Iontophoresis-assisted corneal cross-linking (I-CXL) is an emerging transepithelial technique for treating keratoconus. I-CXL employs a small electric current to enhance the corneal penetration of riboflavin 5'-phosphate sodium (RF), avoiding the de-epithelialization side effects associated with conventional CXL (C-CXL). However, I-CXL is less effective than C-CXL due to insufficient RF surface charge hindering iontophoresis response and absorption efficiency, as well as inadequate reactive oxygen species (ROS) generation in the corneal stroma. This work reports the development of an innovative composite photosensitizer (NS-CQDs-RF), utilizing N, S co-doped carbon quantum dots (NS-CQDs) as nanocarriers, to overcome the bottlenecks facing I-CXL. Experimental results show that the as-synthesized NS-CQDs-RF composites exhibit a remarkable electric field response, enhanced RF corneal permeability, high ROS production efficiency, and excellent biocompatibility. Notably, I-CXL incorporating NS-CQDs-RF significantly improves corneal biomechanical stability, outperforming C-CXL while reducing total UVA irradiation energy without compromising efficacy. Density Functional Theory (DFT) calculations reveal that the optimized electronic structure and favorable energetics between NS-CQDs and RF drive efficient charge transfer, amplifying ROS generation. These findings highlight the superiority of the NS-CQDs-RF composite photosensitizers in I-CXL, offering a novel strategy for future keratoconus treatment.

Dry eye disease is a common condition that affects the eyes. It is caused by problems with the tear film and the tear dynamics. Dry eye can be caused by an increase in the amount of reactive oxygen species (ROS) in the corneal epithelium. The treatment for dry eye typically focuses on relieving the uncomfortable symptoms by using eye drops such as artificial tears, antibiotics, and by using anti-inflammatory/immunosuppressive agents such as cyclosporine, and lifitegrast. However, the recovery of patients with dry eye can take several years particularly if the symptoms are severe. This is because the present treatment approaches for dry eye are not based on its cause, e.g., the oxidative stress arising from the rapid increase in ROS. This work describes a new type of antioxidant made from pterostilbene (PS) and carboxyl-chitosan modified graphene (CG). The use of a hydrophilic two-dimensional CG nanosheet to improve the properties of PS is reported. Superior enhanced properties including better cellular permeability, long sustained release period (over 30 h), and antioxidant properties, were realized by using PS-CG. A hyperosmotic (HS) damaged human corneal epithelial cell (HCEC) model was used for antioxidant tests. This model has an intracellular ROS level 4 times more than that of a control group. The ROS content was declined efficiently to the same amount as normal cells in the PS-CG treated HS group. There was a significant decline in the content of lactate dehydrogenase (LDH) and the apoptosis rate of HCEC in the PS-CG treated HS group when compared to that seen in the HS model. Real-time polymerase chain reaction (PCR) and western blots (WB) were used to understand the antioxidant mechanism of PS-CG. The results showed that the antioxidant was working by activating the Keap1-Nrf2-ARE signalling pathway. In vivo testing testing using a dry eye mouse model suggested that the PS-CG acted as an efficient antioxidant. More tear production and healthier corneal and conjunctival epithelial cells were achieved when PC-CG was applied to this model. The use of PS-CG could be a new strategy for treating dry eye and other ocular diseases caused by ROS.

The efficient regeneration of corneal nerves is of limited success in the field of ophthalmology. This work reports the use of a non-invasive electrical stimulation technique that uses a transparent graphene-based corneal stimulation electrode and that can achieve efficient regeneration of corneal nerves. The corneal stimulation electrode is prepared using electroactive nitrogen-containing conducting polymers such as polyaniline functionalized graphene (PAG). This composite can carry a high capacitive current. It can be used to tune transmembrane signaling pathways including calcium channels and the MAPK signaling pathway. Tuning can lead to the efficient regeneration of corneal damaged nerves after the surgery of laser in-situ keratomileusis (LASIK). The composite and its application reported have the potential to provide a new way to treat nerve-related injuries.

