Simultaneous Imaging and Photodynamic-Enhanced Photothermal Inhibition of Cancer Cells Using a Multifunctional System Combining Indocyanine Green and Polydopamine-Preloaded Upconversion Luminescent Nanoparticles.

This work introduces a novel multifunctional system called UPIPF (upconversion-polydopamine-indocyanine-polyethylene-folic) for upconversion luminescent (UCL) imaging of cancer cells using near-infrared (NIR) illumination. The system demonstrates efficient inhibition of human hepatoma (HepG2) cancer cells through a combination of NIR-triggered photodynamic therapy (PDT) and enhanced photothermal therapy (PTT). Initially, upconversion nanoparticles (UCNP) are synthesized using a simple thermal decomposition method. To improve their biocompatibility and aqueous dispersibility, polydopamine (PDA) is introduced to the UCNP via a ligand exchange technique. Indocyanine green (ICG) molecules are electrostatically attached to the surface of the UCNP-polydopamine (UCNP@PDAs) complex to enhance the PDT and PTT effects. Moreover, polyethylene glycol (PEG)-modified folic acid (FA) is incorporated into the UCNP-polydopamine-indocyanine-green (UCNP@PDA-ICGs) nanoparticles to enhance their targeting capability against cancer cells. The excellent UCL properties of these UCNP enable the final UCNP@PDA-ICG-PEG-FA nanoparticles (referred to as UPIPF) to serve as a potential candidate for efficient anticancer drug delivery, real-time imaging, and early diagnosis of cancer cells. Furthermore, the UPIPF system exhibits PDT-assisted PTT effects, providing a convenient approach for efficient cancer cell inhibition (more than 99% of cells are killed). The prepared UPIPF system shows promise for early diagnosis and simultaneous treatment of malignant cancers.

A new antioxidant made from a pterostilbene functionalized graphene nanocomposite as an efficient treatment for dry eye disease.

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.

Autophagy positively regulates Wnt signaling in mice with diabetic retinopathy.

Diabetic retinopathy (DR) is a microvascular complication of diabetes. Aberrant Wnt signaling activation plays a pathological role in DR. However, the underlying mechanisms of aberrant Wnt signaling in DR remain unknown. Autophagy has been reported to be involved in the pathophysiology of DR. The present study aimed therefore to investigate the regulatory effects of autophagy on Wnt signaling in DR. Wnt signaling was activated in the retina of db/db mice combined with an increase in the expression of the autophagic proteins microtubule-associated protein 1A/1B-light chain 3 and beclin-1 and a decrease in the expression of the autophagic protein P62. Inhibition of autophagy by 3-methyladenin decreased Wnt signaling in diabetic retinas, indicating a potential association between Wnt signaling and autophagy. Rapamycin, an autophagy inducer, upregulated Wnt signaling in the retina of normal C57BL/6J mice. In cultured Müller cells, rapamycin induced autophagy and activated Wnt signaling, while chloroquine, an autophagy inhibitor, inhibited autophagy and downregulated Wnt signaling, suggesting that autophagy could regulate Wnt signaling in mice retina and retinal cells. In summary, this study demonstrated that autophagy may positively regulate Wnt signaling in diabetic retinas, indicating a potential mechanism of Wnt signaling upregulation in DR and a possible novel therapeutic target of DR.

PPARα-Dependent Effects of Palmitoylethanolamide Against Retinal Neovascularization and Fibrosis.

Purpose: Pathological neovascularization and fibrosis are common pathological changes of many retinal diseases, such as proliferative retinopathy (PR) and age-related macular degeneration (AMD). Treatment modalities for these pathological changes are limited. The purpose of the present study was to test the effects of palmitoylethanolamide (PEA), an endocannabinoid mimetic amide, on retinal neovascularization and fibrosis and to determine its molecular mechanism of action. Methods: A rat Müller cell line (rMC-1), a mouse model of oxygen-induced retinopathy (OIR), and the very-low-density lipoprotein receptor (VLDLR) knockout mouse model were used. PEA was intraperitoneally injected or orally administrated in animal models. Inflammation and profibrotic changes were evaluated by western blot analysis. Glial fibrillary acidic protein (GFAP) and peroxisome proliferator-activated receptor alpha (PPARα) were measured by RT-PCR and western blot analysis. Results: Profibrotic changes were present in OIR and Vldlr-/- retinas. PEA significantly alleviated inflammation and inhibited neovascularization in OIR and Vldlr-/- retinas and suppressed profibrotic changes in OIR and Vldlr-/- retinas. Moreover, PEA potently suppressed Müller gliosis in these retinas. In rMC-1 cells, PEA suppressed Müller gliosis, reduced inflammatory cytokines, and attenuated profibrotic changes. Further, both mRNA and protein levels of PPARα were elevated in the retina under PEA treatment, and the effects of PEA were abolished in Pparα-/- OIR mice. Conclusions: PEA reduced retinal neovascularization and fibrotic changes and suppressed Müller gliosis in experimental PR and neovascular AMD by activating PPARα. PEA may be a potential treatment for retinopathies with pathological neovascularization and fibrosis.

Inhibition of acetylcholinesterase attenuated retinal inflammation via suppressing NF-κB activation.

