Sustained Release of Gas6 via mPEG-PLGA Nanoparticles Enhances the Therapeutic Effects of MERTK Gene Therapy in RCS Rats

Previous researches utilizing MER proto-oncogene tyrosine kinase (MERTK) gene therapy in Royal College of Surgeons (RCS) rats evidenced its effectiveness in treating MERTK-associated retinitis pigmentosa (RP). Specific ligands for receptor tyrosine kinases, such as growth arrest-specific 6 (Gas6), may enhance retinal phagocytosis via the MERTK receptor, and consequently, enhance the therapeutic effects of gene therapy. In order to overcome the short life effect of the injected Gas6 protein, we constructed a Gas6 loaded methoxy-poly (ethylene glyeol)-poly (lactic-co-glycolic acid) (mPEG-PLGA) nanoparticles (Gas6 NPs) system which allowed for localized and sustained Gas6 protein release, and therefore, a prolonged biological effect. Our data demonstrated that Gas6 protein release from Gas6 NPs preserved the bioactivity and promoted retinal pigment epithelium (RPE) phagocytosis in vitro. In vivo studies showed that RCS rats in the hMERTK/Gas6 NPs group exhibiting the highest electroretinogram responses and more complete retinal structure than that in other groups, further demonstrating that the co-administration of AAV2-BEST1-hMERTK and Gas6 NPs could protect photoreceptors from degeneration. These findings strongly suggest that Gas6 NPs are a promising method to enable the sustained release of Gas6 protein and could therefore enhance the therapeutic effects of gene therapy for MERTK-associated RP.

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Ocular Toxoplasmosis: Mechanisms of Retinal Infection and Experimental Models

Parasitologia ◽

10.3390/parasitologia1020007 ◽

2021 ◽

Vol 1 (2) ◽

pp. 50-60

Author(s):

Veronica Rodriguez Fernandez ◽

Giovanni Casini ◽

Fabrizio Bruschi

Keyword(s):

Ex Vivo ◽

Cell Damage ◽

Pigment Epithelium ◽

Retinal Pigment ◽

Treatment Strategies ◽

Experimental Models ◽

Ocular Toxoplasmosis ◽

Cell Infection

Ocular toxoplasmosis (OT) is caused by the parasite Toxoplasma gondii and affects many individuals throughout the world. Infection may occur through congenital or acquired routes. The parasites enter the blood circulation and reach both the retina and the retinal pigment epithelium, where they may cause cell damage and cell death. Different routes of access are used by T. gondii to reach the retina through the retinal endothelium: by transmission inside leukocytes, as free parasites through a paracellular route, or after endothelial cell infection. A main feature of OT is the induction of an important inflammatory state, and the course of infection has been shown to be influenced by the host immunogenetics. On the other hand, there is evidence that the T. gondii phenotype also has an impact on the distribution of the pathology in different areas. Although considerable knowledge has been acquired on OT, a deeper knowledge of its mechanisms is necessary to provide new, more targeted treatment strategies. In particular, in addition to in vitro and in vivo experimental models, organotypic, ex vivo retinal explants may be useful in this direction.

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The cytoskeletal elements of human retinal pigment epithelium: in vitro and in vivo

Journal of Cell Science ◽

10.1242/jcs.91.2.303 ◽

1988 ◽

Vol 91 (2) ◽

pp. 303-312

Author(s):

N.M. McKechnie ◽

M. Boulton ◽

H.L. Robey ◽

F.J. Savage ◽

I. Grierson

Keyword(s):

Retinal Pigment Epithelium ◽

Pigment Epithelium ◽

Retinal Pigment ◽

Cytokeratin 18 ◽

Rpe Cells ◽

Human Retinal Pigment Epithelium ◽

Cytoskeletal Elements

The cytoskeletal elements of normal (in situ) and cultured human retinal pigment epithelium (RPE) were studied by a variety of immunocytochemical techniques. Primary antibodies to vimentin and cytokeratins were used. Positive immunoreactivity for vimentin was obtained with in situ and cultured material. The pattern of reactivity obtained with antisera and monoclonals to cytokeratins was more complex. Cytokeratin immunoreactivity could be demonstrated in situ and in cultured cells. The pattern of cytokeratin expression was similar to that of simple or glandular epithelia. A monoclonal antibody that specifically recognizes cytokeratin 18 identified a population of cultured RPE cells that had particularly well-defined filamentous networks within their cytoplasm. Freshly isolated RPE was cytokeratin 18 negative by immunofluorescence, but upon culture cytokeratin 18 positive cells were identifiable. Cytokeratin 18 positive cells were identified in all RPE cultures (other than early primaries), regardless of passage number, age or sex of the donor. In post-confluent cultures cytokeratin 18 cells were identified growing over cytokeratin 18 negative cells, suggesting an association of cytokeratin 18 immunoreactivity with cell proliferation. Immunofluorescence studies of retinal scar tissue from two individuals revealed the presence of numerous cytokeratin 18 positive cells. These findings indicate that RPE cells can be identified by their cytokeratin immunoreactivity and that the overt expression of cytokeratin 18 may be associated with proliferation of human RPE both in vitro and in vivo.

