitic carbon nitride (gCN),
[0128] ii. synthesizing black phosphorus (BP),
[0129] iii. suspending black phosphorus (BP) in N-methyl-2-pyrrolidone (NMP) at a concentration from about 1,6 to about 1,8 mg / ml, and sonicating the suspension at a controlled temperature of 20-25°C to obtain exfoliated BP nanosheets,
[0130] iv. adding mesoporous graphitic carbon nitride, previously dispersed in NMP, into the sonicated BP dispersion wherein a gCN:BP mass ratio ranges from about 4.7:1 to about 5.1:1, wherein the addition is performed under continuous sonication,
[0131] v. continuing the sonication of the combined dispersion for 3, 5-4, 5 hours so as to promote interfacial contact and heterojunction assembly between the gCN and BP phases, vi. isolating the resulting gCN / BP composite by centrifuging the mixed suspension at about 8500-9500 rpm for about 15-20 minutes, followed by washing the precipitate twice with ethanol to remove unbound material,21644.92
[0132] vii. drying the washed composite,
[0133] viii. adjusting the BP loading in the composite, wherein a standard composition comprises about 16,5-17,5 wt % BP relative to the total mass of the gCN / BP binary heterojunction nanosheet, and thereby obtaining the binary heterojunction photosensitizer. As a representative procedure, black phosphorus (BP) was suspended in N-methyl-2-pyrrolidone (NMP) at a BP concentration from about 1.6 to about 1.8 mg / ml, and sonicated at a controlled temperature of 20-25°C to obtain exfoliated BP nanosheets. Subsequently, mesoporous graphitic carbon nitride (gCN), previously dispersed in NMP, was added to the BP dispersion such that a gCN :BP mass ratio ranges from about 4.7: 1 to about 5.1:1, wherein the addition is performed under continuous sonication. The combined suspension was then subjected to an additional 3, 5-4, 5 hours of ultrasonic treatment to promote interfacial contact and heterojunction assembly between the gCN and BP phases. After completion of the assembly process, the resulting gCN / BP composite was isolated by centrifugation at about 8500-9500 rpm for about 18-22 minutes and washed twice with ethanol to remove unbound material. The washed composite was then dried. In the standard composition, the BP loading was adjusted to about 16,5-17,5 wt% relative to the total mass of the gCN / BP binary heterojunction nanosheet, thereby obtaining the binary heterojunction photosensitizer. Heterostructures with alternative BP loading levels were also obtained by varying the amount of BP introduced into the gCN phase.
[0134] The method of synthesizing mesostructured Graphitic Carbon Nitride (gCN) comprises following steps:
[0135] i. dissolving guanidine hydrochloride in deionized water wherein a guanidine hydrochloride concentration ranges from about 0.8 to about 1.2 g / ml, thereby obtaining an aqueous precursor solution,
[0136] ii. introducing the precursor solution gradually into colloidal silica wherein a guanidine hydrochloride to colloidal silica mass ratio ranges from about 0.37: 1 to about 0.47: 1, under vigorous stirring, thereby allowing the silica to function as a hard template,
[0137] iii. stirring the resulting dispersion overnight in an oil bath maintained at about 48-52°C to ensure uniform mixing and precursor infiltration,
[0138] iv. cooling the mixture to room temperature and recovering the solid content, followed by grinding the obtained material into a fine powder,
[0139] v. placing the ground powder into a lidded quartz crucible and calcining the material under argon atmosphere by heating to about 540-560°C over 1,5-2, 5 hours, maintaining this21644.92
[0140] temperature for an additional 1,5-2, 5 hours, and thereafter allowing the furnace to cool naturally to ambient temperature,
[0141] vi. etching the silica template by immersing the calcined yellow material in 190-210 ml of 3,8- 4,2 M ammonium hydrogen difluoride (NH4HF2) solution and stirring the mixture for approximately 46-50 hours,
[0142] vii. collecting the obtained mesostructured gCN by centrifugation, followed by repeatedly washing the solid with distilled water and ethanol to remove residual fluoride ions and reaction byproducts,
[0143] viii. drying the final product under ambient conditions to obtain mesostructured graphitic carbon nitride suitable for subsequent composite formation.