The noninvasive and real-time detection of glucose sugar from tears is promising for the early diagnosis and treatment of chronic diseases such as diabetes. However, its realization is a big challenge. A suitable biosensor electrode that can closely fit the eye and be electrochemically sensitive is still unrealized. In this work, nitrogen-doped graphene (N-G) was used as an ophthalmic electrode in a high-performance intraocular biosensor. The use of N-G has been reported elsewhere before as it is highly electroactive and so has a particular use in biosensors. We hereby present a novel procedure for making carboxylated chitosan-functionalized nitrogen-containing graphene (GC-COOH) by using a one-step ball-milling process. This process does not use toxic chemicals, flammable gases, or a high temperature. It is thus particularly easy to perform. The fabricated nanomaterial had a high electroactivity and was easily assembled as a glucose biosensor by the immobilization of glucose oxidase. The thus constructed biosensor has a high sensitivity at 9.7 μA mM-1 cm-2, a broad linear range at 12 mM, and a good detection limit of 9.5 μM. It was able to maintain this activity after a month of storage. We also report the intraocular use of this constructed biosensor. The as-prepared GC-COOH was found to be highly biocompatible to ophthalmologic cells such as corneal epithelial and retinal pigment epithelium cells. No change in the intraocular pressure or the corneal structure was measured in a New Zealand white rabbit model. The as-assembled sensor was worn by the animals for more than 24 h without undue impact. This result confirmed the biosensor's potential for intraocular application in the clinic. Its assembly into a useful sensor shown here has great potential to provide real-time monitoring of glucose levels in tear fluids of patients with high sugar levels.

The regeneration of neurons is an important goal of neuroscience and clinical medicine. The electrical stimulation of cells is a promising technique to meet this goal. However, its efficiency highly depends on the electrochemical properties of the stimulation electrodes used. This work reports on the preparation and use of a highly electroactive and biocompatible nanoelectrode made from a novel polyaniline functionalized graphene composite. This nanocomposite was prepared using a facile and efficient polymerization-enhanced ball-milling method. It was used to stimulate the growth of PC12 cells under various electrical fields. The enhanced growth of axons and improved wound regeneration of PC12 cells were observed after this treatment, suggesting a promising strategy for neuro traumatology.

Tumorous metastasis is a difficult challenge to resolve for researchers and for clinicians. Targeted delivery of antitumor drugs towards tumor cells’ nuclei can be a practical approach to resolving this issue. This work describes an efficient nuclear-targeting delivery system prepared from trans-activating transcriptional activator (TAT) peptide-functionalized graphene nanocarriers. The TAT peptide, originally observed in a human immunodeficiency virus 1 (HIV-1), was incorporated with graphene via an edge-functionalized ball-milling method developed by the author’s research group. High tumor-targeting capability of the resulting nanocarrier was realized by the strong affinity between TAT and the nuclei of cancer cells, along with the enhanced permeability and retention (EPR) effect of two-dimensional graphene nanosheets. Subsequently, a common antitumor drug, mitomycin C (MMC), was covalently linked to the TAT-functionalized graphene (TG) to form a nuclear-targeted nanodrug MMC-TG. The presence of nanomaterials inside the nuclei of ocular choroidal melanoma (OCM-1) cells was shown using transmission electron microscopy (TEM) and confocal laser scanning microscopy. In vitro results from a Transwell co-culture system showed that most of the MMC-TG nanodrugs were delivered in a targeted manner to the tumorous OCM-1 cells, while a very small amount of MMC-TG was delivered in a non-targeted manner to normal human retinal pigment epithelial (ARPE-19) cells. TEM results further confirmed that apoptosis of OCM-1 cells was started from the lysis of nuclear substances, followed by the disappearance of nuclear membrane and cytoplasm. This suggests that the as-synthesized MMC-TG is a promising nuclear-target nanodrugfor resolution of tumorous metastasis issues at the headstream.

