Biocompatible and injectable photovoltaic retinal prosthesis

Abstract

The present invention relates to hollow graphitic carbon nitride nanospheres (hg- C3N4) for use as medicaments. In particular, the present invention relates to hollow graphitic carbon nitride nanospheres (hg-C3N4) as biocompatible and injectable photovoltaic retinal prosthesis for use in the treatment or alleviation of visual deficit and / or cardiac disease. The invention also relates to pharmaceutical compositions comprising the hollow graphitic carbon nitride nanospheres (hg- C3N4).

Description

[0001]82931PC01 1 Biocompatible and injectable retinal prosthesis Technical field of the invention The present invention relates to hollow graphitic carbon nitride nanospheres (hg- C3N4) for use as medicaments. In particular, the present invention relates to hollow graphitic carbon nitride nanospheres (hg-C3N4) as biocompatible and injectable photovoltaic retinal prosthesis for use in the treatment or alleviation of visual deficit and / or cardiac disease. The invention also relates to pharmaceutical compositions comprising the hollow graphitic carbon nitride nanospheres (hg- C3N4). Background of the invention The possibility to electrically sense and stimulate living tissue opens up unprecedented potential for therapeutic applications. Particularly, neural and cardiac disease treatment can benefit from materials allowing electrical modulation of bioelectric activity. As almost all cells are electroactive and respond to electrical signals, electrical stimulation on specific cells needs to be delivered in a precise and controllable manner with high spatiotemporal control. Optogenetics has proven to be a versatile tool to control transmembrane and intracellular bioelectric activity by light trigger, with high cell-specific selectivity and spatiotemporality. However, clinical translation of the optogenetic approach is hampered by the requirement for genetic modifications and concerning immunogenicity of the introduced exogenous transmembrane proteins of often non-mammalian origin. Interfaces between biological systems and nanomaterials open an array of possibilities for non-genetic modulation of bioelectric activity with similar spatiotemporal control. Semiconductor materials with intriguing optoelectric properties, which can be designed in various shapes and patterns at the nanoscale, are promising candidates to achieve the optically active biointerface. This is especially true for intracellular electrical interfaces, as the natural scale of organelles ranges within tens and several hundred nanometers. Nanoparticles (NPs) have shown to be able to build tight interfaces with both intra- and extracellular membranes. Importantly, light as external power source 82931PC01 2 can trigger electrochemical or effects at the particle / cellular interface acting as a leadless electrophysiological modulator. The organic semiconductor graphitic carbon nitride (g-C3N4) has drawn attention in various fields including biomedical applications. C. A. Müller et al. (Bionic Opto- responsive Fiber for Directing Neurite Growth. Materials Today Nano, 100311 (2023)) have demonstrated that bulk g-C3N4 combined with graphene oxide can be used to control neurite outgrowth of PC12 cells under visible-light stimulation. A band gap of 2.7 eV makes g-C3N4 a potent photocatalyst within the visible-light range. Sun et al. (Bioinspired hollow semiconductor nanospheres as photosynthetic nanoparticles. Nature Communications | (2012) 3:1139) demonstrates a polymeric hollow nanospheres of graphitic carbon nitride (hg-C3N4) with advantages from the inner optical reflection and improved structure condensation provide a remarkably increased photochemical activity. However, the use of hollow g-C3N4 in a biomedical setting has not been explored nor considered. Francia et al. (Light-induced charge generation in polymeric nanoparticles restores vision in advanced-stage retinitis pigmentosa rats. Nature Communications | (2022) 13:3677) and US 2020 / 0113843 A1 demonstrate the functional role of organic photovoltaic nanoparticles being non-hollow P3HT-NPs in restoring visual activities in fully degenerate retinas. P3HT-NPs are non-hollow and heterogenous nanoparticles made of polymerized 3-hexyl-thiophene. Bai et al (Materials Letters 65 (2011) 1101-1104) discloses hollow g-C3N4 nanosphere synthesis by solvothermal technique using silica nanospheres as template and its use as drug delivery carriers. Hence, an improved pharmaceutical composition for stimulating living tissue would be advantageous, and in particular, a more efficient pharmaceutical composition for electrical modulation of bioelectric activity of living tissue without any invasive surgery would be advantageous. 82931PC01 3 Summary of the invention The present invention relates to the identification of hollow graphitic carbon nitride nanoparticle (hg-C3N4) as being very suitable as injectable photovoltaic retinal prosthesis. Particularly, the particles hold potential to help to restore vision loss for diseases characterized by degradation of photoreceptor cells. Activated by visible light, the particles could stimulate retinal ganglion cells via photoelectrochemical and photothermal effects, and thereby reestablish light sensitivity of the eye. Further, the ability of the particles to stimulate cardiac cells could be highly beneficial in the treatment of cardiac disease like heart arrhythmia. As conventional pacemaker therapy goes along with invasive surgery to implant the bulky device and relies on leads for the power transfer, the injectability and the wireless light-based power transfer can help to reduce risks associated with lead- based power transfer. By light stimulation (photostimulation), the particles act on adjacent cardiomyocytes and could enable synchronization and pacing of the cardiomyocytes to the target light pulse frequency. Example 1 shows synthesis and characterization of hollow sphere graphitic carbon nitride particle (hg-C3N4). The results show that a homogeneous hollow spherically shaped hg-C3N4nanoparticle is obtained with an inner diameter of around 110 nm and an outer diameter of 190 nm, leaving a controlled amorphous shell of around 80 nm. hg-C3N3 showed up to 4 times higher light absorbance within a range of 350-600 nm, indicating considerably more absorbance in the visible spectrum compared to bulk non-hollow g-C3N4. Example 2 shows that the hg-C3N4-mediated photostimulation is based on both mechanisms, anodic faradaic currents and evident photothermal effects. Example 3 shows that hg-C3N4 nanoparticles can successfully be internalized. The example further shows that hg-C3N4 is cytocompatible with no considerable toxic effects in cells. Example 4 shows photo stimulation of NIH / 3T3 fibroblasts and HELA cells with internalized hg-C3N4. 82931PC01 4 Example 5 shows that hg-C3N4 associated with or internalized into cardiomyocytes (CMs) and cardiac fibroblasts (CF) were directly stimulated, resulting in immediate intracellular calcium flux and beating. These results are promising because it shows that the light-mediated leadless stimulation approach of the particles can trigger beating in primary cardiomyocytes (CMs), which could be highly useful for cardiac stimulation. Example 6 shows that hg-C3N4 is able to modulate cardiomyocyte (CM) activity by applying LED stimulation with fixed frequency leading to synchronous beating. Example 7 shows the potential of hg-C3N4 particles as an injectable retinal prosthesis that can induce pronounced photo-response in porcine retinal tissue and holds great potential to help patients with photoreceptor degeneration, such as retinitis pigmentosa and age-related macular degeneration. This promising approach may offer hope to those suffering from vision loss. Example 8 shows that intravitreal injection seems to be a safe delivery route for hg-C3N4 NPs. The hg-C3N4 compound has shown several surprising advantages compared to state-of-the-art molecules such as P3HT-NPs. Advantages are: - The hollow structure of hg-C3N4 results in better light harvesting properties, compared to non-hollow structures, such as P3HT. Due to the high degree of tunability, g-C3N4 can achieve higher Power Conversion Efficiency (PCE) than P3HT (up to 15% vs 3% for P3HT) depending on the configuration of the tunable structure. (Chemical Physics Letters Volume 845, 16 June 2024, 141300 and Solar Energy Volume 239, June 2022, Pages 74-87). Hollow structures may also act as a typical drug delivery vehicle, which can be easily modified with targeting molecules for example antibodies. - Internalization: The ability of hg-C3N4 to be internalized into target cells as shown in Example 3 and Example 4, enables photostimulation with subcellular resolution. US 2020 / 0113843 A1 discloses the inability of non- hollow P3HT-NPs to be internalized (see paragraph 0012). Internalization into cells results in prolonged retention times and slower clearance. This 82931PC01 5 characteristic could reduce required dosage and frequency of administration, providing a more patient-friendly treatment regimen. Efficient internalization is vital for achieving subcellular resolution in therapeutic applications, improving overall effectiveness, and minimizing potential side effects. - The melting temperature of hg-C3N4 is much higher than for P3HT. According to ACS Chemistry for Life (https: / / www.acs.org / molecule-of-the- week / archive / c / carbon-nitride.html), the melting temperature of g-C3N4 is 600–700 °C. Trinh Tung Ngo et al. (2012 Adv. Nat. Sci: Nanosci. Nanotechnol. 3045001) describes the melting temperature of P3HT being 233.7 °C (page 3, column 1). Hence, the higher melting temperature of g- C3N4 indicates a higher stability of g-C3N4 compared to P3HT. - Homogeneity: The size uniformity (homogeneity) of hg-C3N4 is greater than that of P3HT (when comparing SEM data shown in Francia et al. (supplementary Figure 2A) and figure 2A presented in here, as well as the SEM data shown in US 2020 / 0113843 A1 (Figure 1A) and Figure 3A presented in here) and is essential for ensuring predictable cell-particle interactions. Therefore, the particles of the present invention should show better control over biodistribution and reduced variability in therapeutic outcomes. This predictability enhances patient safety and makes outcomes more reliable during treatment. (Danaei et al. Pharmaceutics 2018, 10(2), 57). - Chemical stability: The chemical stability of hg-CN makes it practically inert to chemical attacks (Sun et al. (Bioinspired hollow semiconductor nanospheres as photosynthetic nanoparticles. Nat. Commun. 3, 1139, 2012), allowing it stay stable in the acidic condition inside the lysosome. P3HT might not be able to withstand as it is an organic nanoparticle as the highly acidic environment of lysosomes, rich with enzymes, contributes to the degradation of organic nanoparticles. (Blanco et al. (Nanoparticle design to overcome biological barriers in drug delivery. Nat. Biotechnol. 33, 941–951, 2015)). Thus, an object of the present invention relates to the provision of an improved composition comprising hg-C3N4 for use as a medicament and / or for use in a treatment of vision loss and / or cardiac disease. 82931PC01 6 In particular, it is an object of the present invention to provide a pharmaceutical composition that solves the above-mentioned problems of the prior art with an improved composition comprising hg-C3N4 for use as a medicament and treatment of diseases involving excitable cells in order to perform photostimulation of target cells and to induce intracellular bioelectric activity. Thus, one aspect of the invention relates to a composition comprising one or more hollow graphitic carbon nitride (hg-C3N4) nanoparticles for use as a medicament. Another aspect of the present invention relates to a composition comprising one or more hollow graphitic carbon nitride (hg-C3N4) nanoparticles for use in the treatment or alleviation of photoreceptor degeneration, such as retinitis Pigmentosa (RP) or related syndromes, preferably said syndromes being selected from the group consisting of Usher Syndrome, Bardet-Biedl syndrome, Refsum disease, Batten disease, and Jalili syndrome; neuropathy, ataxia, NARP (neuropathy, ataxia, and retinitis pigmentosa) syndrome, inherited retinal degenerations, preferably selected from the group consisting of Stargardt's disease, Leber's congenital amaurosis, and atrophic age-related macular degeneration (AMD). A third aspect of the present invention relates to a composition comprising one or more hollow graphitic carbon nitride (hg-C3N4) nanoparticles for use in the treatment or alleviation of cardiac disease, such as arrhythmia, congestive heart failure, cardiomyopathy, bradycardia, tachycardia. Yet another aspect of the present invention is to provide a pharmaceutical composition comprising one or more hollow graphitic carbon nitride (hg-C3N4) nanoparticles. Another aspect of the invention relates to use of one or more hollow graphitic carbon nitride (hg-C3N4) nanoparticles for in vitro stimulation of one or more cells, such as photo-stimulation and / or light-induced stimulation. 