[0144] Mesostructured gCN was prepared by thermal polymerization of guanidine hydrochloride using a hard-template-assisted strategy. In a typical synthesis, guanidine hydrochloride (GndCl) was dissolved in deionized water to obtain an aqueous solution having a GndcL concentration ranging from about 0,8 to 1,2 g / ml, and the resulting solution was slowly added to colloidal silica (Ludox HS40) wherein a GndCl to colloidal silica mass ratio ranged from about 0,37:1 to about 0,47:1, under vigorous stirring, thereby enabling the silica particles to function as a sacrificial template. The mixture was stirred overnight in an oil bath maintained at 48-52°C (321-325 K) to ensure uniform distribution of the precursor within the silica network. After cooling to room temperature, the solid phase was separated and ground into a fine powder. The obtained material was transferred to a lidded quartz crucible and calcined in a tubular furnace under an argon atmosphere, where the temperature was increased to 540-560°C (813-833 K) over 1,5-2, 5 hours, held for an additional 1,5-2, 5 hours, and then allowed to cool naturally. To remove the silica framework, the yellow calcined powder was immersed in 190-210 ml of 3, 8-4, 2 M ammonium hydrogen difluoride (NH4HF2) solution and stirred for 46-50 hours. Following etching, the product was collected by centrifugation and thoroughly washed with distilled water and ethanol to eliminate residual fluoride species and other byproducts.
[0145] The method of synthesizing Black Phosphorus (BP) comprises following steps:
[0146] i. weighing and introducing red phosphorus, tin (Sn), and tin(IV) iodide (SnL) into a quartz ampoule wherein a red phosphorus :Sn: SnL mass ratio ranges from about 48:2:1 to about 52:2:1, wherein the quartz ampoule has a length of about 18-22 cm and an inner diameter of about 1,5-2 cm,21644.92
[0147] ii. evacuating the ampoule under high-vacuum conditions and sealing it to prevent contamination and oxidative degradation,
[0148] iii. positioning the sealed ampoule horizontally inside a programmable muffle furnace, iv. increasing the temperature of the furnace gradually to about 883-903 K and maintaining this temperature for approximately 4-5 hours to sublimate volatile species and activate mineralizer-assisted phosphorus transport,
[0149] v. decreasing the furnace temperature slowly to about 750-760 K and holding the ampoule at this temperature for about 1,5-2, 5 hours to promote nucleation and controlled crystal growth of black phosphorus,
[0150] vi. cooling the system to about 383-403 K to preserve crystallinity, followed by natural cooling to ambient temperature,
[0151] vii. opening the ampoule under anhydrous toluene to prevent oxidation and isolating the black phosphorus crystals,
[0152] viii. purifying the isolated crystals by subjecting them to ultrasonic treatment in absolute ethanol for about-25-35 minutes, thereby removing residual mineralizers and unreacted red phosphorus and yielding high-purity black phosphorus (BP).
[0153] High-purity black phosphorus (BP) was synthesized using a refined chemical vapor transport (CVT) method adapted from previous studies (3,4). In this approach, red phosphorus (RP) served as the elemental precursor, while tin (Sn) and tin(IV) iodide (SnL) were employed as mineralizing agents known to facilitate crystal nucleation and guide the controlled formation of BP through transient intermediate phases. For the synthesis, RP, Sn, and Snh were precisely weighed in mass ratio ranges from about 48:2: 1 to about 52:2: 1 and loaded into a quartz ampoule measuring 18-22 cm in length and 1-2 cm in inner diameter. The ampoule was then thoroughly evacuated and sealed under high vacuum to prevent oxidation and external contamination. After sealing, it was placed horizontally in a programmable muffle furnace, where the temperature gradually increased to 883-903 K and maintained for 4, 5-5, 5 hours to enable complete sublimation of all volatile components except Sn. The furnace temperature was then slowly reduced to 748-768 K, the established regime for BP nucleation and crystal growth, and held for 1,5-2, 5 hours. Subsequently, the ampoule was cooled to 383-403 K to minimize thermal shock and promote crystallinity, followed by natural cooling to ambient temperature. The ampoule was opened under anhydrous toluene to avoid oxidation, and the resulting BP crystals were collected. To remove residual mineralizers and unreacted RP, the crystals were subjected to 25-35 minutes of ultrasonic treatment in absolute ethanol, yielding purified BP suitable for further use.21644.92
[0154] A method of using the photosensitizer in photodynamic therapy comprises following steps:
[0155] i. administering the photosensitizer in a medium to allow its accumulation in targeted tumor tissues,
[0156] ii. exposing the target area to near-infrared (NIR) light to activate the photosensitizer,
[0157] iii. initiating the generation of reactive oxygen species (ROS) upon light activation.
