Neuroprotective effect of insulin-loaded chitosan nanoparticles/PLGA-PEG-PLGA hydrogel on diabetic retinopathy in rats

Xianfang Rong1 Department of Ophthalmology and The Eye Institute, Eye and Ear, Nose, and Throat Hospital, Fudan University, Shanghai, China, [email protected]; [email protected];2 The Key Laboratory of Myopia, Ministry of Health, Shanghai, China, [email protected]; [email protected];3 Shanghai Key Laboratory of Visual Impairment and Restoration, Shanghai, China, [email protected]; [email protected];4 Key National Health Committee Key Laboratory of Myopia, Fudan University, Shanghai, China, [email protected]; [email protected];5 Laboratory of Myopia, Chinese Academy of Medical Sciences, Shanghai, China, [email protected]; [email protected]

Yinghong Ji1 Department of Ophthalmology and The Eye Institute, Eye and Ear, Nose, and Throat Hospital, Fudan University, Shanghai, China, [email protected]; [email protected];2 The Key Laboratory of Myopia, Ministry of Health, Shanghai, China, [email protected]; [email protected];3 Shanghai Key Laboratory of Visual Impairment and Restoration, Shanghai, China, [email protected]; [email protected];4 Key National Health Committee Key Laboratory of Myopia, Fudan University, Shanghai, China, [email protected]; [email protected];5 Laboratory of Myopia, Chinese Academy of Medical Sciences, Shanghai, China, [email protected]; [email protected]

Xiangjia Zhu1 Department of Ophthalmology and The Eye Institute, Eye and Ear, Nose, and Throat Hospital, Fudan University, Shanghai, China, [email protected]; [email protected];2 The Key Laboratory of Myopia, Ministry of Health, Shanghai, China, [email protected]; [email protected];3 Shanghai Key Laboratory of Visual Impairment and Restoration, Shanghai, China, [email protected]; [email protected];4 Key National Health Committee Key Laboratory of Myopia, Fudan University, Shanghai, China, [email protected]; [email protected];5 Laboratory of Myopia, Chinese Academy of Medical Sciences, Shanghai, China, [email protected]; [email protected]

Jin Yang1 Department of Ophthalmology and The Eye Institute, Eye and Ear, Nose, and Throat Hospital, Fudan University, Shanghai, China, [email protected]; [email protected];2 The Key Laboratory of Myopia, Ministry of Health, Shanghai, China, [email protected]; [email protected];3 Shanghai Key Laboratory of Visual Impairment and Restoration, Shanghai, China, [email protected]; [email protected];4 Key National Health Committee Key Laboratory of Myopia, Fudan University, Shanghai, China, [email protected]; [email protected];5 Laboratory of Myopia, Chinese Academy of Medical Sciences, Shanghai, China, [email protected]; [email protected]

Dongjin Qian1 Department of Ophthalmology and The Eye Institute, Eye and Ear, Nose, and Throat Hospital, Fudan University, Shanghai, China, [email protected]; [email protected];2 The Key Laboratory of Myopia, Ministry of Health, Shanghai, China, [email protected]; [email protected];3 Shanghai Key Laboratory of Visual Impairment and Restoration, Shanghai, China, [email protected]; [email protected];4 Key National Health Committee Key Laboratory of Myopia, Fudan University, Shanghai, China, [email protected]; [email protected];5 Laboratory of Myopia, Chinese Academy of Medical Sciences, Shanghai, China, [email protected]; [email protected]

Xiaofen Mo1 Department of Ophthalmology and The Eye Institute, Eye and Ear, Nose, and Throat Hospital, Fudan University, Shanghai, China, [email protected]; [email protected];2 The Key Laboratory of Myopia, Ministry of Health, Shanghai, China, [email protected]; [email protected];3 Shanghai Key Laboratory of Visual Impairment and Restoration, Shanghai, China, [email protected]; [email protected];4 Key National Health Committee Key Laboratory of Myopia, Fudan University, Shanghai, China, [email protected]; [email protected];5 Laboratory of Myopia, Chinese Academy of Medical Sciences, Shanghai, China, [email protected]; [email protected]Correspondence[email protected] [email protected]