82931PC01 7 Brief description of the figures Figure 1 Figure 1 shows (A) a SEM image of hg-C3N4,which clearly shows the hollow sphere structure of the particles with an inner diameter of around 110 nm and an outer diameter of 190 nm, leaving a controlled amorphous shell of around 80 nm. (B) A TEM image of hg-C3N4,which clearly shows the hollow sphere structure of the particles with an inner diameter of around 110 nm and an outer diameter of 190 nm, leaving a controlled amorphous shell of around 80 nm. (C) An AFM topographical height line profile of single hg-C3N4particle (1-1’) and aggregated hg-C3N4 particles and a particle height distribution of (2-2’). (D) shows a Stokes diameter distribution derived from particle tracking analysis of hg-C3N4. (E) A UV- vis absorption spectrum of hg-C3N4 and bulk g-C3N4. Figure 2 Figure 2 shows (A) an illustration of a patch-clamp photo-response measurement set-up used to measure photocurrent. (B) shows photocurrent traces measured for a range of holding currents I0 while applying 100 ms LED illumination. (C) shows photocurrent amplitude for a range of holding currents 100 ms LED illumination. (D) shows quantification of hg-C3N4 produced H2O2 under visible- light LED illumination. Data (n=7) is shown as means ± s.d. Statistical analysis was performed using unpaired two-tailed t-test where p<0.0001 is denoted as ****. Figure 3 Figure 3 shows (A) a scheme of the proposed internalization mechanism. After coming in contact with the cell membrane, the particles could be engulfed in a phagosome. The directed transport towards the perinuclear region of cells could be enabled by active transport with the cytoskeleton proteins, where particles form clusters and aggregate. (B) shows particle tracking of eleven internalized particles over 16h. Dashed circle in the middle represents nucleus. (C) shows quantification of directedness of internalized and non-internalized particles shown as boxplots (n=8). Statistical analysis was performed using unpaired two-tailed t- test where p<0.0001 is denoted as ****. (D) shows LDH cytotoxicity assay. Data (n=6) is shown as means ± s.d. (E) shows percentage of dead cells based on live 82931PC01 8 dead staining. Statistical analysis performed with one-way ANOVA and Tukey’s test for multiple comparisons where p<0.0001 is denoted as **** with n=7 and ns=no significance (p>0.05). Figure 4 Figure 4 shows (A) intracellular electrical interrogation of NIH / 3T3 fibroblast with corresponding vector map. The arrow indicates the location of 200 ms laser pulse, 473 nm, spot size 3 µm, 8.8 mW / µm2. The scale bar is 10 µm. (B) shows intracellular electrical interrogation of a HELA cell with corresponding vector map. The arrow indicates the location of 200 ms laser pulse, 473 nm, spot size 3 µm, 8.8 mW / µm2. The scale bar is 10 µm. (C) shows the intercellular calcium propagation with corresponding ΔF / F0 plots between CFs. (D) shows the intercellular calcium propagation with corresponding ΔF / F0 plots between CF and CM. (E) shows the intercellular calcium propagation with corresponding ΔF / F0 plots between CM and CM. Figure 5 Figure 5 shows (A) a scheme of the MEA retina measurement setup. (B) shows tracings of light-evoked potentials with 32 channel MEA while applying 1 ms 450 nm LED pulse, with 30 mW / cm2 (left). Quantification of peak-peak amplitudes recorded for each channel of the 32 channel MEA. Horizontal line indicates 25% and 75% percentile of TTX silenced firing. Statistical analysis was performed using the unpaired two-tailed t-test where p<0.0001 is denoted as **** (right). (C) shows average tracings of light-evoked potentials of 32 channels for different light doses of 100 ms 450 nm LED pulse (left). Quantification of peak-peak amplitudes of light dose-response recordings. Statistical analysis was performed with one- way ANOVA and Tukey’s test for multiple comparisons where p<0.0001 is denoted as ****(right). Figure 6 Figure 6 shows Tri-s-triazine, the building blocks making up g-C3N4 (Sun et al. 2012). 82931PC01 9 Figure 7 Figure 7 shows intravitreal delivery of hg-C3N4 NPs is safe and results in NP adherence to the retinal surface near the RGCs and their axons. (A) Schematic drawing of the intravitreal injection procedure and experimental timeline. (B) Representative fundus images and optical coherence tomography scans from eyes intravitreally injected with hg-C3N4 NPs (NP IVT) or buffer (PBS IVT). The integrity of retinal layers is preserved and hyperreflective spots corresponding to aggregated NPs (white arrowheads) can be observed in the vitreous and on the retinal surface. Scale bars are 200 µm. (C) Position of retinal borders in the NP IVT eyes at baseline and D14 (mean ± sd) with comparisons of the mean thickness of interposed retinal layers. Paired t-test between mean thickness of specified retinal layers, baseline vs. D14. n = 6. NGI = combined thickness of retinal nerve fiber layer, ganglion cell layer and inner plexiform layer; INL = inner nuclear layer; OPL = outer plexiform layer; ONL = outer nuclear layer. The present invention will now be described in more detail in the following. Detailed description of the invention Definitions Prior to discussing the present invention in further details, the following terms and conventions will first be defined: Graphitic carbon nitride (g-C3N4) The organic semiconductor known as graphitic carbon nitride (g-C3N4) has gained significant attention in the field of optoelectronics due to several advantageous characteristics. These include its ease of synthesis, affordability, remarkable structural and chemical stability, excellent biocompatibility, minimal cytotoxicity, and exceptional photocatalytic performance. Structurally, g-C3N4 is composed of building blocks consisting of heptazine and s-triazine units (Figure 6). With its inherent band gap of 2.7 eV (approximately 460 nm), it serves as a highly efficient photocatalyst within the visible light spectrum. The simplicity of these building units renders g-C3N4 highly customizable, enabling adjustments of its physicochemical properties based on its specific structure, morphology, and chemical composition. 82931PC01 10 Hollow graphitic carbon nitride (hg-C3N4) In the present context the terms “hollow or “polymeric hollow nanosphere of graphitic carbon carbon nitride nanoparticle” refer to hollow graphitic carbon nitride nanospheres. In the present context, these terms may be used interchangeably. Sun et al. (2012) synthesized hollow g-C3N4 nanospheres (hg-C3N4) using silica nanoparticles as templates. These nanoparticles possess a unique hollow architecture, offering a greater surface area that directly improves catalytic efficiency. Additionally, the separation of the exterior and interior of the nanospheres allows for localized photo oxidation and photo reduction reactions, which must be strictly separated in nanospace. Retinitis Pigmentosa (RP) Retinitis Pigmentosa (RP) arises from mutations in numerous genes associated with the process called photo transduction. It primarily entails the degeneration of rod cells, with cone cell survival being affected later in the disease progression. Consequently, RP patients experience an early deterioration in their scotopic vision (vision of the eye in low light conditions), leading eventually to complete blindness. Scotopic vision relies solely on the functionality of rod cells, which exhibit the highest sensitivity to light wavelengths at around 498 nm (green-blue) and are unresponsive to wavelengths longer than approximately 640 nm (red). Age-related macular degeneration (AMD) Age-related macular degeneration (AMD) is a condition characterized by the selective degeneration of foveal cone cells and is prevalent among a significant portion, potentially up to 20%, of individuals aged 75 and older. In the context of AMD, the primary impairment occurs in the foveal photoreceptors, leading to a notable loss of high-resolution vision in the center of the visual field. The fovea, also known as the central fovea or fovea centralis, is a small depression within the macula of the retina, and it plays a pivotal role in providing the clearest and most precise vision. Activatable 82931PC01 11 In the present context, to be understood as the hg-C3N4 nanoparticles being able to induce bioelectric activity in target cells, where hg- C3N4 nanoparticles are associated with or internalized in, upon activation by an external source, such as light and / or radiation. The hg-C3N4 nanoparticles are able to modulate cells electrophysiology on the subcellular level, intercellular level, and tissue level. The hg-C3N4 nanoparticles will, upon activation, modulate single cell signalling across a broad spectrum of excitable and non-excitable cell types, and notably endow intercellular signal propagation. Hollow sphere graphitic carbon nitride particles (hg-C3N4) for use as a medicament The ability of g-C3N4 to be fabricated as hollow nanospheres with improved optoelectronic properties, broadens its potential towards 0D optical biointerfaces. The inventors have demonstrated the versatility of hg-C3N4 particles for modulating electrophysiology on the subcellular level, intercellular level, and tissue level upon light trigger. As seen in Examples 4, 5 and 6, hg-C3N4 as organic semiconductor nanoparticles are able to modulate single cell signalling across a broad spectrum of excitable and non-excitable cell types, and notably endow intercellular signal propagation by applying laser stimulation with subcellular resolution. The fact that the photo-response of hg-C3N4is triggered within the visible light range, holds great potential for hg-C3N4 being applied as a medicament and for use in the treatment of visual deficiencies, such as retinitis pigmentosa and age-related macular degeneration, as an injectable retinal prosthesis (See Example 7). Thus, an aspect of the invention relates to a composition comprising one or more hollow graphitic carbon nitride (hg-C3N4) nanoparticles for use as a medicament. As shown in Examples 4-7, hg-C3N4 induces intracellular bioelectric activity and thereby holds great potential for being applied as a medicament. Example 7 shows that the composition comprising hg-C3N4 can induce pronounced photo-response in porcine retinal tissue and holds great potential to help patients with photoreceptor degeneration. Hence, a second aspect of the invention is directed to a composition comprising one or more hollow graphitic carbon nitride (hg-C3N4) nanoparticles for use in the treatment or alleviation of photoreceptor degeneration, such as retinitis Pigmentosa (RP) or related syndromes, preferably 82931PC01 12 said syndromes being selected group consisting of Usher Syndrome, Bardet-Biedl syndrome, Refsum disease, Batten disease, and Jalili syndrome; neuropathy, ataxia, NARP (neuropathy, ataxia, and retinitis pigmentosa) syndrome, inherited retinal degenerations, preferably selected from the group consisting of Stargardt's disease, Leber's congenital amaurosis, and atrophic age- related macular degeneration (AMD). A third aspect of the invention relates to a composition comprising one or more hollow graphitic carbon nitride (hg-C3N4) nanoparticles for use in the treatment or alleviation of cardiac disease, such as arrhythmia, congestive heart failure, cardiomyopathy, bradycardia, tachycardia. As seen in Example 5, the composition comprising hg-C3N4 particles can electrically modulate cardiomyocytes (CMs). hg- C3N4 nanoparticles associated with or internalized into CMs were directly stimulated, resulting in immediate intracellular calcium flux and beating. The calcium flux of the stimulated cell led to calcium flux and beating of three adjacent coupled CMs. Cell-cell signal propagation was observed not only between the CMs, but also cardiac fibroblasts (CF). In an embodiment, the composition is administered by injection. The route of administration is to ensure that hg-C3N4 nanoparticles reach the desired location. In an embodiment, the composition is administered and / or applied and / or injected intraorbitally or intracapsular, preferably by microinjection into a subretinal or epiretinal space, wherein said microinjection is preferably performed through the sclera and / or through the vitreous chamber. This is to ensure that hg-C3N4 nanoparticles reach and are internalized in the excitable cells in the eye. In an embodiment, the composition is administered intrapericardially, intramyocardially, epicardially or intravenously to the heart. This is to ensure that hg-C3N4 nanoparticles reach and are internalized in the excitable cells in the heart. In an embodiment, the composition is activatable by a light emitting device implanted in or near the heart. As seen in Examples 5 and 6, the hg-C3N4 nanoparticles in the cardiomyocytes are activatable by a laser and LED light source respectively. In practice, hg-C3N4 nanoparticles injected into the heart of a 82931PC01 13 subject requires activation by a device near or on site. Therefore, such light emitting device may be implanted in or near the heart. In an embodiment, the