[0158] The invention works on the principle of PDT, where a photosensitizing agent, the binary heterojunction photosensitizer, is activated by light to produce ROS. These ROS induce apoptosis (programmed cell death) in retinoblastoma cells, while healthy retinal cells remain unharmed. The binary heterojunction photosensitizer are activated by NIR light, which can penetrate deeper into biological tissues compared to visible light. This allows the treatment of retinoblastoma tumors located deeper within the eye. Once the photosensitizer is activated by NIR light, the heterojunction efficiently generates ROS at the tumor site. The heterojunction minimizes electronhole recombination, improving the efficiency of ROS generation, which is crucial for effectively destroying cancer cells. Additionally, the binary heterojunction photosensitizer selectively accumulates in tumor tissues, reducing the risk of collateral damage to healthy retinal cells. A key improvement of this invention is the enhanced stability and biocompatibility of the photosensitizer. BP, when used alone, tend to degrade rapidly in biological environments. However, when combined with gCN, stability improves, enhancing effectiveness during treatment. The binary heterojunction photosensitizer, composed of carbon, nitrogen, and phosphorus, are biocompatible and non-toxic, minimizing the risk of immune responses or side effects. The treatment is non-invasive, as the binary heterojunction photosensitizers are administered intravenously, accumulate around the tumor, and are activated by NIR light. This procedure is painless and takes 30 minutes, with the photosensitizer eventually broken down and cleared from the body.
[0159] In vitro experiments were conducted using RPE- 1 cells (Retinal Pigment Epithelial Cell), a healthy control cell line, and retinoblastoma (RB) cells, a cancer cell line. RPE-1 cells were cultured in Dulbecco's Modified Eagle's Medium Nutrient Mixture F-12 (DMEM-F12), supplemented with 10% Bovine Calf Serum (BCF), penicillin, and streptomycin, under controlled conditions of 37 °C with 5% CO2. Similarly, RB cells were grown in RPMI 1640 Medium with 20% BCF, penicillin, and streptomycin. To prepare nanosheets for these cell culture experiments, 3200 pg / ml BP, gCN, and gCN / BP binary heterojunction nanosheet were dissolved in ddHiO and sonicated for 24 hours at 17 °C, protected from light. After sonication, the dispersion was centrifuged at 200021644.92
[0160] rpm for 15 minutes at 4 °C, and the supernatant containing the nanosheets was transferred to a new tube for use. For the cell culture experiment, 100 pL of the stock solution was diluted in 900 pL of the cell medium, followed by serial dilutions to obtain varying concentrations as 320 pg / ml, 160 pg / ml, 80 pg / ml, 40 pg / ml, and 20 pg / ml, which were applied to RPE-1 and RB cells seeded in 96-well plates.
[0161] RPE-1 cells were seeded at a density of 5xl03cells per well, and RB cells at 2xl04cells per well, both incubated overnight at 37 °C. Following incubation, 100 pL of the nanosheet dilutions were added to each well, and the treatment lasted for 24 hours. Cell viability after treatment was assessed using an MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) assay. In another set of experiments to evaluate vitro PDT, after overnight incubation, the culture medium was replaced with 100 pL of the nanosheet solutions, followed by 810 nm NIR laser irradiation for 30 minutes with 38 mW. After irradiation, cells were incubated for another 24 hours to quantify cell viability using the MTT assay. The experimental groups included 40 pg / ml, 80 pg / ml, and 320 pg / ml nanosheet solutions, with and without NIR exposure, as well as control groups receiving only light exposure or no treatment.
[0162] Before combining BP with gCN, the BP and gCN nanosheets alone were tested for cytotoxicity. As shown in Figure 1A, contrary to expectations, increasing concentrations of BP nanosheets resulted in increased RB cell viability. After irradiation, the results remained inconsistent as seen in Figure IB. These unexpected outcomes were attributed to the aggregation of BP nanosheets in the cell medium, as evidenced by brightfield microscopy as shown in Figure 3. Additionally, as shown in Table 1, BP nanosheets exhibited intrinsic absorbance at 570 nm, interfering with the MTT assay results. These issues suggested that the BP nanosheets were not suitable for further experiments due to their interference with the assay. On the other hand, Table 2 showed that gCN nanosheets had no intrinsic absorbance at 570 nm. However, they did not induce RB cell death, even under light exposure as seen in Figure 4A-C. The absorbance (OD570) represents the optical density measured at a wavelength of 570 nm, which reflects the extent of light absorption by the sample and is commonly used as an indicator of cell viability or metabolic activity in bioassays.