Yi Lu1 Department of Ophthalmology and The Eye Institute, Eye and Ear, Nose, and Throat Hospital, Fudan University, Shanghai, China, [email protected]; [email protected];2 The Key Laboratory of Myopia, Ministry of Health, Shanghai, China, [email protected]; [email protected];3 Shanghai Key Laboratory of Visual Impairment and Restoration, Shanghai, China, [email protected]; [email protected];4 Key National Health Committee Key Laboratory of Myopia, Fudan University, Shanghai, China, [email protected]; [email protected];5 Laboratory of Myopia, Chinese Academy of Medical Sciences, Shanghai, China, [email protected]; [email protected]Correspondence[email protected] [email protected]

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Figures & data

Figure 1 Scotopic ERG in rats at 4 weeks after subconjunctival injection.

Notes: Normal group: no injection with any drugs; ICNPH group: subconjunctival injection of 20 µL ICNPH containing 80 µg insulin; Insulin group: subconjunctival injection of 80 µg insulin in 20 µL deionized water; Blank group: subconjunctival injection of 20 µL blank nanosized hydrogel; Sham group: an empty syringe without any drugs was punctured into the conjunctiva of the diabetic rats.

Abbreviations: A, A-wave of the ERG; B, B-wave of the ERG; ERG, electroretinography; ICNPH, insulin-loaded chitosan nanoparticles/PLGA-PEG-PLGA composite hydrogel; LE, left eye; PLGA-PEG-PLGA, poly(lactic-co-glycolic acid)-poly(ethylene glycol)-poly(lactic-co-glycolic acid); RE, right eye.

Figure 1 Scotopic ERG in rats at 4 weeks after subconjunctival injection.Notes: Normal group: no injection with any drugs; ICNPH group: subconjunctival injection of 20 µL ICNPH containing 80 µg insulin; Insulin group: subconjunctival injection of 80 µg insulin in 20 µL deionized water; Blank group: subconjunctival injection of 20 µL blank nanosized hydrogel; Sham group: an empty syringe without any drugs was punctured into the conjunctiva of the diabetic rats.Abbreviations: A, A-wave of the ERG; B, B-wave of the ERG; ERG, electroretinography; ICNPH, insulin-loaded chitosan nanoparticles/PLGA-PEG-PLGA composite hydrogel; LE, left eye; PLGA-PEG-PLGA, poly(lactic-co-glycolic acid)-poly(ethylene glycol)-poly(lactic-co-glycolic acid); RE, right eye.

Figure 2 HE staining and TEM observation of the retinal microstructure and ultrastructure changes in rats at 2 weeks after subconjunctival injection.

Notes: (A) The representative images of HE staining in retina in different groups. (B) The changes of retinal microvascular structure in different groups. (C) The changes of retinal ganglion cells structure in different groups. Retinal microvascular or retinal ganglion cells are identified by arrows. Normal group: no injection with any drugs; ICNPH group: subconjunctival injection of 20 µL ICNPH containing 80 µg insulin; Insulin group: subconjunctival injection of 80 µg insulin in 20 µL deionized water; Blank group: subconjunctival injection of 20 µL blank nanosized hydrogel; Sham group: an empty syringe without any drugs was punctured into the conjunctiva of the diabetic rats.

Abbreviations: ICNPH, insulin-loaded chitosan nanoparticles/PLGA-PEG-PLGA composite hydrogel; PLGA-PEG-PLGA, poly(lactic-co-glycolic acid)-poly(ethylene glycol)-poly(lactic-co-glycolic acid); TEM, transmission electron microscope.