hg-C3N4 nanoparticles are internalized in one or more target cells of the subject. As seen in Example 3, the hg-C3N4 nanoparticles are successfully internalized in the target cells. Further, the study confirms that hg- C3N4 is cytocompatible with no toxic effects in cells. In an embodiment, the target cells are excitable cells such as retinal ganglion cells and cardiomyocytes. Examples 5 and 6 show that hg-C3N4 nanoparticles can be internalized in cardiomyocytes. Example 7 shows that hg-C3N4 nanoparticles can be internalized in retinal ganglion cells. Example 4 even shows that hg-C3N4 nanoparticles can be internalized in the non-excitable cell types NIH / 3T3 fibroblasts and HELA cells. In an embodiment, the subject suffers from visual deficit and / or cardiac disease. As shown in Examples 5, 6, and 7 the hg-C3N4 nanoparticles are able to induce intracellular bioelectric activity in retinal cells and cardiac cells, thus having great potential in the treatment of subjects suffering from visual deficit and / or cardiac disease. In an embodiment, the visual deficit is associated with degeneration of photoreceptors in the eye, preferably cone degeneration, more preferably foveal cone degeneration; and / or the degeneration of photoreceptors is selective degeneration of photoreceptors, preferably cone degeneration and / or macular degeneration. Cone degeneration is typical of retinal degenerative diseases. The result of such hg-C3N4 nanoparticles for use in the treatment of visual deficit for subjects in need thereof, is regaining or improving light sensitivity and thereby restoring vision completely or partially. In a preferred embodiment, the subject is mammal, preferably human. As also described above, when it comes to treatment of heart diseases, an implanted light emitting source may be required. Thus, in an embodiment, the subject has an implanted light source capable of activating hg-C3N4 nanoparticles delivered to the heart. It could also be foreseen that the light source is implanted after the hg- 82931PC01 14 C3N4 nanoparticles are the subject. Thus, in another embodiment, said subject is scheduled for having a light source implanted capable of activating hg-C3N4 nanoparticles delivered to the heart. In a preferred embodiment, the composition is a pharmaceutical composition. In an embodiment, the composition is administered with a concentration of hg- C3N4 nanoparticles in the range 0.01 – 100 mg / ml, such as 0.05 – 50 mg / ml, preferably such as 0.1 – 10 mg / ml, more preferably such as 0.3 -5 mg / ml. In another embodiment the composition is administered with a concentration of hg-C3N4 nanoparticles in the range 2.5 – 500 µg / ml, such as 10 – 300 µg / ml, such as 15 – 200 µg / ml, such as 20 – 100 µg / ml, preferably 25-50 µg / ml. The composition comprising hg-C3N4 nanoparticles should be administered in a therapeutic effective amount to produce a biological response. More specifically, the amount and number of hg-C3N4 nanoparticles administered to a subject should be sufficient to produce bioelectric activity in the target cells. Thus, in yet an embodiment, the composition is administered with an amount of hollow graphitic carbon nitride (hg-C3N4) nanoparticles in the range 2 – 5000 µg, such as 20 – 2000 µg, such as 50-1000 µg, preferably 100-500 µg, more preferably 150-250 µg. These amounts are based on comparing the human eye volume to the mouse eye volume. The human eye has a diameter of around 24 mm, resulting in a volume of approximately 7238 mm³. In contrast, the mouse eye has a diameter of about 4 mm, giving it a volume of 33.5 mm³. This results in a ratio of approximately 212. In mouse experiments (see example 8), 1 µL of NP at a concentration of 1 mg / mL (1 µg) was injected. Based on this ratio, for humans, the corresponding dosage could be around 212 µg of hg-C3N4. Pharmaceutical composition comprising hg-C3N4 The use of hg-C3N4 as a pharmaceutical composition in a biomedical setting has not previously been explored nor considered. Thus, another aspect of the invention relates to a pharmaceutical composition comprising one or more hollow graphitic carbon nitride (hg-C3N4) nanoparticles. 82931PC01 15 In an embodiment, the pharmaceutical composition further comprises pharmaceutically acceptable saline or hydrogel. In an embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, solvent and / or diluent. In an embodiment, the pharmaceutical composition further comprises an osmotic agent. Especially, when the pharmaceutical composition is administered to the eye, the osmotic pressure must be suitable for treatment and uptake. Hence, an osmotic agent will ensure a correct ocular pressure. In an embodiment, the pharmaceutical composition further comprises a pharmaceutically effective amount of at least one additional ingredient preferably selected from the group consisting of anti-inflammatory, astringent drugs, preferably selected from the group consisting of neostigmine methylsulfate, ε- amino caproic acid, allantoin, berberine chloride, zinc sulfate, lysozyme chloride, sodium azulene sulfonate, dipotassium glycyrrhizinate and combinations thereof; antiallergic agents, preferably selected from the group consisting of diphenhydramine hydrochloride, isopenzyl hydrochloride, chlorpheniramine maleate, sodium cromoglycate and combinations thereof; vitamins other than pyridoxine hydrochloride, preferably selected from the group consisting of vitamin B2, vitamin B12, vitamin A, vitamin E, calcium pantothenate and combinations thereof, amino acids, preferably selected from the group consisting of potassium L-aspartate, magnesium L-aspartate, aminoethylsulfonic acid and combinations thereof; sulfa drugs, preferably selected from the group consisting of sulfamethoxazole, sulfisoxazole, sulfisomidine and combinations thereof, bacteriocides, preferably selected from the group consisting of sulfur, isopropylmethyl phenol, hinokithiol and combinations thereof; topical anesthetics, preferably selected from: lidocaine, lidocaine hydrochloride, procaine hydrochloride, dibucaine hydrochloride and combinations thereof, inorganic salts, preferably selected from the group consisting of potassium chloride, sodium chloride, sodium bicarbonate and combinations thereof, thickening agents, preferably selected from the group consisting of polyvinyl alcohol, polyvinyl 82931PC01 16 pyrrolidone, carboxymethyl hyaluronic acid, glucose and combinations thereof. In an embodiment, the pharmaceutical composition further comprises at least one additive, preferably solubilization auxiliary agents, isotonizing agents, stabilizing agents, chelating agents, pH adjusting agents, refrigerants, preservatives, and thickening agents; and / or a carrier, preferably buffer agents or ointment bases or a combination thereof. In an embodiment, the pharmaceutical composition is administered with a concentration in the range 2.5 – 500 µg / ml, such as 10 – 300 µg / ml, such as 15 – 200 µg / ml, such as 20 – 100 µg / ml, preferably 25-50 µg / ml. The pharmaceutical composition comprising hg-C3N4 nanoparticles should be administered in a therapeutic effective amount to produce a biological response. More specifically, the amount and number of hg-C3N4 nanoparticles administered to a subject (to an eye) should be sufficient to produce bioelectric activity in the target cells. In example 8 (in a mice study), 1 µL NP (1 mg / mL) were injected. Thus, in an additional embodiment, the pharmaceutical composition is administered with a concentration in the range 0.01 – 100 mg / ml, such as 0.05 – 50 mg / ml, preferably such as 0.1 – 10 mg / ml, more preferably such as 0.3 - 5 mg / ml. In another embodiment, the pharmaceutical composition is administered with an amount of hg-C3N4 nanoparticles in the range 2 – 5000 µg, such as 20 – 2000 µg, such as 50-1000 µg, preferably 100-500 µg, more preferably 150-250 µg. Preferably the indicated amounts and / or concentrations are per eye, more preferably a human eye. In a preferred embodiment, the pharmaceutical composition being a photovoltaic retinal prosthesis. Example 7 demonstrates the potential of the pharmaceutical composition being a photovoltaic retinal prosthesis. In a preferred embodiment, the pharmaceutical composition being an ophthalmic pharmaceutical composition, preferably an injectable ophthalmic composition. 82931PC01 17 Example 7 demonstrates the of the pharmaceutical composition being an ophthalmic pharmaceutical composition. In an embodiment, the pharmaceutical composition has a pH value in the range 6.6-7.8, more preferably in the range 7.0-7.5. The pH value must be generally acceptable for ophthalmic applications to optimize ocular comfort; the pH value of the ophthalmic formulations should preferably match the tear film or at least be in the ocular range of 6.6-7.8, as an acidic or alkaline pH value may induce lacrimation, ocular pain, and discomfort. In an embodiment, the pharmaceutical composition has an osmotic pressure value is in the range 280 and 310 mOsm / L. The osmotic pressure value must be generally acceptable for ophthalmic applications, preferably in the range 290 and 300 mOsm / L. In an embodiment, the pharmaceutical composition is in an ophthalmic acceptable composition, preferably selected from aqueous solutions, aqueous suspensions, aqueous dispersion, emulsions, gelatinous materials, ointments and combinations thereof. In an embodiment, the hg-C3N4 nanoparticles have an inner diameter in the range 50- 500 nm, such as 100 – 120 nm, preferably 105-115 nm, more preferably 110 nm and / or an outer diameter in the range 60-600 nm, such as, 170 – 210 nm, preferably 180 – 200 nm, more preferably 190 nm. As seen in Example 1, the hg- C3N4 nanoparticles tested have an inner diameter of around 110 nm and an outer diameter of 190 nm, leaving a controlled amorphous shell of around 80 nm. However, the inner and outer diameters of the hg-C3N4 nanoparticles are adjustable depending on the desired size and shell thickness of the final hg-C3N4 nanoparticles. For example, the size is adjustable, by adjusting the size of silica template used. In an embodiment, the hg-C3N4 nanoparticles comprise at least one surface modification and / or at least one cargo agent. As the hg-C3N4 nanoparticles have hollow structure, it is possible to load the hg-C3N4 nanoparticles with a cargo of interest. 82931PC01 18 In a preferred embodiment, the surface modification is a peptide or oligonucleotide, such as an aptamer or polymerized cathecholamines. The surface modification is for the hg-C3N4 nanoparticles to be delivered to the site of interest, to further functionalize the hg-C3N4 nanoparticles, as a bioadhesive to bind further peptides, and / or to increase photoelectrochemical and photothermal effects. In an embodiment, the cargo agent is a drug. The potential for the hollow hg-C3N4 nanoparticles to be loaded with a cargo agent provides the possibility of loading the hg-C3N4 nanoparticles with a drug of interest for delivery to a subject. Further, it is possible to provide hg-C3N4 nanoparticles with surface modifications and loaded with a drug, for delivery to a predetermined site in the subject. In an embodiment, the hg-C3N4 nanoparticles are activatable by an external light source. As seen in Example 1, the hg-C3N4 nanoparticles showed up to 4 times higher light absorbance within a range of 350-600 nm, indicating considerably more absorbance in the visible spectrum. This effect could be attributed to multiple reflection sites residing in the hollow structure, prolonging the light path length for absorption within the spheres. In Examples 4-7, it is seen that the hg- C3N4 nanoparticles induce light-triggered intracellular bioelectric activity, which is evident from the observed calcium flux and pulse measurements. In an embodiment, the external source is a light source, such as natural light and radiation. As seen in Examples 4-7, the tested external sources for activating the hg-C3N4 nanoparticles were a laser or LED light. In an embodiment, the external source is implantable in the heart. As seen in Examples 5 and 6, the hg-C3N4 nanoparticles in the cardiomyocytes are activatable by a laser and light source. In practice, hg-C3N4 nanoparticles injected into the heart of a subject requires activation by an external source near or on site. Therefore, such external source, such as a light emitting device, may be implanted in the heart, such as before, simultaneous or after providing the hg- C3N4 nanoparticles to the heart. 