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JAM-C maintains VEGR2 expression to promote retinal pigment epithelium cell survival under oxidative stress

Thrombosis and Haemostasis ◽

10.1160/th16-11-0885 ◽

2017 ◽

Vol 117 (04) ◽

pp. 750-757

Author(s):

Xin Jia ◽

Chen Zhao ◽

Qishan Chen ◽

Yuxiang Du ◽

Lijuan Huang ◽

...

Keyword(s):

Oxidative Stress ◽

Retinal Pigment Epithelium ◽

Cell Survival ◽

Pigment Epithelium ◽

Retinal Pigment ◽

Retinal Pigment Epithelium Cell ◽

Photoreceptor Cells ◽

Rpe Cells

SummaryJunctional adhesion molecule-C (JAM-C) has been shown to play critical roles during development and in immune responses. However, its role in adult eyes under oxidative stress remains poorly understood. Here, we report that JAM-C is abundantly expressed in adult mouse retinae and choroids in vivo and in cultured retinal pigment epithelium (RPE) and photoreceptor cells in vitro. Importantly, both JAM-C expression and its membrane localisation are downregulated by H2O2-induced oxidative stress. Under H2O2-induced oxidative stress, JAM-C is critically required for the survival of human RPE cells. Indeed, loss of JAM-C by siRNA knockdown decreased RPE cell survival. Mechanistically, we show that JAM-C is required to maintain VEGFR2 expression in RPE cells, and VEGFR2 plays an important role in keeping the RPE cells viable since overexpression of VEGFR2 partially restored impaired RPE survival caused by JAM-C knockdown and increased RPE survival. We further show that JAM-C regulates VEGFR2 expression and, in turn, modulates p38 phosphorylation. Together, our data demonstrate that JAM-C plays an important role in maintaining VEGR2 expression to promote RPE cell survival under oxidative stress. Given the vital importance of RPE in the eye, approaches that can modulate JAM-C expression may have therapeutic values in treating diseases with impaired RPE survival.

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Preparation of Liposomal Formulations for Ocular Delivery of Thymoquinone: In Vitro Evaluation in HCEC-2 e HConEC Cells

Pharmaceutics ◽

10.3390/pharmaceutics13122093 ◽

2021 ◽

Vol 13 (12) ◽

pp. 2093

Author(s):

Elisa Landucci ◽

Francesca Bonomolo ◽

Chiara De Stefani ◽

Costanza Mazzantini ◽

Domenico Edoardo Pellegrini-Giampietro ◽

...

Keyword(s):

Epithelial Cells ◽

Therapeutic Potential ◽

Nigella Sativa ◽

Pigment Epithelium ◽

Retinal Pigment ◽

Fold Increase ◽

Prolonged Release ◽

Liposomal Formulations

Thymoquinone (TQ) is the main constituent of Nigella sativa L. essential oil. In vitro studies have shown its protective effect against H2O2-induced oxidative stress in human retinal pigment epithelium cells, and in vivo experiments have demonstrated its effect in decreasing corneal neovascularization and reducing the inflammation in an experimental dry eye model in mice. Its therapeutic use is limited by poor bioavailability, low solubility, and scarce permeability. In this study, two liposomal formulations have been developed, both of which consist of phosphatidylcholine and Plurol Oleique, a liquid lipid, and one of which is coated with 0.1% w/v hyaluronic acid (HA) to increase both TQ solubility and its ocular therapeutic potential. Each formulation has a size <200 nm and an EE% around 70%, determined by scattering techniques and the HPLC-DAD analytical method, respectively, and they result in a 2-fold increase in TQ solubility. HA-coated liposomes are stable over 2 months at +4 °C, and coated and uncoated liposomes present a gradual and prolonged release of TQ. Two cell lines, human corneal epithelial cells (HCEC-2) and human conjunctival epithelial cells (HConEC) were used to investigate the safety of the liposomal formulations. Uptake studies were also performed using fluorescent liposomes. Both liposomes and, in particular, HA-coated liposomes reduce the TQ toxicity observed at high doses in both HCEC-2 and HConEC cells, and both formulations increase the absorption at the cellular level and especially at the nucleus level, with a more pronounced effect for HA-coated liposomes.