Interpenetrating network structures from Graphene foam (GF) and 58S bioactive glass (BG) are synthesized to combine the highly mechanical stability and conductivity from graphene with the superb bioactivity and biocompatibility from 58S BG. GF/58S BG scaffolds were prepared via multiple steps including chemical vapor deposition (CVD), spin-coating, and freeze drying methods. Simulated body fluid test confirms the highly bioactivity of the as-synthesized GF/58S BG scaffold after incorporating of sol-gel derived 58S BG. The GF/58S BG scaffold also remains good electrical conductivity of graphene after combination of 58S BG. Biocompatibility of both GF and GF/58S BG scaffold against the rabbit mesenchymal stem cells (rMSCs) is studied. Both GF and GF/58S BG scaffold facilitate the adhesion and extension of rMSCs, while the GF/58S BG scaffold shows a higher proliferation. Electrical stimulation was further applied on the both GF and GF/58S BG scaffold. Both scaffolds promote the osteogenic differentiation of rMSCs, while GF is more sensitive to the alternating electrical current. In vivo results based on the critical-sized radius defect rabbit model confirmed that the resulting GF/58S BG scaffold considerably promoted the formation of new bone. Our studies suggest that the as-synthesized GF/58S BG scaffolds are the promising candidates for bone tissue engineering and electrically stimulated regeneration considering unique bioactive, biocompatible, conductive and stable properties of the resulting nanoscaffolds.

The synthesis of transferrin (Tf)‐modified pegylated graphene (PG) and its application as a highly efficient drug delivery carrier for therapy of Ocular Choroidal Melanoma‐1 (OCM‐1) cells is presented. For the first reported time, nanoscaled PG is prepared using an environmentally friendly ball‐milling technique. The unique 2D nanostructure obtained using this PG synthesis approach offers considerable advantages in terms of drug loading and delivery, as well as the conjugation of Tf to PG providing a more targeted delivery vehicle. A highly efficient targeted pathway toward OCM‐1 cells triggered by an affinity between Tf and Tf receptors expressed on the surface of OCM‐1 cells is reported first here. PG‐Tf is observed to easily anchor anticancer drugs such as doxorubicin via π–π stacking. This work performs a Transwell two cells coculture experiment, a 3D in vitro tumor model, and an in vivo mouse model with OCM‐1 tumors to demonstrate the composite's therapeutic superiority over conventional systems for the targeted delivery and controlled release of antitumor drugs.

An efficient and targeted treatment for tumor cells is demonstrated. This targeting is based upon the strong affinity between hydroxyl-functional groups on graphene and acidic tumors. The hydroxylated graphene (GOH) with a unique 2D architecture further improve the targeting capacity of the system via an enhanced permeability and retention (EPR) process. Polyethylene glycol (PEG) was employed for better biocompatibility and the antitumor drug doxorubicin (DOX) was then incorporated. These additions created a biocompatible system with a superior pH-dependent drug release property. Its proficiency was due to its ability to pass through cell membranes via a process of endocytosis and exocytosis. The results from a Transwell co-culture system discovered that the PEG-GOH-DOX system had a large impact on tumor cell viability (less than 10% survived after treatment) and little influence on normal cells (more than 80% survived). An in vitro 3D tumor model study demonstrated that the size of the PEG-GOH-DOX treated tumor was 50% less than that of the pristine DOX treated tumor. In vivo data indicated that the PEG-GOH-DOX system was able to inhibit the size of tumors by a factor of 6.5 when compared to the untreated tumors.

The controlled synthesis of La2O2SO4: Eu3+ nanofibres (NF) for luminescent applications is presented here for the first time. Three dimensional nanofibrous structures with average diameter of 90 nm were prepared successfully. Eu3+ ions were further doped to study the luminescent property and phase structure. A strong red luminescence was obtained at the as-prepared NF. High asymmetry ratio (R) in the emission spectrum indicated that the Eu3+ ions were located in a distorted cation environment and the luminescent NF obtained possessed a good colour purity. The emission intensity of the resulting NF varied with different Eu3+ contents with the best emission brightness obtained at 20% Eu3+ doping. An 18% improved luminescence decay time at a relative lower annealing temperature (650 degrees C) was obtained for the nanofibrous structure when compared to the corresponding bulk materials (BM). The quantum efficiency (QE) of the as-synthesised NF was found to be 6.3%, which was 17% enhanced from the QE of BM (5.4%). Oxidation activity of NF compared to BM was measured using Temperature-Programmed Reduction (TPR). The TPR peak temperature for the consumption of hydrogen was decreased 12% with the employment of NF, suggesting well improved oxygen storage capability of the resulting NF. Our results indicate the multifunctional applications of the resulting NF in different fields such as optics and oxygen storage. (C) 2016 Elsevier B.V. All rights reserved.