82931PC01 19 Use of hg-C3N4 nanoparticles The use of hg-C3N4 nanoparticles for the purpose of internalization and stimulation of cells has not previously been explored nor considered. Thus, another aspect of the invention relates to use of one or more hollow graphitic carbon nitride (hg- C3N4) nanoparticles for in vitro stimulation of one or more cells. It is indeed evident from Examples 4-7 that hg-C3N4 nanoparticles can be in vitro internalized in cells, such as NIH / 3T3 fibroblasts, HELA cells, cardiomyocytes, and ex vivo in retinal cells, and afterwards the hg-C3N4 nanoparticles are in vitro activated for stimulation of the cells. In an embodiment, the cells are excitable cells such as retinal ganglion cells and cardiomyocytes. Examples 5 and 6 show that hg-C3N4 nanoparticles can be internalized in cardiomyocytes. Example 7 shows that hg-C3N4 nanoparticles can be internalized ex vivo in retinal ganglion cells. Example 4 even shows that hg- C3N4 nanoparticles can be internalized in the non-excitable cell types, NIH / 3T3 fibroblasts and HELA cells. In an embodiment, the stimulation is photo stimulation. As seen in Examples 4-7, the stimulation, such as generation of photo electrochemical and photo thermal effects, of the cells is achieved by photo stimulation using laser photo stimulation and / or LED photo stimulation. Retinal prosthesis can also be used in vitro. Thus, an aspect also relates to the in vitro use of hg-C3N4 nanoparticles as a retinal prosthesis. The hg-C3N4 can induce a light-evoked response when incubated with cells or tissue as described above. Thus, an additional aspect relates to the in vitro use of hollow graphitic carbon nitride (hg-C3N4) for inducing a light-evoked response in cells, preferably in retinal cells or cardiac cells. In an embodiment, the light-evoked response is an intracellular calcium flux and / or an action potential. In yet an embodiment, the light-evoked response is synchronization of beating behavior of cardiac cells, such as cardiomyocytes. 82931PC01 20 In yet another embodiment, the light-evoked response is activation of retinal cells, such as ganglion cells, amacrine cells and / or photoreceptors. Method for inducing an (in vitro) light-evoked response A further aspect relates to a method for inducing an (in vitro) light-evoked response in one or more target cells or tissue, the method comprising - providing of hollow graphitic carbon nitride (hg-C3N4) nanoparticles to the target cells or tissue; and - applying light, such as light pulses, to the target cells or tissue. In an embodiment, the target cells or tissue are retinal cells or tissue or cardiac cells or tissue. Other aspects of the invention A further aspect of the invention relates to a method for treating or alleviating a subject in the need thereof, the method comprising administering to the subject a composition comprising one or more hollow graphitic carbon nitride (hg-C3N4) nanoparticles. In a preferred embodiment, the composition is administered to the eye of the subject. In an embodiment, the hg-C3N4 nanoparticles are internalized in one or more target cells of the subject. In a preferred embodiment, the subject is mammal, preferably human. In an embodiment, the target cells are excitable cells such as retinal ganglion cells, cardiac cells, cardiomyocytes, cardiac fibroblasts. Yet another aspect of the invention relates to a method for modulating cell activity, the method comprising 82931PC01 21 - administering a composition comprising one or more hollow graphitic carbon nitride (hg-C3N4) nanoparticles according to a previous aspect to a subject, and - activating the pharmaceutical composition using an external source. Again, activation may be by LED light or laser light, such as for “training” the cells. However, it may also be by natural light, such that a subject may regain at least some light sensibility or improve remaining light sensibility. In an embodiment, the subject suffers from visual deficit and / or cardiac disease. It should be noted that embodiments and features described in the context of one of the aspects of the present invention also apply to the other aspects of the invention. All patent and non-patent references cited in the present application, are hereby incorporated by reference in their entirety. The invention will now be described in further details in the following non-limiting examples. Items of the invention 1. A composition comprising one or more hollow graphitic carbon nitride (hg-C3N4) nanoparticles for use as a medicament. 2. A composition comprising one or more hollow graphitic carbon nitride (hg-C3N4) nanoparticles for use in the treatment or alleviation of photoreceptor degeneration, such as retinitis Pigmentosa (RP) or related syndromes, preferably said syndromes being selected from the group consisting of Usher Syndrome, Bardet-Biedl syndrome, Refsum disease, Batten disease, and Jalili syndrome; neuropathy, ataxia, NARP (neuropathy, ataxia, and retinitis pigmentosa) syndrome, inherited retinal degenerations, preferably selected from the group consisting of Stargardt's disease, Leber's congenital amaurosis, and atrophic age- related macular degeneration (AMD). 82931PC01 22 3. A composition comprising one hollow graphitic carbon nitride (hg-C3N4) nanoparticles for use in the treatment or alleviation of cardiac disease, such as arrhythmia, congestive heart failure, cardiomyopathy, bradycardia, tachycardia. 4. The composition for use according to any of the preceding items, wherein the composition is administered by injection. 5. The composition for use according to any of the preceding items 1-2 and 4, wherein the composition is administered and / or applied and / or injected intraorbitally or intracapsular, preferably by microinjection into a subretinal or epiretinal space, wherein said microinjection is preferably performed through the sclera and / or through the vitreous chamber. 6. The composition for use according to any of the preceding items 1 and 3-4, wherein the composition is administered intrapericardially, intramyocardially, epicardially or intravenously to the heart. 7. The composition for use according to any of the preceding items 1 and 3-5, wherein the composition is activatable by a light emitting device implanted in or near the heart. 8. The composition for use according to any of the preceding items, wherein the hg-C3N4 nanoparticles are internalized in one or more target cells of the subject. 9. The composition for use according to item 8, wherein the target cells are excitable cells such as retinal ganglion cells and cardiomyocytes. 10. The composition for use according to any of the preceding items, wherein the subject suffers from visual deficit and / or cardiac disease. 11. The composition for use according to any of the preceding items, wherein the visual deficit is associated with degeneration of photoreceptors in the eye, preferably cone degeneration, more preferably foveal cone degeneration; and / or the degeneration of photoreceptors is selective degeneration of photoreceptors, preferably cone degeneration and / or macular degeneration. 82931PC01 23 12. The composition for use according to any of the preceding items, wherein the subject is mammal, preferably human. 13. The composition for use according to any of the preceding items, wherein the composition is a pharmaceutical composition. 14. The composition for use according to any of the preceding items, wherein the composition ^ is administered with a concentration in the range 2.5 – 500 µg / ml, such as 10 – 300 µg / ml, such as 15 – 200 µg / ml, such as 20 – 100 µg / ml, preferably 25-50 µg / ml; or ^ is administered with an amount of hg-C3N4 nanoparticles in the range 2 – 5000 µg, such as 20 – 2000 µg, such as 50-1000 µg, preferably 100-500 µg, more preferably 150-250 µg; or ^ is administered with a concentration in the range range 0.01 – 100 mg / ml, such as 0.05 – 50 mg / ml, preferably such as 0.1 – 10 mg / ml, more preferably such as 0.3 - 5 mg / ml. 15. A pharmaceutical composition comprising one or more hollow graphitic carbon nitride (hg-C3N4) nanoparticles. 16. The pharmaceutical composition according to item 15, further comprising pharmaceutically acceptable saline or hydrogel. 17. The pharmaceutical composition according to any of items 15 and 16, further comprising a pharmaceutically acceptable carrier, solvent and / or diluent. 18. The pharmaceutical composition according to any of items 15 to 17, further comprising an osmotic agent. 19. The pharmaceutical composition according to any of items 15 to 18, further comprising a pharmaceutically effective amount of at least one additional ingredient preferably selected from the group consisting of anti-inflammatory, astringent drugs, preferably selected from the group consisting of neostigmine 82931PC01 24 methylsulfate, ε-amino caproic berberine chloride, zinc sulfate, lysozyme chloride, sodium azulene sulfonate, dipotassium glycyrrhizinate and combinations thereof; antiallergic agents, preferably selected from the group consisting of diphenhydramine hydrochloride, isopenzyl hydrochloride, chlorpheniramine maleate, sodium cromoglycate and combinations thereof; vitamins other than pyridoxine hydrochloride, preferably selected from the group consisting of vitamin B2, vitamin B12, vitamin A, vitamin E, calcium pantothenate and combinations thereof, amino acids, preferably selected from the group consisting of potassium L-aspartate, magnesium L-aspartate, aminoethylsulfonic acid and combinations thereof; sulfa drugs, preferably selected from the group consisting of sulfamethoxazole, sulfisoxazole, sulfisomidine and combinations thereof, bacteriocides, preferably selected from the group consisting of sulfur, isopropylmethyl phenol, hinokithiol and combinations thereof; topical anesthetics, preferably selected from: lidocaine, lidocaine hydrochloride, procaine hydrochloride, dibucaine hydrochloride and combinations thereof, inorganic salts, preferably selected from the group consisting of potassium chloride, sodium chloride, sodium bicarbonate and combinations thereof, thickening agents, preferably selected from the group consisting of polyvinyl alcohol, polyvinyl pyrrolidone, carboxymethyl cellulose, hyaluronic acid, glucose and combinations thereof. 20. The pharmaceutical composition according to any of items 15 to 19, further comprising at least one additive, preferably solubilization auxiliary agents, isotonizing agents, stabilizing agents, chelating agents, pH adjusting agents, refrigerants, preservatives, and thickening agents; and / or a carrier, preferably buffer agents or ointment bases or a combination thereof. 21. The pharmaceutical composition according to any of items 15 to 20, wherein the pharmaceutical composition being a photovoltaic retinal prosthesis. 22. The pharmaceutical composition according to any of items 15 to 21, wherein the pharmaceutical composition being an ophthalmic pharmaceutical composition, preferably an injectable ophthalmic composition. 82931PC01 25 23. The pharmaceutical to any of items 15 to 22, wherein the pharmaceutical composition has a pH value in the range 6.6-7.8, more preferably in the range 7.0-7.5. 24. The pharmaceutical composition according to any of items 15 to 23, wherein the pharmaceutical composition has an osmotic pressure value is in the range 280-310 mOsm / L. 25. The pharmaceutical composition according to any of items 15 to 24, wherein the pharmaceutical composition is in an ophthalmic acceptable composition, preferably selected from aqueous solutions, aqueous suspensions, aqueous dispersion, emulsions, gelatinous materials, ointments and combinations thereof. 26. The pharmaceutical composition according to any of items 15 to 25, wherein the hg-C3N4 nanoparticles have an inner diameter in the range 50-500 nm, such as 100–120 nm, preferably 105-115 nm, more preferably 110 nm and / or an outer diameter in the range 60-600 nm, such as, 170–210 nm, preferably 180–200 nm, more preferably 190 nm. 27. The pharmaceutical composition according to any of items 15 to 26, wherein the hg-C3N4 nanoparticles comprise at least one surface modification and / or at least one cargo agent. 28. The pharmaceutical composition according to item 27, wherein the surface modification is a peptide or oligonucleotide, such as an aptamer or polymerized cathecholamines. 29. The pharmaceutical composition according to item 27, wherein the cargo agent is a drug. 30. The pharmaceutical composition according to any of items 15 to 29, wherein the hg-C3N4 nanoparticles are activatable by an external source. 31. The pharmaceutical composition according to item 30, wherein the external source is a light source, such as natural light and radiation. 82931PC01 26 32. The pharmaceutical composition according to any of item 30 and 31, wherein the external source is implantable in the heart. 33. Use of one or more hollow graphitic carbon nitride (hg-C3N4) nanoparticles for in vitro stimulation of one or more cells. 34. The use according to item 40, wherein the cells are excitable cells such as retinal ganglion cells and cardiomyocytes. 35. The use according to item 41, wherein the stimulation is photo stimulation and / or light induced stimulation. 36. A method for treating or alleviating a subject in the need thereof, the method comprising administering to the subject a composition comprising one or more hollow graphitic carbon nitride (hg-C3N4) nanoparticles. 37. The method according to item 36, wherein the composition is administered to the eye of the subject. 38. The method according to any of items 36 and 37, wherein the hg-C3N4 nanoparticles are internalized in one or more target cells of the subject. 39. The method according to any of items 36 to 38, wherein the subject is mammal, preferably human. 