[0163]
[0164] 21644.92
[0165] Table 1
[0166]
[0167] Table 2
[0168] To resolve these problems, binary heterojunction photosensitizers were fabricated, which significantly improved the stability of BP and eliminated unwanted signals. As shown in Figure 6A, binary heterojunction photosensitizer alone did not affect RB cell viability without NIR stimulation. However, after dual therapy with nanosheets and NIR, RB cell viability significantly decreased as shown in Figure 6C, with no toxic effects observed on healthy RPE-1 cells as shown in Figure 6B-D.
[0169] Table 3 shows absorbance measurement of binary heterojunction photosensitizer at varying concentrations.
[0170]
[0171] Table 3
[0172] The role of reactive oxygen species (ROS) production was then assessed, a key mechanism in PDT- induced cell death. ROS production was evaluated 24 hours after treatment, and as shown in Figure 8A-C, dual therapy increased oxidative stress in RB cells, while no significant changes were observed in RPE-1 cells as shown in Figure 8B-D. The apoptotic pathway was further examined by Western blot analysis, showing a decrease in the anti- apoptotic protein BCL-2 and an increase in Caspase-9 as shown in Figure 9, confirming the activation of apoptosis in RB cells.21644.92
[0173] Interestingly, Bax expression remained unchanged, suggesting that apoptosis might be driven through other pathways.
[0174] In terms of cell morphology and proliferation, immunofluorescence staining was performed using 4',6-diamidino-2-phenylindole (DAPI) to label cell nuclei and ZO-1 to assess tight junction activity. No damage to healthy RPE-1 cells was observed after treatment as shown in Figure 13. Proliferation assays showed that dual therapy disrupted RB cell integrity and reduced clustering as shown in Figure 11, while no significant effects were seen in healthy cells as shown in Figure 12.
[0175] The TEM micrographs reveal the morphology of the gCN / BP binary heterojunction nanosheets. As shown in Figure 14A-C, the observed structures exhibit irregular, sheet-like domains with varying contrast, indicating the coexistence of thin gCN nanosheets and relatively thicker BP flakes. The uniform dispersion and close contact between the two phases suggest successful heterojunction formation. The presence of distinct interface boundaries supports the interfacial interaction between the components, which is critical for promoting charge transfer pathways in photocatalytic applications. The high-angle annular dark-field (HAADF) STEM image further confirms the hybrid nature of the composite. As shown in Figure 14D, the contrast differences across the image reflect variations in elemental composition and density, where the brighter regions correspond to BP domains due to their higher atomic number, and the darker regions represent the carbon- and nitrogen-rich gCN matrix. The clear structural integration indicates that BP nanosheets are embedded within or anchored onto the gCN framework. Elemental distribution maps for phosphorus (P), carbon (C), and nitrogen (N) further validate the successful integration of the two components. As shown in Figure 14E-H, the phosphorus signal is primarily localized in the brighter HAADF domains, confirming the presence of BP, whereas carbon and nitrogen are homogeneously distributed throughout the composite, consistent with the structural characteristics of gCN. The superimposed elemental map demonstrates the spatial co-localization of BP and gCN, confirming the formation of a well-interfaced heterojunction without significant phase segregation. Overall, the microscopic and elemental analyses confirm the successful assembly of BP and gCN into a uniform heterostructure, which is expected to facilitate efficient charge separation and charge transfer, thereby enhancing photocatalytic performance under visible-light irradiation.