A pH-sensitive, nanostructured antitumor drug, GO-CONH-Schiff base (GCS), was prepared from the chitosan-xanthone Schiff base (CS)-modified graphene oxide (GO) complex. The successful synthesis of GCS was confirmed using various spectroscopic techniques, including FT-IR, XPS, UV-vis and TGA. The resulting GCS showed superb antitumor activity with the pH-sensitive release of the antitumor part, CS, and decreased cytotoxicity of GCS to normal human cells. The release of CS was stable and thorough in the solution at pH 1 (the pH value for gastric juice), suggesting that the as-synthesized, pH-sensitive drug could provide new insights into the design of advanced nanostructured oral drugs.

A facile, but effective, method has been developed for large-scale preparation of NaLa(MoO4)2 nanorods and microflowers co-doped with Eu(3+) and Tb(3+) ions (abbreviated as: NLM:Ln(3+)). The as-synthesized nanomaterials possess a pure tetragonal phase with variable morphologies from shuttle-like nanorods to microflowers by controlling the reaction temperature and the amount of ethylene glycol used. Consequently, the resulting nanomaterials exhibit superb luminescent emissions over the visible region from red through yellow to green by simply changing the relative doping ratios of Eu(3+) to Tb(3+) ions. Biocompatibility study indicates that the addition of NLM:Ln(3+) nanomaterials can stimulate the growth of normal human retinal pigment epithelium (ARPE-19) cells. Therefore, the newly-developed NaLa(MoO4)2 nanomaterials hold potentials for a wide range of multifunctional applications, including bioimaging, security protection, optical display, optoelectronics for information storage, and cell stimulation.

We have presented our recent efforts on genotoxicity and intraocular biocompatibility of hydroxylated graphene (G-OH) prepared by ball milling. We have previously demonstrated that the as-synthesized G-OH could be considered as an excellent alternative for graphene oxide which had been applied widely. Following our last report on G-OH, we carried out detailed studies on genotoxicity and in vivo biocompatibility of G-OH in this work. Less than 5% enhanced caspase-3 level was observed for cells exposed to more than 50 μg/mL G-OH over 72 h, suggesting G-OH caused cell apoptosis was slight. The G-OH induced DNA damage was also found to be mild since expression of p53 and ROS regeneration level was quite low even at high concentration of G-OH over a long time. Cell viability was found to be higher than 90% with 50 μg/mL G-OH and 80% with 100 μg/mL G-OH using flow cytometry. Comet results suggested that less than 5% tail could be found with 100 μg/mL G-OH. TEM results confirmed that G-OH could penetrate into and out of the cytoplasm by means of endocytosis and exocytosis without causing damage on cell membranes. In vivo biocompatibility of G-OH was studied by intravitreal injection of G-OH into rabbits. The ocular fundus photography results showed that G-OH could be diffused in the vitreous body gradually without any damage caused. Injection of G-OH had caused few damages on eyesight related functions such as intraocular pressure, electroretinogram and histological structures of the retina.

Hydroxyl-functionalized graphene (G–OH) was efficiently produced from exfoliation of graphite powder by ball milling the presence of potassium hydroxide (KOH). While the nanoscale graphene sheets were confirmed by various characterization techniques, including atomic force microscopy (AFM), scanning electron microscopy (SEM), transmission electron microscopy (TEM), and UV-vis spectroscopy, the formation of hydroxyl groups in G–OH was revealed by Fourier transform infrared (FTIR), Nuclear Magnetic Resonance analysis (NMR), Raman and X-ray photoelectron spectroscopic (XPS) measurements. The as-prepared G–OH showed strong hydrophilicity with good solubility in water, excellent electrochemical activity and no influence on the growth of human retinal pigment epithelium (RPE) cells. More than 80% cell survival rate and less than 7% lactate dehydrogenase (LDH) release were observed for RPE cells treated with G–OH, indicating excellent biocompatibility.