40. The method according to any of items 38 and 39, wherein the target cells are excitable cells such as retinal ganglion cells, cardiac cells, cardiomyocytes, cardiac fibroblasts. 41. A method for modulating cell activity, the method comprising - administering a pharmaceutical composition according to any of items 15 to 32 to a subject, and - activating the pharmaceutical composition using an external source. 82931PC01 27 42. The method according to item wherein the subject suffers from visual deficit and / or cardiac disease. 43. in vitro use of hg-C3N4 nanoparticles as a retinal prosthesis. 44. in vitro use of hollow graphitic carbon nitride (hg-C3N4) for inducing a light- evoked response in cells, preferably in retinal cells or cardiac cells. 45. The in vitro use according to item 44, wherein the light-evoked response is an intracellular calcium flux and / or an action potential. 46. The in vitro use according to item 44 or 45, wherein the light-evoked response is synchronization of beating behavior of cardiac cells, such as cardiomyocytes. 47. The in vitro use according to any of items 44-45, wherein the light-evoked response is activation of retinal cells, such as ganglion cells, amacrine cells and / or photoreceptors. 48. A method for inducing an (in vitro) light-evoked response in one or more target cells or tissue, the method comprising - providing of hollow graphitic carbon nitride (hg-C3N4) nanoparticles to the target cells or tissue; and - applying light, such as light pulses, to the target cells or tissue. 49. The method according to item 48, wherein the target cells or tissue are retinal cells or tissue or cardiac cells or tissue. Examples Example 1 – Synthesis and characterization of hollow sphere graphitic carbon nitride particle (hg-C3N4) Aim of study To synthesize homogeneous hollow spherically shaped hg-C3N4 nanoparticles. 82931PC01 28 Methods and material Synthesis of silica template The silica template was synthesized according to the classical Stöber method. In detail, the mixed solution containing 4.0 mL of aqueous ammonia (28 wt%), 74.0 mL of ethanol and 10.0 mL of deionized water were stirred vigorously at 30 °C for 1h. Then, 5.6 mL of tetraethoxysilane (TEOS) was slowly added to the above mixture under continuous stirring and stood still for 1 h to yield uniform monodisperse silica cores. Next, 3.56 mL of TEOS and 1.71 mL of n- octadecyltrimethoxysilane (C18TMOS) were added dropwise to the above solution while stirring. After the solution was dropped, the mixture was continued to stir for 15 min to form a thin silica shell around the dense silica core. Subsequently, the mixed solution was allowed to stand for 3 hours to promote the cohydrolysis and condensation of the TEOS and C18TMOS. The mixed solution was centrifuged, washed in DI water and ethanol, dried at 80 °C and calcined at 550 °C for 6h in air. Finally, the obtained silica template was neutralized with a 1 M HCl solution and dried at 80 °C overnight. Synthesis of hollow sphere graphitic caron nitride particle (hg-C3N4) A solution of 1.0 g of the silica template and 5.0 mL of cyanamide aqueous solution (50 wt%) was mixed for 30 min in a flask, then the mixture was ultrasonicated in vacuum at 60 °C for 3 h and stirred at 60 °C overnight. After centrifugation and drying, the products were transferred to CVD furnace for calcination and heated to 550 °C under flowing N2 for 4 h with a heating rate of 4.4 °C min−1. The obtained products were treated with 4 M NH4HF2 for 12 h to remove the silica template. In the last, the yellow powders were centrifuged, washed three times in DI water and once in ethanol, and then dried in vacuum at 60 °C for 10 h to obtain the final HCNS products. Electron microscopy The particles morphology was analyzed by SEM (TM3030Plus, Tabletop microscope, HITACHI). Transmission electron microscopy (TEM, Tecnai G2 Spirit) was used to visualize the hollow-sphere structure. TEM copper grids (Merck) were employed for analysis with an acceleration voltage of 120 kV AFM characterization 82931PC01 29 AFM measurements were commercial Dimension Icon (Bruker, Santa Barbara, USA) using AC160TS-R3 (k=26 N / m) probe. The topography of hg-C3N4 sphere was captured by tapping mode in ambient air. The data processing was completed in Nanoscope Analysis software. The particle analysis was performed in SPIP software. UV-vis spectroscopy UV-Vis spectra were acquired with the GENESYS 150 UV-Visible Spectrophotometer (Thermo Fisher Scientific). Particle tracking The ONI Nanoimager was employed for single particle tracking in cell free media to investigate stokes diameter and diffusion coefficient. The maximal frame gab was set to 5, maximal distance between frames was 0.7 µm and the minimum number of steps was set to 20. For particle tracking of internalized particles in 3T3 cells the Manual Tracking plugin for ImageJ was used. Based on the tracking data acquired with Manual Tracking plugin, the directedness was calculated as a parameter for directional movement. The angle Ѳ was defined as the angle between particle movement direction and direction of the center of an individual cell. The directedness is defined as the cosine of this angle. If particle movement is directed exactly towards the center of the cell the value is 1, whereas the value 0 indicates perpendicular movement, and -1 movement in the opposite direction. The average directedness was calculated as ΣcosѲi / n, where Ѳ was the angle between the individual movement directionality vector and the directionality vector towards the center of the cell relative to the particle position (n=8). Results To achieve hollow spherically shaped hg-C3N3 nanoparticles, a silica template with subsequent hydrofluoric acid (HF) etching was employed to achieve submicron shape and size requirements inspired by intracellular organelles and vesicles. The particles morphology was analyzed by SEM (TM3030Plus, Tabletop microscope, HITACHI). Transmission electron microscopy (TEM, Tecnai G2 Spirit) was used to visualize the hollow-sphere structure. TEM copper grids (Merck) were employed for analysis with an acceleration voltage of 120 kV. SEM and TEM 82931PC01 30 clearly show the hollow sphere of the particles with an inner diameter of around 110 nm and an outer diameter of 190 nm, leaving a controlled amorphous shell of around 80 nm (Figure 1A and 1B). The great light harvesting properties of hg-C3N3 nanoparticle could be confirmed by UV-Vis spectroscopy, where hg-C3N3 showed up to 4 times higher light absorbance within a range of 350-600 nm, indicating considerably more absorbance in the visible spectrum compared to bulk non-hollow g-C3N3 (Figure 1E). This effect could be attributed to multiple reflection sites residing in the hollow structure, prolonging the light path length for absorption within the spheres. The AFM topography height line profile revealed a few single hg-C3N4 nanoparticles with a height around 200 nm (Figure 1C) together with some aggregates, which is similar to the TEM results. Via particle analysis (Figure 1D), the statistic height of the stacked layer is (383 ± 79) nm, indicating the possible bilayer-stacking. The further particle tracking analysis revealed a mean stokes diameter of (636 ± 209) nm (Figure 1D), showcasing the possible aggregation of the nanoparticles. Conclusion The results show that a homogeneous hollow spherically shaped hg-C3N4 nanoparticle is obtained with an inner diameter of around 110 nm and an outer diameter of 190 nm, leaving a controlled amorphous shell of around 80 nm. hg- C3N3 showed up to 4 times higher light absorbance within a range of 350-600 nm, indicating considerably more absorbance in the visible spectrum compared to bulk non-hollow g-C3N3. Example 2 – Photocurrent characterization of hg-C3N4. Aim of study To investigate the nature and magnitude of the photo-response of hg-C3N4. Methods and material See Example 1 for synthesis of hg-C3N4. 82931PC01 31 The setup was based on an electrophysiology patch-clamp aperture. A P-97 micropipette puller was used to pull glass pipettes and achieve a resistance of 2-4 MΩ. A micromanipulator was used to bring a silver chloride electrode inserted in the glass pipette in close proximity to the particles ( <10 µm). The sample as well as the objective is immersed in PBS. The upright microscope (Olympus, BX61WI) allows to illuminate the sample with a 365 nm LED light, with a pulse duration of 100 ms. An AxoPatch 200B amplifier (Molecular devices), and Digidata 1550 digitizer (Molecular devices), and Clampex software (Molecular devices), worked together to conduct the measurement and control the light source. A patch-clamp electrophysiology apparatus with integrated photocurrent measurement set-up was employed as shown in Figure 2A. H2O2 quantification Hydrogen Peroxide (H2O2) production was evaluated using different concentrations of particles in PBS 5 µg / cm2. The solutions were added to 24-wells and particles were allowed to settle for 2 hours before being irradiated with 450 nm LED light (75 mW / cm2), pulsed at 100 ms duration and 1 Hz frequency for 30 minutes. Control groups were kept light-protected under the same conditions. Subsequently, 3x109 µl were transferred from each well to a black 96-well plate. The aliquots were stained with a solution containing 91 µl of horseradish peroxidase (HRP; 4250 units / L, Thermofischer), p-hydroxyphenyl acid (pOHPAA; 1.0e-3 M, Sigma), in 0.25 M Tris buffer (Sigma). Standards with known H2O2 concentrations were treated in the same way. Fluorescence intensity was measured with an excitation of 320 nm and an emission of 405 nm using a microplate reader (CLARIOstar Plus Microplate Reader). Results The relationship of photocurrent trace amplitude and applied holding currents I0 provides insight to the nature of the generated photocurrents. As photo thermal effects go along with temperature increase, the increased ion mobility leads to reduced pipette resistance and subsequently increased current, directly proportional to the amperage of I0. On the other hand, photo electrochemical processes, like capacitive and faradaic currents, are independent from the applied holding current. 82931PC01 32 Figure 2B, together with the non-zero intercept of Figure 2C, show that both photo electrochemical and photo thermal effects are generated. The sustained current with negative polarity over the entire pulse length and low amplitude around 30 pA for I0=0 nA, is evident of an anodic faradaic current, as opposed to capacitive currents, characterized by the fast charge and discharge processes. Since faradic photocurrents go along with redox reactions at the semiconductor / electrolyte interface, the generation of reactive oxygen species is probable. The anodic currents lead to a predominately oxidative reaction, and subsequent generation of H2O2 e.g. by the oxidation of H2O. However, the interfacial reactions are complex and there are other plausible pathways leading ROS generation. The quantification of H2O2 (Figure 2D) revealed that indeed 10 µmol / mg of H2O2 is produced upon visible light exposure, confirming an electrochemical process. ROS like H2O2 is involved in many essential physiological processes acting as signal molecules, even interacting with neuronal ion channels. Together with the photocurrent traces, the hg-C3N4 mediated photo stimulation is based on both mechanisms, anodic faradaic currents and evident photo thermal effects. Conclusion The hg-C3N4-mediated photo stimulation is based on both mechanisms, anodic faradaic currents and evident photo thermal effects. Example 3 – Internalization and toxicity of hg-C3N4 Aim of study To investigate internalization and toxicity of hg-C3N4 nanoparticles in cells. Methods and material See Example 1 for synthesis of hg-C3N4. Cell culture and material seeding NIH / 3T3 mouse embryonic fibroblasts cells were cultured in DMEM supplemented with 10% FBS, 1% P / S, and 4 mM L-glutamine. All cells were kept at 37 °C and 5% CO2 and media were changed every second day. 82931PC01 33 hg-C3N4 internalization A 0.5 mg / ml hg-C3N4 stock was prepared in PBS by sonication with 20 W for 15 min. The stock dispersion was diluted in media and added to the cells to achieve a final particle concentration of 0.5 µg / cm2. After 6-8 h of incubation at 37 °C the media was removed, and cells were trypsinized and passaged to new well plate. The internalization videos were recorded using the Nikon TI2-E inverted microscope. Particle tracking of internalized particles was done using Manual Tracking plugin in ImageJ. Particle tracking For particle tracking of internalized particles in 3T3 cells, the Manual Tracking plugin for ImageJ was used. Based on the tracking data acquired with Manual Tracking plugin, the directedness was calculated as a parameter for directional movement. The angle Ѳ was defined as the angle between particle movement direction and direction of the center of an individual cell. The directedness is defined as the cosine of this angle. If particle movement is directed exactly towards the center of the cell the value is 1, whereas the value 0 indicates perpendicular movement, and -1 movement in the opposite direction. The average directedness was calculated as ΣcosѲi / n, where Ѳ was the angle between the individual movement directionality vector and the directionality vector towards the center of the cell relative to the particle position (n=8). LDH cytotoxicity assay The cytotoxicity assay was conducted following the manufacturer's instructions (LDH cytotoxicity kit, Roche). Briefly, 2500 3T3 cells were evenly seeded into sextuplicate wells of a 96-well plate with 100 µl of complete medium. Two groups were examined: one with particles and one without. For the wells with particles, 0.8 µg / cm2 of hg-C3N4 particles were added. After 24 hours of incubation, 80 µl of the cell culture medium was collected and centrifuged at 1700 rpm for 5 minutes at 4 °C. The activity levels of lactate dehydrogenase (LDH) were then measured by transferring 50 µl of the supernatant to a new 96-well plate and adding reaction mix containing catalyst and dye in a 1:1 ratio. Subsequently, the plate was incubated in the dark at room temperature for 30 minutes, followed by analysis of absorbance on a Victor 82931PC01 34 Multilabel Reader (PerkinElmer, 490 nm. To establish the toxicity levels, the LDH activity of cells cultured without particles was regarded as the low toxicity control, while that of cells treated with 1% Triton X-100 was marked as high- toxicity control. The relative toxicity levels were calculated using the below equation: ^^^. ^^^^^ − ^^^ ^^^^^^^ %^^^^^^^^^^^^ = ^^^^^^^ − ^^^ ^^^^^^^ CCK-8 proliferation assay The effect of particles on cell proliferation was assessed using a Cell Counting Kit- 8 (CCK-8, Dojindo). 