[0176] The XRD pattern of pristine mesoporous g-CsN4 (gCN), as shown in Figure 15, exhibits a broad and intense diffraction peak centered at 29 ~ 28°, which is assigned to the (002) plane21644.92
[0177] corresponding to the interlayer stacking of conjugated aromatic frameworks. This characteristic peak indicates the formation of a layered graphitic structure, which is typical for polymeric carbon nitride materials and is consistent with previously reported mesoporous gCN structures based on 7t— 7t conjugated planar stacking. The XRD pattern of crystalline black phosphorus (BP), as also shown in Figure 15, displays three sharp and intense diffraction peaks located at 29 ~ 17°, 34.5°, and 52.5°, which are indexed to the (020), (040), and (060) planes of the orthorhombic BP crystal structure, respectively. The sharpness and symmetry of these reflections confirm the high crystallinity and phase purity of the synthesized BP, in agreement with previously reported bulk orthorhombic BP structures. The XRD pattern of the gCN / BP binary heterojunction nanosheet hybrid material, as shown in Figure 15, clearly presents the characteristic diffraction features of both gCN and BP, confirming the successful formation of a binary heterostructure. The main gCN diffraction peak near 29 ~ 27.4°, corresponding to the (992) plane, is preserved in the composite, although with slightly reduced intensity, which can be attributed to lattice perturbation caused by BP incorporation. Likewise, the BP (929) reflection at approximately 17° is also retained in the hybrid structure but appears broadened and weakened, indicating partial exfoliation of BP into thinner nanosheets and a corresponding decrease in long-range crystallinity. Importantly, the coexistence of the characteristic diffraction peaks of both gCN and BP without the appearance of new impurity phases demonstrates that the heterostructure is formed predominantly through physical interfacial interaction rather than chemical phase transformation. This preservation of the individual crystal structures within the composite confirms the formation of a well-defined gCN / BP binary heterojunction nanosheet architecture suitable for subsequent photonic and photocatalytic applications.
[0178] The optical absorption properties of pristine gCN and the gCN / BP binary heterojunction nanosheets were investigated by UV-vis-NIR diffuse reflectance spectroscopy (DRS). As shown in Figure 16A, pristine gCN exhibits a distinct absorption edge at approximately 455 nm, which is characteristic of its intrinsic band gap as a polymeric semiconductor. After incorporation of BP, the gCN / BP binary heterojunction nanosheet displays a significant enhancement in light absorption across the entire visible-light region. This pronounced broadening and red shift of the absorption edge indicate successful hybridization between BP and gCN, resulting in improved visible-light harvesting capability. To further quantify the optical band gap energies, the reflectance data were transformed using the Kubelka-Munk function and analyzed by Tauc plots assuming an indirect electronic transition. As shown in Figure 16B, the band gap energy of pristine gCN was estimated to be approximately 2.87 eV, which is consistent with literature values for mesoporous21644.92
[0179] graphitic carbon nitride. In contrast, the gCN / BP binary heterojunction nanosheet exhibited a reduced band gap of approximately 2.75 eV, indicating that the incorporation of BP effectively narrows the energy gap. This band gap narrowing is attributed to interfacial electronic interactions and the formation of intermediate energy levels at the gCN / BP binary heterojunction nanosheet interface. The enhanced light-harvesting capability of the hybrid structure is expected to promote the generation of a larger population of photoexcited charge carriers under visible-light irradiation, which is highly advantageous for improving photocatalytic and photodynamic performance in light-driven applications.
[0180] The steady- state photoluminescence (PL) spectra of pristine gCN and the gCN / BP binary heterojunction nanosheets were recorded to evaluate the radiative recombination behavior of photogenerated charge carriers. As shown in Figure 17A, pristine gCN exhibits a strong emission peak, which is indicative of intense radiative recombination resulting from the limited charge separation efficiency within the bulk polymeric framework. In contrast, the gCN / BP binary heterojunction nanosheet shows a markedly quenched PL intensity, demonstrating a pronounced suppression of the recombination process. This significant reduction in emission intensity indicates the presence of interfacial electronic interactions that promote more efficient charge carrier separation within the heterostructure. To further investigate the carrier dynamics, time-resolved photoluminescence (TRPL) spectroscopy was performed. As shown in Figure 17B, the fluorescence decay curves were fitted using a biexponential decay model to resolve the fast and slow lifetime components. The average carrier lifetime (r_avg) of pristine gCN was determined to be approximately 7.1 ns, which is consistent with values reported for mesoporous carbon nitride systems. Upon formation of the gCN / BP binary heterojunction nanosheet, the average lifetime increased to approximately 7.8 ns, indicating a noticeable delay in charge carrier recombination. This prolongation of carrier lifetime clearly demonstrates that the formation of the gCN / BP binary heterojunction nanosheet enables more effective spatial separation of photoinduced electron-hole pairs. The improved charge transport and suppressed recombination losses are expected to significantly enhance the photocatalytic and photodynamic performance of the composite under visible-light irradiation.