Elevated inflammatory cytokines contribute to the pathogenesis of various retinal diseases such as diabetic retinopathy, retinal vasculitis and retinitis. However, the underlying mechanism of retinal inflammation remains largely unknown. Recent studies demonstrated that acetylcholinesterase (ACHE) is an inflammatory indicator in central neural system. This study was aimed to dissect the role of ACHE in retinal inflammation, and its mechanism of action. Retinal inflammation was induced by intravitreal injection of tumor necrosis factor-α (TNF-α) in heterozygous ACHE knockdown mice (ACHE+/-) and wild type mice (ACHE+/+). Donepezil, a well-known ACHE inhibitor, was administrated by daily gavage. Expression of ACHE and intercellular adherent molecule-1 (ICAM-1), infiltration of CD11b+ inflammatory cells, retinal leukostasis and vascular leakage was determined in both ACHE+/- and ACHE+/+ mice. ARPE-19 cells, a human retinal pigment epithelial cell line, were cultured for in vitro assay. Knockdown of ACHE was achieved by lipofectamine-mediated siRNA transfection and pharmaceutical suppression of ACHE was manipulated by donepezil. Cellular expression and distribution of ACHE, ICAM-1, and phosphorylation of NF-κB, IκB and IKKα/ß were detected by western-blot analysis or immunocytochemistry. Retinal expression of ACHE was dramatically upregulated, in parallel with increased ICAM-1 expression, enhanced leukostasis and augmented CD11b+ inflammatory cell infiltration as well as vascular hyperpermeability in ACHE+/+ mice injected with TNF-α. However, TNF-α-injected ACHE+/- mice showed lower level of ICAM-1, less leukostasis and fewer infiltrated CD11b+ cells. Moreover, TNF-α-induced retinal vascular leakage was significantly reduced in ACHE+/- mice. Similarly, TNF-α-induced retinal inflammatory response were also attenuated by donepezil intervention. In addition, TNF-α treatment resulted in significant induction of ACHE, upregulation of ICAM-1 and nuclear translocation of NF-κB, phosphorylation of IκB and IKKα/ß in ARPE-19 cells. However, inhibition of ACHE reduced TNF-α-induced phosphorylation of NF-κB, IκB and IKKα/ß in ARPE-19 cells. The present study reveals a pivotal role of ACHE in retinal inflammation. Inhibition of ACHE attenuates retinal inflammation and retinal leakage likely through suppressing NF-κB signaling activation.

Fenofibrate Inhibits Subretinal Fibrosis Through Suppressing TGF-ß-Smad2/3 signaling and Wnt signaling in Neovascular Age-Related Macular Degeneration.

Subretinal fibrosis is a common pathological change that causes vision loss in neovascular age-related macular degeneration (nAMD). Treatment modalities for subretinal fibrosis are limited. In the present study, the effects of fenofibrate, a specific peroxisome proliferator-activated receptor alpha agonist, on subretinal fibrosis of nAMD were tested, and its molecular mechanisms of action were delineated. Collagen deposition and protein expression of fibrotic markers, such as vimentin, collagen-1, alpha-smooth muscle actin, and fibronectin, were increased in very low-density lipoprotein receptor (VLDLR) knockout mouse, indicating Vldlr -/- mice can be used as a model for subretinal fibrosis. Fenofibrate suppressed subretinal fibrosis of Vldlr -/- mice by reducing collagen deposition and protein expression of fibrotic markers. Two fibrotic pathways, TGF-ß-Smad2/3 signaling and Wnt signaling, were significantly up-regulated, while inhibited by fenofibrate in Vldlr -/- retinas. Moreover, fenofibrate significantly reduced the downstream connective tissue growth factor (CTGF) expression of these two pathways. Müller cells were a major source of CTGF in Vldlr -/- retinas. Fenofibrate was capable of suppressing Müller cell activation and thus reducing the release of CTGF in Vldlr -/- retinas. In cultured Müller cells, fenofibrate reversed TGF-ß2-induced up-regulation of Wnt signaling and CTGF expression. These findings suggested that fenofibrate inhibits subretinal fibrosis by suppressing TGF-ß-Smad2/3 signaling and Wnt signaling and reducing CTGF expression, and thus, fenofibrate could be a potential treatment for nAMD with subretinal fibrosis.

0.005% Preservative-Free Latanoprost Induces Dry Eye-Like Ocular Surface Damage via Promotion of Inflammation in Mice.

Purpose: To investigate the side effects of preservative-free 0.005% latanoprost on the murine ocular surface. Methods: We applied 0.005% latanoprost or vehicle in mice in two patterns for 14 to 28 days. Tear production was measured by phenol red cotton test, and corneal epithelial barrier function was assessed by Oregon-green-dextran (OGD) staining. Periodic acid-Schiff (PAS) staining was used to quantify conjunctival goblet cells (GCs). The expression of matrix metalloproteinase (MMP)-3 and -9, occludin-1 and zonula occludens (ZO)-1 in corneal epithelium was assessed by immunofluorescent staining and/or quantitative real-time PCR (qRT-PCR). Inflammation in conjunctiva was assessed by activation of P38 and NF-κB, infiltration of CD4+ T cells, and production inflammatory cytokines including TNF-α, IL-1ß, IFN-γ, IL-17A, and IL-13. Apoptosis in ocular surface was assessed by TUNEL and immunofluorescent staining for activated caspase-3 and -8. Cell viability assay was performed in human corneal epithelial cells. Results: Topical latanoprost treatment decreased tear production, induced conjunctival GC loss, disrupted the corneal epithelial barrier, and promoted cell apoptosis in the ocular surface. Topical latanoprost treatment increased the expression of MMP-3 and -9, and decreased the expression of ZO-1 and occludin-1 in the corneal epithelium. Topical application of latanoprost promoted activation of P38-NF-κB signaling and production of TNF-α and IL-1ß in conjunctiva. Topical application of latanoprost increased CD4+ T cells infiltration, with increased production of IFN-γ and IL-17A and decreased production of IL-13 in conjunctiva. Conclusion: 0.005% latanoprost induced dry eye-like ocular surface damage via promotion of inflammation in mice.