5000 NIH / 3T3 cells were seeded in 96-well plates in sextuplicate. 2.0 µg / cm2 of hg-C3N4 particles were added. The CCK-8 assay was performed on days 1, 3, and 8 following the manufacturer’s protocol. Briefly, the culture medium in each well was replaced with CCK-8 solution diluted 1:20 in medium. The cells were then incubated for 2 hours, after which the medium was transferred to a new 96-well plate and fresh culture medium was replenished. Subsequently, the absorbance at 450 nm was analyzed on Victor Multilabel Reader (PerkinElmer, US).. The proliferation index is calculated as: ^^ ^^^^^^^^^^^^^ ^^^^^ =^with Ab being the measured on day x (x = 1, 3, 8). Live cell staining Live cell staining of 3T3 cells was conducted using CellBrite Steady Dye 550 diluted 1:1000 (Biotium) for membrane staining, and Calcein AM (ab141420, abcam) as viability stain. Imaging and Z-stacks were generated using the Nikon TI2-E inverted microscope. Cell viability upon exposure to particles and light was assessed using Live / Dead staining using NIH / 3T3 cells cultured for 24 h. The particles (2.0 µg / cm2) were introduced 6 hours prior to the light stimulation. The staining solution, a combination of 1:1000 Calcein-AM (green dye) and 1:500 Ethidiom iodide (red dye) medium, was added after aspiration of the culture medium and a single rinse with serum-free medium. After adding the staining solution, cells were then incubated for 30 min at 37 °C. Afterwards, the staining 82931PC01 35 solution was discarded, wells were with PBS, and cells could be examined using a fluorescent microscope (Invitrogen EVOS M7000). Results NIH / 3T3 mouse embryonic fibroblasts were chosen to study the internalization process of hg-C3N4 nanoparticles, as efficient internalization is highly important to achieve subcellular resolution. To characterize the spontaneous internalization of hg-C3N4, the inherent blue fluorescence property of hg-C3N4 is used to track the particles over time while single cells were continuously imaged. The proposed internalization mechanism is illustrated in Figure 3A. Tracking of the particles demonstrates a clear directionality of the particles from the extracellular space towards the center of the cell, close to the nucleus (Figure 3B). The directedness is used as a quantitative indicator for the loss of random mobility of internalized particles compared to non-internalized particles (Figure 3C). The angle Ѳ of the directionality vector of the particle relative to the direction of the nucleus is used as a measure for directionality. Here, the median cos(Ѳ) for internalized particles is 0.91, indicating movement angle Ѳ ± 25° in the direction of the nucleus. Non-internalized particles show random mobility with cos(Ѳ) distributed broadly between 1 (directed movement, Ѳ =0°), 0 (perpendicular movement, Ѳ =90°), and -1 (movement in opposite direction, Ѳ = -180°). This decrease in random mobility is thus a strong indicator of successful internalization. The clear directedness quantified as cos Ѳ (Figure 3C) is indicative of a phagocytosis or endocytosis driven mechanism (Figure 3A). To investigate hg-C3N4 toxicity and influence on cell growth, a lactate dehydrogenase (LDH) assay, live / dead staining, and cell counting kit 8 (CCK-8) proliferation assay was conducted. The LDH assay could not show any considerable toxic effects of hg-C3N4 particles against NIH / 3T3 cells (Figure 3D). The CCK8 shows that hg-C3N4 particles do not influence the proliferation rate of NIH / 3T3 cells, confirming the cytocompatibility of hg-C3N4 (Figure 3E). Conclusion hg-C3N4 nanoparticles were successfully internalized, and the study confirms that hg-C3N4 is cytocompatible with no considerable toxic effects in cells. 82931PC01 36 Example 4 – Photo stimulation of NIH / 3T3 fibroblasts and HELA cells with internalized hg-C3N4Aim of study Subcellular photo stimulation of non-excitable cells. The ability of the particles to induce light-triggered intracellular bioelectric activity with subcellular resolution was investigated by monitoring calcium flux under focused laser beam stimulation of single internalized particles. Methods and material See Example 1 for synthesis of hg-C3N4.Cell culture and material seeding NIH / 3T3 and HELA cells were cultured in DMEM supplemented with 10% FBS, 1% P / S, and 4 mM L-glutamine. All cells were kept at 37 °C and 5% CO2 and media were changed every second day. hg-C3N4 internalization A 0.5 mg / ml hg-C3N4 stock was prepared in PBS by sonication with 20 W for 15 min. The stock dispersion was diluted in media and added to the cells to achieve a final particle amount of 0.5 µg / cm2. After 6-8 h of incubation at 37 °C the media was removed, and cells were trypsinized and passaged to new well plate. Calcium imaging and laser stimulation To visualize intracellular calcium Cal-520, AM (ab171868, abcam) at 4 µM was used. After incubating the cells with dye in glass bottom dishes at 37 °C for 30 min, the cells were rinsed and incubated for another 30 min at 37 °C in medium. The cells were observed with a 60x oil immersion objective using a Nikon TI2-E inverted microscope. Laser (473 nm) power was calibrated using the PM100D optical power meter (ThorLabs) and S120C photodiode power sensor (ThorLabs). Isochronal and vector maps were generated using the ImageJ plugin Spiky (60). An online available macro was used to generate Δ F / F movies (61). Calcium flux videos were quantified with ImageJ by defining regions of interest around cells and measuring mean gray values (F). As background value (F0) the mean gray value of an ROI without cells was defined. The mean fluorescence intensity 82931PC01 37 normalized to the background was calculated as ΔF / F0 for each individual frame. Results A video showed that a laser pulse alone could not provoke any change in calcium dynamics in NIH / 3T3 fibroblast cells without internalized hg-C3N4 particles (data not shown). hg-C3N4 nanoparticles showed strong fluorescence in the green channel, enabling simplified identification of internalized hg-C3N4. A focused 473 nm (8.8 mW / µm2) laser beam with a pulse width of 200 ms and spot size of 3 µm was employed to stimulate the particles. The laser pulse alone could not provoke any change in calcium dynamics in NIH / 3T3 fibroblast cells. When particles were present, photo stimulation led to an immediate calcium flux through the cytosol originating from the stimulated particle location (Figure 4A). A video also showed that photo stimulation led to an immediate calcium flux, as evident by green fluorescently stained calcium through the cytosol originating from the stimulated particle location (data not shown). For 3T3 cells, the average calcium flux velocity was (2.4 ± 3.8) µm / s. The versatility of hg-C3N4 particles to study intracellular bioelectric activity of non- excitable cells was also demonstrated by HELA cells (Figure 4B). A video showed that a single 200 ms laser pulse led to an immediate intracellular calcium flux with a prolonged increase in intracellular calcium, evident by a sustained increase in fluorescence intensity (data not shown). The average velocity of the calcium flux in HELA cells was determined to be (1.3 ± 2.7) µm / s. The mechanism behind the observed intracellular calcium transient might be attributed to hg-C3N4 interacting with calcium storing organelles like the endoplasmic reticulum (ER) (Figure 4A). Conclusion As shown in Example 2, the generated faradaic photocurrents go along with ROS production (Figure 2D), ROS might interact with organelles leading to 82931PC01 38 intracellular calcium release. It shown that photo thermal effects can cause membrane poration, and may lead to release of calcium. It is thus plausible that both faradaic electrochemical and photo thermal effects act together to achieve intracellular membrane depolarization, as both effects have been found to modulate bioelectrical behavior independently. In sum, the example shows photo stimulation of NIH / 3T3 fibroblasts and HELA cells with internalized hg-C3N4. Example 5 – Photo stimulation of cardiomyocyte (CM) with internalized hg-C3N4 Aim of study To test the ability of hg-C3N4 particles to electrically modulate primary rat CMs, which play a key role in heart tissue muscle contraction. Electrical modulation of primary CFs were also investigated. Methods and material See Example 1 for synthesis of hg-C3N4. Cardiac cell culture. Primary cardiomyocytes (CMs) and cardiac fibroblasts (CFs) were isolated and harvested from P0-5 neonatal rats following literature (39) and reagents were obtained from Pierce™ primary cardiomyocyte isolation kit (Thermo Fisher Scientific). Basically, the heart tissues were excised into ice cold Hanks' Balanced Salt solution (HBSS) medium without Ca2+ or Mg2+ and digested with reconstituted papain and thermolysin enzymes. Isolated CFs / CMs hybrid cells were washed with HBSS, seeded onto fibronectin (Sigma)-coated glass bottom imaging dishes, and cultured in high glucose DMEM plus 10% FBS, 1% penicillin– streptomycin, and 1% GlutaMAX. Animal procedures were approved by the University of Chicago Institutional Animal Care and Use Committee (IACUC) and conducted in complete compliance with the IACUC Animal Care and Use Protocol. Particle co-culture. Before seeding, hg-C3N4 particles were sonicated in ethanol (or treated with UV lights for 15 min) to sterilize and washed and resuspended in PBS. Harvested CFs / CMs hybrid cells were enriched in 24-well plates and proliferated until ~70% 82931PC01 39 confluency and around 0.6 µg / cm2C3N4 nanoparticles were seeded into each well. Calcium imaging and laser stimulation To visualize intracellular calcium Cal-520, AM (ab171868, abcam) at 4 µM was used. After incubating the cells with dye in glass bottom dishes at 37 °C for 30 min, the cells were rinsed and incubated for another 30 min at 37 °C in medium. The cells were observed with a 60x oil immersion objective using a Nikon TI2-E inverted microscope. Laser (473 nm) power was calibrated using the PM100D optical power meter (ThorLabs) and S120C photodiode power sensor (ThorLabs). Isochronal and vector maps were generated using the ImageJ plugin Spiky. An online available macro was used to generate Δ F / F movies. Calcium flux videos were quantified with ImageJ by defining regions of interest around cells and measuring mean gray values (F). As background value (F0) the mean gray value of an ROI without cells was defined. The mean fluorescence intensity normalized to the background was then calculated as ΔF / F0 for each individual frame. Results Particles associated with CMs were directly stimulated, resulting in immediate intracellular calcium flux and beating (Figure 4E). Intriguingly, the calcium flux of the stimulated cell led to calcium flux and beating of three adjacent coupled CMs. The mechanism of this signal propagation is typical for coupled CMs and relies on interconnected connexin gap junctions, enabling synchronous beating essential for the physiological function of the heart. Cell-cell signal propagation was observed not only between the CMs, but also cardiac fibroblasts (CF). CFs express a plethora of ion channels, mainly potassium (K+) channels and mechanosensitive channels, that contribute to their resting membrane potential. Indeed, a stimulated primary CF propagated the calcium transient to an adjacent CF with a velocity of (8.8 ± 16.2) µm / s (Figure 4C). Direct coupling of cardiomyocytes and fibroblasts via connexin-based gap junctions is supported by compelling evidence. 