[0181] All statistical analyses were conducted using GraphPad Prism 8.0, with p-values less than 0.05 and 0.01 indicating significance. The analysis confirmed the efficacy and safety of binary heterojunction photosensitizer combined with NIR for retinoblastoma therapy. These results demonstrated that while BP nanosheets alone caused instability and assay interference, the binary21644.92
[0182] heterojunction photosensitizer resolved these issues and significantly enhanced apoptotic effects in RB cells without harming healthy retinal cells.
[0183] The combination of gCN and BP in a binary heterojunction structure enhances light absorption in the NIR spectrum, improving the depth of penetration and making the treatment more effective for deeper tumors. The design also optimizes charge carrier separation, resulting in greater ROS production, which enhances the destruction of tumor cells. Furthermore, by stabilizing BP and ensuring biocompatibility, the invention offers a more durable and patient-friendly solution with fewer side effects. The targeted activation of the photosensitizer in tumor cells reduces damage to healthy tissues, improving overall safety and efficacy.
[0184] In conclusion, the invention provides a significant advancement in the field of photodynamic therapy for retinoblastoma, providing an efficient, stable, and biocompatible treatment option. The use of binary heterojunction photosensitizers offers a novel and powerful tool for the selective destruction of cancer cells while minimizing harm to surrounding healthy tissues.
[0185] References
[0186] [1] Kim, J. W, Jacobsen, B., Zolfaghari, E., Ferrario, A., Chevez-Barrios, P., Berry, J. L., Lee, D.
[0187] K„ Rico, G., Madi, I., Rao, N., Stachelek, K„ Wang, L. C., & Gomer, C. (2017). Rabbit model of ocular indirect photodynamic therapy using a retinoblastoma xenograft. Graefe's archive for clinical and experimental ophthalmology = Albrecht von Graefes Archiv fur klinische und experimentelle Ophthalmologic, 255(12), 2363-2373. https: / / doi.org / 10.1007 / s00417-017-3805-8
[0188] [2] Gomer, C. J., Ferrario, A., & Murphree, A. L. (1987). The effect of localized porphyrin photodynamic therapy on the induction of tumour metastasis. British journal of cancer, 56(1), 27-32. https: / / doi.org / 10.1038 / bjc.1987.147
[0189] [3] Yamamoto, N., Sery, T. W., Hoober, J. K., Willett, N. P., & Lindsay, D. D. (1994). Effectiveness of photofrin II in activation of macrophages and in vitro killing of retinoblastoma cells. Photochemistry and photobiology, 60(2), 160-164. https: / / doi.org / 10.1111 / j.1751- 1097.1994.tb05084.x
[0190] [4] Walther, J., Schastak, S., Dukic-Stefanovic, S., Wiedemann, P., Neuhaus, J., & Claudepierre, T. (2014). Efficient photodynamic therapy on human retinoblastoma cell lines. PloS one, 9(1), e87453. https: / / doi.org / 10.1371 / journal.pone.0087453
Claims
21644.92CLAIMS1. A binary heterojunction nanosheet photosensitizer for use in photodynamic therapy (PDT) for treating retinoblastoma, comprising:• graphitic carbon nitride (gCN), a two-dimensional (2D) polymeric semiconductor, and• black phosphorus (BP).
2. The photosensitizer of claim 1, wherein graphitic carbon nitride (gCN) is a two- dimensional (2D) polymeric semiconductor with a bandgap of 2.7 eV.
3. The photosensitizer of claim 1, wherein bandgap of black phosphorus (BP) is 1.4 eV.
4. The photosensitizer of any of preceding claim, wherein the heterojunctions generate reactive oxygen species (ROS) upon activation by near- infrared (NIR) light, thereby inducing apoptosis in retinoblastoma cells.
5. The photosensitizer of claim 4, wherein the heterojunction is capable of absorbing nearinfrared (NIR) light at a wavelength of 810 nm, enabling deeper tissue penetration for effective treatment of retinoblastoma.