As graphene becomes one of the most exciting candidates for multifunctional biomedical applications, contact between eyes and graphene-based materials is inevitable. On the other hand, eyes, as a special organ in the human body, have unique advantages to be used for testing new biomedical research and development, such as drug delivery. Intraocular biocompatible studies on graphene-related materials are thus essential. Here, we report our recent studies on intraocular biocompatibility and cytotoxicity of graphene oxide (GO) both in vitro and in vivo. The successful preparation of GO nanosheets was confirmed using atomic force microscopy, contact angle analyzer, Fourier transform infrared spectroscopy, and Raman spectroscopy. The influence of GO on human retinal pigment epithelium (RPE) cells in terms of the cell morphology, viability, membrane integrity, and apoptosis was investigated using various techniques, including optical micrography, cell counting kit-8 (CCK-8) assay, lactate dehydrogenase (LDH) assay, and apoptosis assay. The addition of GO had little influence on cell morphology, but the change was visible after long-time culturing. RPE cells showed higher than 60% cell viability by CCK-8 assay in GO solutions and less than 8% LDH release, although a small amount of apoptosis (1.5%) was observed. In vitro results suggested good biocompatibility of GO to RPE cells with slight adverse influence, on the cell viability and morphology in long-time periods, along with aggregation of GO. Thus, some further studies are needed to clarify the cytotoxicity mechanism of GO. GO intravitreally injected eyes showed few changes in eyeball appearance, intraocular pressure (IOP), eyesight, and histological photos. Our results suggested that GO did not cause any significant toxicity to the cell growth and proliferation. Intravitreal injection of GO into rabbits' eyes did not lead to much change in the eyeball appearance, IOP, electroretinogram, and histological examination.

PURPOSE:Eyelid development is a dynamic process involving cell proliferation, differentiation, and migration regulated by a number of growth factors and cytokines. Mice deficient in the orphan G protein-coupled receptor 48 (GPR48) showed an eye open at birth (EOB) phenotype. In this study, the authors attempted to clarify the role of GPR48 in eyelid development and the molecular mechanisms leading to the EOB phenotype.METHODS:Phenotypic analysis of the eyelids of Gpr48(-/-) mice was carried out using histology and scanning electron microscopy. GPR48 expression pattern was determined using X-gal staining. In vitro scratch assay was used to determine cell motility defects in Gpr48(-)(/)(-) keratinocytes. The molecular mechanism underlying GPR48-mediated eyelid closure was explored using Western blot and immunostaining analyses. Expression levels of EGFR and its phosphorylated counterpart were examined in Gpr48(-/-) and wild-type keratinocytes and in eyelids.RESULTS:GPR48 is highly expressed in the epithelium and apical mesenchymal cells of eyelids during embryonic development. Detailed analysis revealed that Gpr48(-/-) mice exhibited delayed leading-edge extension, reduced filopodia formation, and decreased rounded periderm cell formation around eyelid margins. Keratinocytes lacking GPR48 are defective in cell proliferation and migration with reduced F-actin staining. In addition, the phosphorylation of EGFR was dramatically decreased in cultured keratinocytes and developing eyelids in the absence of GPR48.CONCLUSIONS:Inactivation of GPR48 induces the EOB phenotype by reducing epithelial cell proliferation and migration, indicating that GPR48 plays an essential role in eyelid development. Furthermore, GPR48 contributes to eyelid development through the regulation of the EGFR signaling pathway.

目的：通过动物实验观察羧基单壁碳纳米管的眼内生物安全性。方法：选择健康成年雄性SD大鼠，随机分为3组，每组5只，羧基单壁碳纳米管剂量分别为 0.05 、0.1、 0.2 mg/ml，各5μl注入实验眼，各组均以右眼为实验眼，左眼注入等量的磷酸盐缓冲液（PBS）为对照，共观察1个月。采用裂隙灯和间接眼底镜等方法进行临床观察，玻璃体腔注入羧基单壁碳纳米管后观察眼压变化，视网膜电图评价注入前及注入后各时间点视网膜功能状态，光镜观察视网膜各层结构改变。结果：玻璃体腔注入羧基单壁碳纳米管前后眼压没有明显的变化，视网膜电图未见明显的改变，光镜观察视网膜各层结构未见明显改变。结论：实验剂量的羧基单壁碳纳米管注入SD大鼠玻璃体腔未见明显毒性。