Figure 2 HE staining and TEM observation of the retinal microstructure and ultrastructure changes in rats at 2 weeks after subconjunctival injection.Notes: (A) The representative images of HE staining in retina in different groups. (B) The changes of retinal microvascular structure in different groups. (C) The changes of retinal ganglion cells structure in different groups. Retinal microvascular or retinal ganglion cells are identified by arrows. Normal group: no injection with any drugs; ICNPH group: subconjunctival injection of 20 µL ICNPH containing 80 µg insulin; Insulin group: subconjunctival injection of 80 µg insulin in 20 µL deionized water; Blank group: subconjunctival injection of 20 µL blank nanosized hydrogel; Sham group: an empty syringe without any drugs was punctured into the conjunctiva of the diabetic rats.Abbreviations: ICNPH, insulin-loaded chitosan nanoparticles/PLGA-PEG-PLGA composite hydrogel; PLGA-PEG-PLGA, poly(lactic-co-glycolic acid)-poly(ethylene glycol)-poly(lactic-co-glycolic acid); TEM, transmission electron microscope.

Figure 3 TUNEL staining for apoptotic cells in retinas at 2 weeks after subconjunctival injection.

Figure 4 Immunofluorescence staining for GFAP, VEGF, and Occludin expression in retina at 2 weeks after subconjunctival injection.

Notes: (A) Immunofluorescence staining showing GFAP expression in retina in different groups. (B) Immunofluorescence staining showing VEGF expression in retina in different groups. (C) Immunofluorescence staining showing Occludin expression in retina in different groups. Normal group: no injection with any drugs; ICNPH group: subconjunctival injection of 20 µL ICNPH containing 80 µg insulin; Insulin group: subconjunctival injection of 80 µg insulin in 20 µL deionized water; Blank group: subconjunctival injection of 20 µL blank nanosized hydrogel; Sham group: an empty syringe without any drugs was punctured into the conjunctiva of the diabetic rats.

Abbreviations: GFAP, glial fibrillary acidic protein; ICNPH, insulin-loaded chitosan nanoparticles/PLGA-PEG-PLGA composite hydrogel; PLGA-PEG-PLGA, poly(lactic-co-glycolic acid)-poly(ethylene glycol)-poly(lactic-co-glycolic acid); VEGF, vascular endothelial growth factor.

Figure 4 Immunofluorescence staining for GFAP, VEGF, and Occludin expression in retina at 2 weeks after subconjunctival injection.Notes: (A) Immunofluorescence staining showing GFAP expression in retina in different groups. (B) Immunofluorescence staining showing VEGF expression in retina in different groups. (C) Immunofluorescence staining showing Occludin expression in retina in different groups. Normal group: no injection with any drugs; ICNPH group: subconjunctival injection of 20 µL ICNPH containing 80 µg insulin; Insulin group: subconjunctival injection of 80 µg insulin in 20 µL deionized water; Blank group: subconjunctival injection of 20 µL blank nanosized hydrogel; Sham group: an empty syringe without any drugs was punctured into the conjunctiva of the diabetic rats.Abbreviations: GFAP, glial fibrillary acidic protein; ICNPH, insulin-loaded chitosan nanoparticles/PLGA-PEG-PLGA composite hydrogel; PLGA-PEG-PLGA, poly(lactic-co-glycolic acid)-poly(ethylene glycol)-poly(lactic-co-glycolic acid); VEGF, vascular endothelial growth factor.

Figure 5 Western blot and real-time PCR assays for GFAP, VEGF, and Occludin expression levels in retina at 2 weeks after subconjunctival injection.

Notes: (A) The changes of GFAP expression at protein or mRNA levels in retinas in different groups. (B) The changes of VEGF expression at protein or mRNA levels in retinas in different groups. (C) The changes of Occludin expression at protein or mRNA levels in retinas in different groups. Normal group: no injection with any drugs; ICNPH group: subconjunctival injection of 20 µL ICNPH containing 80 µg insulin; Insulin group: subconjunctival injection of 80 µg insulin in 20 µL deionized water; Blank group: subconjunctival injection of 20 µL blank nanosized hydrogel; Sham group: an empty syringe without any drugs was punctured into the conjunctiva of the diabetic rats. *P<0.05; **P<0.01.

Neuroprotective effect of insulin-loaded chitosan nanoparticles/PLGA-PEG-PLGA hydrogel on diabetic retinopathy in rats

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Neuroprotective effect of insulin-loaded chitosan nanoparticles/PLGA-PEG-PLGA hydrogel on diabetic retinopathy in rats

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