6. A method of synthesizing the binary heterojunction nanosheet photosensitizer for use in photodynamic therapy (PDT) comprising the following steps:i. synthesizing mesostructured graphitic carbon nitride (gCN),ii. synthesizing black phosphorus (BP),iii. suspending black phosphorus (BP) in N-methyl-2-pyrrolidone (NMP) at a concentration from about 1,6 to about 1,8 mg / ml, and sonicating the suspension at a controlled temperature of 20-25°C to obtain exfoliated BP nanosheets,iv. adding mesoporous graphitic carbon nitride, previously dispersed in NMP, into the sonicated BP dispersion wherein a gCN:BP mass ratio ranges from about 4.7:1 to about 5.1:1, wherein the addition is performed under continuous sonication, v. continuing the sonication of the combined dispersion for 3, 5-4, 5 hours so as to promote interfacial contact and heterojunction assembly between the gCN and BP phases,21644.92vi. isolating the resulting gCN / BP composite by centrifuging the mixed suspension at about 8500-9500 rpm for about 15-20 minutes, followed by washing the precipitate twice with ethanol to remove unbound material,vii. drying the washed composite,viii. adjusting the BP loading in the composite, wherein a standard composition comprises about 16,5-17,5 wt % BP relative to the total mass of the gCN / BP binary heterojunction nanosheet, and thereby obtaining the binary heterojunction photosensitizer.
7. The method of claim 6, wherein the method of synthesizing mesostructured Graphitic Carbon Nitride (gCN) comprises following steps:i. dissolving guanidine hydrochloride in deionized water wherein a guanidine hydrochloride concentration ranges from about 0.8 to about 1.2 g / ml, thereby obtaining an aqueous precursor solution,ii. introducing the precursor solution gradually into colloidal silica wherein a guanidine hydrochloride to colloidal silica mass ratio ranges from about 0.37: 1 to about 0.47: 1, under vigorous stirring, thereby allowing the silica to function as a hard template, iii. stirring the resulting dispersion overnight in an oil bath maintained at about 48-52°C to ensure uniform mixing and precursor infiltration,iv. cooling the mixture to room temperature and recovering the solid content, followed by grinding the obtained material into a fine powder,v. placing the ground powder into a lidded quartz crucible and calcining the material under argon atmosphere by heating to about 540-560°C over 1,5-2, 5 hours, maintaining this temperature for an additional 1,5-2, 5 hours, and thereafter allowing the furnace to cool naturally to ambient temperature,vi. etching the silica template by immersing the calcined yellow material in 190-210 ml of 3, 8-4, 2 M ammonium hydrogen difluoride (NH4HF2) solution and stirring the mixture for approximately 46-50 hours,vii. collecting the obtained mesostructured gCN by centrifugation, followed by repeatedly washing the solid with distilled water and ethanol to remove residual fluoride ions and reaction byproducts,viii. drying the final product under ambient conditions to obtain mesostructured graphitic carbon nitride suitable for subsequent composite formation.21644.
928. The method of claim 6, wherein the method of synthesizing Black Phosphorus (BP) comprises following steps:i. weighing and introducing red phosphorus, tin (Sn), and tin(IV) iodide (SnL) into a quartz ampoule wherein a red phosphorus :Sn:SnI4 mass ratio ranges from about 48:2:1 to about 52:2:1, wherein the quartz ampoule has a length of about 18-22 cm and an inner diameter of about 1,5-2 cm,ii. evacuating the ampoule under high-vacuum conditions and sealing it to prevent contamination and oxidative degradation,iii. positioning the sealed ampoule horizontally inside a programmable muffle furnace, iv. increasing the temperature of the furnace gradually to about 883-903 K and maintaining this temperature for approximately 4-5 hours to sublimate volatile species and activate mineralizer-assisted phosphorus transport,v. decreasing the furnace temperature slowly to about 750-760 K and holding the ampoule at this temperature for about 1,5-2, 5 hours to promote nucleation and controlled crystal growth of black phosphorus,vi. cooling the system to about 383-403 K to preserve crystallinity, followed by natural cooling to ambient temperature,vii. opening the ampoule under anhydrous toluene to prevent oxidation and isolating the black phosphorus crystals,viii. purifying the isolated crystals by subjecting them to ultrasonic treatment in absolute ethanol for about 25-35 minutes, thereby removing residual mineralizers and unreacted red phosphorus and yielding high-purity black phosphorus (BP).
9. A method of using the photosensitizer of claim 1 in photodynamic therapy, comprising following steps:i. administering the photosensitizer in a medium to allow its accumulation in targeted tumor tissues,ii. exposing the target area to near-infrared (NIR) light to activate the photosensitizer,iii. initiating the generation of reactive oxygen species (ROS) upon light activation.21644.9210. The method of using the photosensitizer of claim 7, wherein the activation procedure involves non-invasive light exposure lasting-30 minutes, with the photosensitizer breaking down naturally after the procedure.