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Targeted Delivery of Chloroquine to Plasmacytoid Dendritic Cells Enhances Inhibition of the Type I Interferon Response

10.1101/2021.06.09.447773 ◽

2021 ◽

Author(s):

Marilyn E Allen ◽

Amit Golding ◽

Violeta Rus ◽

Nicholas B Karabin ◽

Sophia Li ◽

...

Keyword(s):

Lupus Erythematosus ◽

Targeted Delivery ◽

Pigment Epithelium ◽

Retinal Pigment ◽

Interferon Response ◽

Type I ◽

Healthy Human ◽

Biodistribution Studies

Systemic lupus erythematosus (SLE) causes damaging inflammation in multiple organs via the accumulation of immune complexes. These complexes activate plasmacytoid DCs (pDCs) via TLR7 and TLR9, contributing to disease pathogenesis by driving secretion of inflammatory type I IFNs. Antimalarial drugs, such as chloroquine (CQ), are TLR antagonists used to alleviate inflammation in SLE. However, they require ~3 months of continuous use before achieving therapeutic efficacy and can accumulate in the retinal pigment epithelium with chronic use resulting in retinopathy. We hypothesized that poly(ethylene glycol)-b-poly(propylene sulfide) (PEG-b-PPS) filamentous nanocarriers, filomicelles (FMs) could improve drug activity and reduce toxicity by directly delivering CQ to pDCs via passive, morphology-based targeting. Healthy human PBMCs were treated with soluble CQ or CQ-loaded FMs, stimulated with TLR agonists or SLE patient sera, and type I IFN secretion was quantified via multi-subtype IFN-α ELISA and MX1 gene expression using real-time RT-qPCR. Our results showed that 50 µg CQ/mg FM decreased MX1 expression and IFN-α production after TLR activation with either synthetic nucleic acid agonists or immune complex rich sera from SLE patients. Cellular uptake and biodistribution studies showed that FMs preferentially accumulate in human pDCs in vitro and in tissues frequently damaged in SLE patients (i.e., liver and kidneys) while sparing the eye in vivo. These results showed that nanocarrier morphology enables drug delivery, and CQ-FMs may be equally effective and more targeted than soluble CQ at inhibiting SLE-relevant pathways.

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Targeting CD146 using folic acid–conjugated nanoparticles and suppression of tumor growth in a mouse glioma model

Journal of Neurosurgery ◽

10.3171/2020.4.jns193078 ◽

2020 ◽

pp. 1-11

Author(s):

Naoki Fukui ◽

Toshio Yawata ◽

Takahito Nakajo ◽

Yu Kawanishi ◽

Youichirou Higashi ◽

...

Keyword(s):

Gene Therapy ◽

Folic Acid ◽

Tumor Growth ◽

In Vivo Imaging ◽

Water Soluble ◽

Therapeutic Effects ◽

Glioma Model ◽

Mouse Glioma

OBJECTIVEGlioma stem cells (GSCs) are responsible for tumor initiation, therapeutic resistance, and recurrence. CD146 is mainly expressed in dividing GSCs and regulates cell cycle progression. However, the evaluation of the efficacy of targeted therapy against CD146 in vivo remains to be investigated. In this study, the authors aimed to develop gene therapy targeting GSCs using chitosan oligosaccharide lactate (COL) nanoparticles (NPs) conjugated with folic acid–polyethylene glycol (FA-PEG-COL NPs) for in vitro and in vivo delivery of CD146 small-interfering RNA (siCD146) and to determine the effect of CD146 knockdown on tumor growth.METHODSTo examine the uptake of NPs by tumor cells, immunofluorescence staining, flow cytometry, and in vivo imaging were performed. The knockdown effect of siCD146 was measured by western blot and water-soluble tetrazolium salt–8 assay in mouse glioma cells. The efficacy of siRNA therapy–targeted GSCs was evaluated by monitoring tumor growth through in vivo imaging and histological analysis.RESULTSIn vivo accumulation of the FA-PEG-COL NPs in subcutaneous and intracranial gliomas following NP administration via a mouse tail vein was observed. Additionally, in vitro delivery of siCD146 ionically cross-linked NPs, reduced CD146 levels, and suppressed growth in the glioma tumor sphere. Evaluation of the in vivo therapeutic effects of siCD146–cross-linked NPs in a mouse glioma model revealed significant suppression of intracranial tumor growth, with complete removal of the tumor observed in some mice on histological examination. Furthermore, delivery of siCD146 significantly reduced the Ki-67 index in residual tumor tissues relative to that in control mice.CONCLUSIONSCD146 is a potential therapeutic target, and folic acid–conjugated NPs delivering siRNA may facilitate gene therapy in malignant gliomas.