82931PC01 40 Stimulating of a primary CF CM led to a similar observation, when the intracellular calcium flux of the fibroblast reached the outer cell membrane with (4.4 ± 8.5) µm / s, the adjacent CM started with continues beating (Figure 4D). Conclusion It is observed that hg-C3N4 nanoparticles associated with or internalized into CMs were directly stimulated, resulting in immediate intracellular calcium flux and beating. The calcium flux of the stimulated cell led to calcium flux and beating of three adjacent coupled CMs. Cell-cell signal propagation was observed not only between the CMs, but also cardiac fibroblasts (CF). A stimulated primary CF propagated the calcium transient to an adjacent CF. Propagation between CF and CM was also observed. The results on cardiac cells are intriguing because it can be foreseen that the compounds of the invention can be used to achieve cardiac pacing in vivo. By injecting the hg-C3N4 nanoparticles into target cardiac cells and implanting a light source at site for activation of the administered hg-C3N4 nanoparticles. The ability of the particles to stimulate cardiomyocytes through light stimulation can be highly beneficial to achieve leadless, and therefore less invasive cardiac pacing compared to conventional leadless pacemakers. Example 6 – LED photo stimulation and synchronization of cardiomyocytes Aim of study The injectability of nanoparticles makes them an attractive non-invasive alternative to conventional electronic pacemaker therapy. As the stimulation of single cardiomyocytes via laser has proven feasible, we next wanted to apply interval training to many cardiomyocytes simultaneously in order to synchronize beating and align to common beating frequency using low intensity (10 mW / cm2, λ=450 nm) LED as light source. Methods and material See Example 1 for synthesis of hg-C3N4. HL-1 pacing 82931PC01 41 HL-1 cells were cultured in 24-well at a seeding density of 100,000 cells per well. Upon reaching 90% confluency, particles were added at concentrations of 2.0 µg / cm2 followed by a 6-hour settling period before initiating calcium imaging. For calcium flux imaging, cells were stained with Fluo-4 AM (Invitrogen, F14201). Samples were rinsed with Hanks’ Buffer (Sigma H6648) supplemented with 0.1 mM norepinephrine (Sigma, A0937), 5 mM glutamax, and 20 mM Hepes (Sigma H0887). Subsequently, cells were incubated with 5 µM Fluo-4 AM diluted in the same buffer for 30 min at 37 °C, 5% CO2. After rinsing twice, the cells were incubated in the buffer for an additional 20 minutes. Following the aspiration of the buffer, supplemented Claycomb Medium was added and used during imaging. Stimulation was performed using light irradiation at a power of 10 mW / cm2, frequency of 1 Hz, and pulse width of 100 ms. Calcium flux was monitored and recorded via a 10x objective on the EVOS microscope (EVOS m7000) equipped with an onstage incubator (37 °C, 5% CO2). Results When HL-1 cardiomyocytes were cultured together with hg-C3N4 particles and LED photostimulation with a frequency of 1 Hz and 100 ms pulse width was applied for 10 min, the beating frequency changed from an irregular non-synchronized pattern between 0.5 and 2 Hz to a more synchronized pattern of 2 Hz (Data not shown). This effect was even more pronounced after 30 min of stimulation. Further, the calcium flux appeared to travel as a directional synchronized wave through the cardiomyocyte cell sheet, when both hg-C3N4 and photostimulation was applied (Data not shown). Cardiomyocytes cultured without hg-C3N4 remained in a state of unsynchronized beating behavior. Conclusion It was demonstrated that hg-C3N4 was able to modulate CM activity by applying LED stimulation with fixed frequency leading to synchronous beating. Thus, “training” of cardiomyocytes beating frequency is feasible. Example 7 – Photo stimulation of retinal tissue with internalized hg-C3N4 Aim of study Retinal tissue is an essential part of this system playing a critical role in transmitting light mediated visual information to the brain. This complex process 82931PC01 42 relies on the interplay of different of neurons within the retina. Among these, retinal ganglion cells are responsible for receiving input from photoreceptors, through bipolar cells, and then transmitting this information directly to the visual areas of the brain. In this study, the potential of hg-C3N4 particles as an injectable retinal prosthesis using porcine retina is explored. Methods and material See Example 1 for synthesis of hg-C3N4. Retina dissection Eyes from Danish Land Race pigs (Sus domesticus) were collected at a local slaughterhouse (Danish Crown, Horsens, Denmark) immediately after the animals had been anesthetized with carbon dioxide and killed by exsanguination. The eyes were transported to AUH laboratory in PSS at 4°C, and the time from the collection of the eyes to the commencement of the dissection procedure never exceeded 5 h. Dissection was performed in 4°C PSS0.0 as follows: each eye was bisected at the equator with a double-edged blade, and the anterior segment was removed. The posterior segment containing the optic disk was placed under a stereo microscope, the vitreous body was removed. A few mm from the optic disk a segment of approximately 3 x 3 mm2 neuroretinal tissue was cut out using a self-locking chisel blade handle (VWR International, Herlev, Denmark) equipped with a 30 ^ microblade (BD Beaver, D.J. Instruments, Billerica, USA). Chemical Solutions for storage and transportation: Physiological saline solution (PSS) with the following composition (in mM): 118 NaCl, 4.8 KCl, 1.14 MgSO4, 25 NaHCO3, 5 Hepes, 1.5 CaCl2, 5.5 glucose. MEA electrophysiology A micro-electrode array (MEA) with 32 channels from Blackrock Microsystems was used to measure electrical signals from the retinal tissue. The MEA was connected via an Omnetics connector to a Blackrock Microsystems Cereplex Direct dedicated data acquisition. Data was sampled at 30 kHz, high pass filtered at 250 Hz and further filtered for 50 Hz line noise by a built-in Blackrock Line Noise Cancelation filter (filtering 50 Hz, 1 second, soft synchronization). Data was analyzed using custom Matlab software. 82931PC01 43 After placing the retinal tissue on with the retinal ganglion cells facing upwards, 5 µg of hg-C3N4 dispersed in 20 µl of PBS we added to tissue and allowed to settle for around 15 min. Afterwards, light stimulation was applied using a 450 nm homemade LED system with 1 ms pluses with one pulse per second (1 Hz). The recording length was at least 30 s. The resulting whole data trace for each of the 32 channels were segmented into traces of 1 sec duration (minimum 30 traces), initiating at the stimulus artefact. These traces were then averaged in order to improve the signal / noise ratio. All electrophysiological experiments were carried out inside a Faraday cage. Chemical Solutions for experiments: PSS1.6 with the following composition (in mM): 119 NaCl, 4.7 KCl, 1.17 MgSO4, 25 NaHCO3, 5 Hepes, 1.6 CaCl2, 5.5 glucose, 1.18 KH2PO4, 0.026 EDTA. PSS0.0 refers to PSS1.6 where CaCl2 has been omitted. PSS1.6 was heated to 374°C and oxygenated prior to all experiments in order to ensure a physiological environment during electrical measurements. Tetrodotoxin (TTX) citrate was obtained from Hello Bio Reagents (Bristol, United Kingdom, HB1035). TTX solutions were prepared by creating a stock solution of TTX in PSS1.6 of 1.0 M concentration. From this solution 1 mL aliquots of 2.25 mM concentration were created for use in future experiments. All TTX solutions were stored at -20 °C between experiments. For TTX control experiments, a volume of 50 µL of the TTX solution was applied directly to the tissue sample. Results The retinal tissue was placed on a microelectrode array with the retinal ganglion cells facing up (Figure 5A). To test the viability of the retina, we applied a 1 ms visible light LED pulse (450 nm) and were able to record light evoked potentials with mean peak-peak amplitude of 11.0 µV that are typical of retina response across all channels. Retinal ganglion cell (RGCs) produce action potentials in order of several mV, recordable through the photoreceptor layer of around 100 µm thickness. hg-C3N4 particles were added to the tissue and allowed them to settle on the retinal ganglion cell layer. With the same light dose (30 mW / cm2), a drastic increase in mean peak-peak amplitude from 11 to 47.7 µV is observed (Figure 5B). The observed augmentation in peak-peak amplitude can be ascribed to synergy from the activation of photoreceptor cells stimulated solely by the light stimulus and the supplementary sensitization facilitated by hg-C3N4 particles. The 82931PC01 44 photo stimulation of hg-C3N4 activate the RGCs and possibly other retinal cell types e. g. amacrine cells and photoreceptors, cumulating in clearly recordable RGC action potentials. To confirm that the recorded signal was indeed of biological nature and consisted of compound action potentials created by the ganglion cells, Tetrodotoxin (TTX) was applied to prevent the ganglion cells from producing action potentials. TTX is a highly effective neurotoxin mainly found in different marine organisms, binding to voltage gated sodium channels and inhibiting neuron firing. No action potential could be detected when TTX was present, confirming that the recorded action potentials are indeed of RGC origin (Figure 5B). A dose response test was conducted, which showed that even light intensities as low as 8.9 mW / cm2can produce light-evoked potentials when hg-C3N4 particles are present (+ P), while peak-peak amplitudes recorded with 3.2 mW / cm2+ P were not distinguishable from peak-peak amplitudes recorded with 26.1 mW / cm2 without particles (- P) (Figure 5C). Conclusion The results demonstrate the potential of hg-C3N4 particles as an injectable retinal prosthesis that can induce pronounced photo-response in porcine retinal tissue and holds great potential to help patients with photoreceptor degeneration, such as retinitis pigmentosa and age-related macular degeneration. This promising approach may offer hope to those suffering from vision loss. Example 8 - C3N4 Nanoparticle delivery to the retina Aim of study To test the feasibility of safe hg-C3N4 NP delivery to the retina, biodistribution and retinal integrity after intravitreal delivery to mice. Materials and methods See Example 1 for synthesis of hg-C3N4. Experimental animals: 82931PC01 45 The use of animals was approved Danish Animal Inspectorate (authorization no. 2020-15-0201-00745). Eight-week-old C57BL / 6JRj male mice were purchased from Janvier Labs (Le Genest-Saint-Isle, France). Animals were maintained under a 12– / 12-hour light / dark cycle at the Animal Facilities at the Department of Biomedicine, Aarhus University, Denmark with free access to autoclaved tap water and standard chow (Altromin, Brogaarden, Denmark). The animals were handled in accordance with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research. For all procedures mice were anesthetized with an intraperitoneal injection of a mixture of ketamine and medetomidine hydrochloride (Ketador [60–100 mg / kg; Richter Pharma AG, Wels, Austria] and Cepetor [0.5–1 mg / kg; ScanVet Animal Health A / S, Fredensborg, Denmark]). Pupils were dilated with a drop of 1% tropicamide (Mydriacyl; Alcon Nordic A / S, Copenhagen, Denmark) and eyes lubricated with carbomer eye gel (Viscotears 2 mg / mL, Alcon Nordic). Anesthesia was reversed by atipamezole 0.5–1 mg / kg (Antisedan, Orion Pharma, openhagen, Denmark). Intravitreal injections: Intravitreal injections (Fig. 7A) were performed under an OPMI 1 FR PRO Surgical microscope (Zeiss, Jena, Germany). A 30G disposable needle was used to puncture the sclera near the limbus, and a 33G blunt-ended needle connected to a Hamilton syringe (Hamilton Company, Reno, NV, USA) was then inserted into the opening followed by injection of 1 µL NP (1 mg / mL) or PBS buffer solution. Both eyes of three animals were injected with NP solution and one animal with buffer solution. Results hg-C3N4 NP were delivered via intravitreal delivery to mice (Fig. 7B). The intravitreal injection of hg-C3N4 NPs into mouse eyes resulted in the visible settlement of particle aggregates on the retinal surface immediately adjacent to the RGCs and their axons as observed in fundus images and optical coherence tomography scans 14 days post-injection (Fig. 7B). Additionally, the hg-C3N4 NPs aggregates could be clearly identified on top of the RGCs using fluorescent imaging of retinal cross sections (Data not shown). Single hg-C3N4 NPs may have 82931PC01 46 penetrated into the retinal layers clear identification was hindered by the background autofluorescence signal. The integrity and thickness of retinal layers were preserved (Fig. 7B,C) with no significant differences in total retinal thickness (p = 0.57) or the thickness of individual layers between baseline and day 14 post-injection. Conclusion Intravitreal injection is a safe delivery route for hg-C3N4 NPs.