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Clinical-grade stem cell–derived retinal pigment epithelium patch rescues retinal degeneration in rodents and pigs

Science Translational Medicine ◽

10.1126/scitranslmed.aat5580 ◽

2019 ◽

Vol 11 (475) ◽

pp. eaat5580 ◽

Cited By ~ 57

Author(s):

Ruchi Sharma ◽

Vladimir Khristov ◽

Aaron Rising ◽

Balendu Shekhar Jha ◽

Roba Dejene ◽

...

Keyword(s):

Stem Cell ◽

Retinal Pigment Epithelium ◽

Pigment Epithelium ◽

Retinal Pigment ◽

Age Related Macular Degeneration ◽

Clinical Grade ◽

Functional Validation ◽

Age Related

Considerable progress has been made in testing stem cell–derived retinal pigment epithelium (RPE) as a potential therapy for age-related macular degeneration (AMD). However, the recent reports of oncogenic mutations in induced pluripotent stem cells (iPSCs) underlie the need for robust manufacturing and functional validation of clinical-grade iPSC-derived RPE before transplantation. Here, we developed oncogenic mutation-free clinical-grade iPSCs from three AMD patients and differentiated them into clinical-grade iPSC-RPE patches on biodegradable scaffolds. Functional validation of clinical-grade iPSC-RPE patches revealed specific features that distinguished transplantable from nontransplantable patches. Compared to RPE cells in suspension, our biodegradable scaffold approach improved integration and functionality of RPE patches in rats and in a porcine laser-induced RPE injury model that mimics AMD-like eye conditions. Our results suggest that the in vitro and in vivo preclinical functional validation of iPSC-RPE patches developed here might ultimately be useful for evaluation and optimization of autologous iPSC-based therapies.

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Heparin-Modified Amniotic Membrane Combined With Growth Factors for Promoting Corneal Wound Healing After Alkali Burn

Frontiers in Bioengineering and Biotechnology ◽

10.3389/fbioe.2020.599800 ◽

2020 ◽

Vol 8 ◽

Author(s):

Xuan Zhao ◽

Xin Zuo ◽

Jing Zhong ◽

Bowen Wang ◽

Saiqun Li ◽

...

Keyword(s):

Wound Healing ◽

Growth Factors ◽

Sustained Release ◽

Corneal Epithelial Cell ◽

Control Group ◽

Therapeutic Effects ◽

Cell Growth And Migration ◽

Corneal Epithelial

Ocular chemical burns are potentially blinding ocular injuries and require urgent management. Amniotic membrane (AM) transplantation is an effective surgical treatment, one of the reasons is because AM is a rich source of growth factors that can promote epithelialization and wound healing. However, growth factors will be gradually lost and insufficient after preparation process and long-time storage, leading to unsatisfactory therapeutic effects. Herein, we present a modified AM (AM-HEP) for the supplement and sustained release of growth factor by surface grafting heparin for treatment of ocular chemical burns. Heparin grafting rate and stability, microstructure, physical property, and sustained release of epithelial growth factor (EGF) of AM-HEP were characterized. Biocompatibility and ability to promote corneal epithelial cell growth and migration were evaluated and compared with a biological amnion, which is available on the market in vitro. The therapeutic effects of AM-HEP combined with EGF (AM-HEP@EGF) in vivo had been evaluated in a model of mouse corneal alkali burn. The results indicated that heparin was introduced into AM and maintain stability over 3 weeks at 37°C. The modification process of AM-HEP did not affect microstructure and physical property after comparing with non-modified AM. EGF could be combined quickly and effectively with AM-HEP; the sustained release could last for more than 14 days. AM-HEP@EGF could significantly promote corneal epithelial cell growth and migration, compared with non-modified AM and control group. Faster corneal epithelialization was observed with the transplantation of AM-HEP@EGF in vivo, compared with the untreated control group. The corneas in the AM-HEP@EGF group have less inflammation and were more transparent than those in the control group. The results from in vitro and in vivo experiments demonstrated that AM-HEP@EGF could significantly enhance the therapeutic effects. Taken together, AM-HEP@EGF is exhibited to be a potent clinical application in corneal alkali burns through accelerating corneal epithelial wound healing.

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