Claims

82931PC01 47 Claims 1. A composition comprising one or more hollow graphitic carbon nitride (hg-C3N4) nanoparticles for use in the treatment or alleviation of photoreceptor degeneration.

2. The composition according to claim 1, wherein the photoreceptor degeneration is retinitis Pigmentosa (RP) or related syndromes, such as the related syndromes being selected from the group consisting of Usher Syndrome, Bardet-Biedl syndrome, Refsum disease, Batten disease, and Jalili syndrome; neuropathy, ataxia, NARP (neuropathy, ataxia, and retinitis pigmentosa) syndrome, inherited retinal degenerations, preferably selected from the group consisting of Stargardt's disease, Leber's congenital amaurosis, and atrophic age-related macular degeneration (AMD).

3. The composition for use according to any of claims 1-2, wherein the composition is administered and / or applied and / or injected intraorbitally or intracapsular, preferably by microinjection into a subretinal or epiretinal space, wherein said microinjection is preferably performed through the sclera and / or through the vitreous chamber.

4. The composition for use according to any of the preceding claims, wherein the hg-C3N4 nanoparticles are internalized in one or more target cells of the subject.

5. The composition for use according to claim 4, wherein the target cells are excitable cells, such as retinal ganglion cells.

6. The composition for use according to any of the preceding claims, wherein the subject suffers from visual deficit.

7. The composition for use according to claim 6, wherein the visual deficit is associated with degeneration of photoreceptors in the eye, preferably cone degeneration, more preferably foveal cone degeneration; and / or the degeneration of photoreceptors is selective degeneration of photoreceptors, preferably cone degeneration and / or macular degeneration.82931PC01 48 8. The composition for use according to any of the preceding claims, wherein the subject is mammal, preferably human.

9. The composition for use according to any of the preceding claims, wherein the composition is a pharmaceutical composition.

10. The composition for use according to any of the preceding claims, wherein the composition is administered - with a concentration of hg-C3N4 nanoparticles in the range 0.01 – 100 mg / ml, such as 0.05 – 50 mg / ml, preferably such as 0.1 – 10 mg / ml, more preferably such as 0.3 -5 mg / ml; and / or - is administered with an amount of hg-C3N4 nanoparticles in the range 2 – 5000 µg, such as 20 – 2000 µg, such as 50-1000 µg, preferably 100-500 µg, more preferably 150-250 µg.

11. The composition for use according to any of the preceding claims, further comprising pharmaceutically acceptable saline or hydrogel.

12. The composition for use according to any of the preceding claims, further comprising a pharmaceutically acceptable carrier, solvent and / or diluent.

13. The composition for use according to any of the preceding claims, further comprising an osmotic agent.

14. The composition according to any of the preceding claims, further comprising a pharmaceutically effective amount of at least one additional ingredient preferably selected from the group consisting of anti-inflammatory, astringent drugs, preferably selected from the group consisting of neostigmine methylsulfate, ε-amino caproic acid, allantoin, berberine chloride, zinc sulfate, lysozyme chloride, sodium azulene sulfonate, dipotassium glycyrrhizinate and combinations thereof; antiallergic agents, preferably selected from the group consisting of diphenhydramine hydrochloride, isopenzyl hydrochloride, chlorpheniramine maleate, sodium cromoglycate and combinations thereof; vitamins other than pyridoxine hydrochloride, preferably selected from the group consisting of vitamin82931PC01 49 B2, vitamin B12, vitamin A, calcium pantothenate and combinations thereof, amino acids, preferably selected from the group consisting of potassium L-aspartate, magnesium L-aspartate, aminoethylsulfonic acid and combinations thereof; sulfa drugs, preferably selected from the group consisting of sulfamethoxazole, sulfisoxazole, sulfisomidine and combinations thereof, bacteriocides, preferably selected from the group consisting of sulfur, isopropylmethyl phenol, hinokithiol and combinations thereof; topical anesthetics, preferably selected from: lidocaine, lidocaine hydrochloride, procaine hydrochloride, dibucaine hydrochloride and combinations thereof, inorganic salts, preferably selected from the group consisting of potassium chloride, sodium chloride, sodium bicarbonate and combinations thereof, thickening agents, preferably selected from the group consisting of polyvinyl alcohol, polyvinyl pyrrolidone, carboxymethyl cellulose, hyaluronic acid, glucose and combinations thereof.

15. The composition for use according to any of the preceding claims, further comprising at least one additive, preferably solubilization auxiliary agents, isotonizing agents, stabilizing agents, chelating agents, pH adjusting agents, refrigerants, preservatives, and thickening agents; and / or a carrier, preferably buffer agents or ointment bases or a combination thereof.

16. The composition for use according to any of the preceding claims, wherein the composition is a photovoltaic retinal prosthesis.

17. The composition for use according to any of the preceding claims, wherein the composition is an ophthalmic pharmaceutical composition, preferably an injectable ophthalmic composition.

18. The composition for use according any of the preceding claims, wherein the pharmaceutical composition has a pH value in the range 6.6-7.8, more preferably in the range 7.0-7.

5.

19. The composition for use according to any of the preceding claims, wherein the pharmaceutical composition has an osmotic pressure value is in the range 280- 310 mOsm / L.82931PC01 50 20. The composition for use according to any of the preceding claims, wherein the pharmaceutical composition is in an ophthalmic acceptable composition, preferably selected from aqueous solutions, aqueous suspensions, aqueous dispersion, emulsions, gelatinous materials, ointments and combinations thereof.

21. The composition for use according to any of the preceding claims, wherein the hg-C3N4 nanoparticles have an inner diameter in the range 50-500 nm, such as 100–120 nm, preferably 105-115 nm, more preferably 110 nm and / or an outer diameter in the range 60-600 nm, such as, 170–210 nm, preferably 180–200 nm, more preferably 190 nm.

22. The composition for use according to any of the preceding claims, wherein the hg-C3N4 nanoparticles comprise at least one surface modification and / or at least one cargo agent.

23. The composition for use according to claim 22, wherein the surface modification is a peptide or oligonucleotide, such as an aptamer or polymerized cathecholamines.

24. The composition for use according to claim 22, wherein the cargo agent is a drug.

25. The composition for use according to any of the preceding claims, wherein the hg-C3N4 nanoparticles are activatable by an external source.

26. The composition for use according to claim 25, wherein the external source is a light source, such as natural light and radiation.

27. Use of one or more hollow graphitic carbon nitride (hg-C3N4) nanoparticles for in vitro stimulation of one or more cells.

28. The use according to claim 27, wherein the cells are excitable cells such as retinal ganglion cells.82931PC01 51 29. The use according to claim 27 wherein the stimulation is photo stimulation and / or light induced stimulation.

30. in vitro use of hg-C3N4 nanoparticles as a retinal prosthesis.

31. in vitro use of hollow graphitic carbon nitride (hg-C3N4) for inducing a light- evoked response in cells, preferably in retinal cells.

32. The in vitro use according to claim 31, wherein the light-evoked response is an intracellular calcium flux and / or an action potential.

33. The in vitro use according to any of claims 31-32, wherein the light-evoked response is activation of retinal cells, such as ganglion cells, amacrine cells and / or photoreceptors.

34. A method for inducing an in vitro light-evoked response in one or more target cells or tissue, the method comprising - providing of hollow graphitic carbon nitride (hg-C3N4) nanoparticles to the target cells or tissue; and - applying light, such as light pulses, to the target cells or tissue.

35. The method according to claim 34, wherein the target cells or tissue are retinal cells or retinal tissue.

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