TPEN loaded poly (lactide-co-glycolide) nanoparticles promote neuroprotection and optic nerve regeneration

Abstract

Development of novel therapeutics for retinal ganglion cells (RGCs) protection and axon regeneration in neurodegenerative diseases, for example, glaucoma, are critical challenges in clinical treatment. Utilization of N, N, N', N'-tetrakis-(2-Pyridylmethyl) ethylenediamine (TPEN), a specific chelator of Zn²⁺, revealed positive medical potentials. However, its therapeutic effect in promoting RGCs survival and axon regeneration is restricted due to the inefficient drug delivery and limited absorption. To address this, this work developed a novel nanoparticles (NPs)-based drug delivery system with sustained release of TPEN, using oil-in-water (O/W) single-emulsion solvent evaporation method with various surfaces coatings. Optic nerve crush (ONC) and acute ocular hypertension (AOH) animal models were carried out to investigate the neuroprotective and axon regenerative effects of TPEN-loaded NPs. RGCs protection was systematically assessed through whole-mount retina immunostaining and hematoxylin eosin staining for histological changes. Electroretinography was used for evaluating visual function changes. Axon protection and regeneration were evaluated by SMI32 stain and intravitreal administration of cholera Toxin Subunit B (CTB), respectively. In vivo, TPEN-loaded NPs achieved a comparable therapeutic effect on neuroprotection and axon regeneration after ONC, with a reduced frequency of vitreous injection and half of TPEN dosage compared to its solution. In the meantime, visual function was also more effectively preserved with TPEN-loaded NPs. Additionally, RGCs survival and axon protection after AOH were significantly enhanced after treating with TPEN-loaded NPs. The developed TPEN-loaded NPs show promise for pre-clinical testing of neuroprotective and neuro-regenerative therapies in glaucoma and other neurodegenerative diseases.

Keywords: Glaucoma; N, N, Nʹ, Nʹ-tetrakis-(2-Pyridylmethyl) ethylenediamine (TPEN); Nanoparticles; Neuroprotection; Optic nerve regeneration; Polylactic acid-hydroxyacetic acid (PLGA); Zinc.

TPEN-loaded nanoparticles fabricated by single emulsion solvent evaporation method with oil-in-water (O/W) promoted RGCs survival and axon regeneration in glaucomatous animal models.

Fig. 1

Characteristics of NPs. (A) Schematic of the formation of TPEN-loaded NPs. (B) SEM image of TP-NP. (C) SEM image of TPDA-NP. (D) Particle size distribution of TP-NP and TPDA-NP. (E) Survey XPS spectra of TP-NP and TPDA-NP. (F) High-resolution core-level XPS spectra of TP-NP. (G) High-resolution core-level XPS spectra of TPDA-NP.

Fig. 2

Effect of different PLGA molecular weight on drug release rate in vitro. The data were analyzed by one-way ANOVA with Bonferroni’s post hoc test. Green ∗, blue ∗ and yellow ∗ mean the difference between groups of TP-NP (molecular weight of PLGA was 48 kDa), TP-NP (molecular weight of PLGA was 30 kDa), TP-NP (molecular weight of PLGA was 32 kDa) and TPDA-NP (molecular weight of PLGA was 48 kDa 10 kDa), respectively. ∗∗P＜0.01, ∗∗∗P＜0.001 between indicated two groups. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

Toxicity of TPEN-loaded nanoparticles in vitro. (A) Cell viability of aRPE-19 cells treated with different concentration of TPEN solution after 24 h. (B) Cell viability of aRPE-19 cells in different groups containing 1 μM TPEN. (C) Cell viability of aRPE-19 cells in different groups containing 10 μM TPEN. (D) Cell viability of 661W cells treated with different concentration of TPEN solution after 24 h. (E) Cell viability of 661W cells in different groups containing 1 μM TPEN. (F) Cell viability of 661W cells in different groups containing 10 μM TPEN. (G) Cell viability of BV2 cells treated with different concentration of TPEN solution after 24 h. (H) Cell viability of BV2 cells in different groups containing 1 μM TPEN. (I) Cell viability of BV2 cells in different groups containing 10 μM TPEN. The data were analyzed by one-way ANOVA with Bonferroni’s post hoc test and unpaired Student’s t-test (2 tailed). ∗P＜0.05, ∗∗∗∗P＜0.0001 between indicated two groups. ###P＜0.001 between indicated two groups.# means unpaired Student’s t-test (2 tailed) between indicated two groups. ns, no significant difference in indicated two groups. Each point shown in the bar charts indicates an individual data point.

Fig. 4

Toxicity of TPEN-loaded nanoparticles in-vivo. (A) Representative images of hematoxylin and eosin staining in different groups. Scale bar, 50 μm. (B-G) The thickness of retina (B), GCL+NFL (C), IPL (D), INL (E), OPL (F) and ONL (G) in various treatments; (H-J) The cell density of GCL (H), INL (I) and ONL (J).The data were analyzed by one-way ANOVA with Bonferroni’s post hoc test and unpaired Student’s t-test (2 tailed). ns means no significant difference in indicated two groups.

Fig. 5

TPEN-loaded nanoparticles decreased concentration of Zn²⁺ in retinas. (A) Representative images of Zn²⁺ concentration in different groups after 1 day of ONC. Scale bar, 100 μm. (B) Bar charts showed the relative intensity of Zn²⁺ in IPL of retinas in different groups. All the data were analyzed by one-way ANOVA with Bonferroni’s post hoc test and unpaired Student’s t-test (2 tailed). ∗∗P＜0.01, ∗∗∗P＜0.001 between indicated two groups. #P＜0.05 between indicated two groups. ##P＜0.01 between indicated two groups.# means unpaired Student’s t-test (2 tailed) between indicated two groups. ns, no significant difference in indicated two groups. Each point shown in the bar charts indicates an individual data point. GCL: Ganglion cell layer; IPL: Inner plexiform layer; INL: Inner nuclear layer.

Fig. 6

TPEN-loaded nanoparticles promoted remarkable more RGCs survival. (A) Schematic of the timeline of performing AOH and ONC injury, intravitreal injection of drugs, and subsequent experiments. (B) Image of flat mount of control retina, which showed the 12 areas that were captured from the center, middle, and periphery of the retina for counting number of RGCs. Scale bar, 1 mm. (C) Representative images of retinal flat mounts labeled with Rbpms (red) and βⅢ tubulin (green) after 2 weeks of ONC injury in different groups. Scale bar, 20 μm. (D) Bar charts showed the average number of both Rbpms and βⅢ tubulin positive RGCs/mm² in the retinas. All the data were analyzed by one-way ANOVA with Bonferroni’s post hoc test and unpaired Student’s t-test (2 tailed). ∗∗P＜0.01, ∗∗∗∗P＜0.0001 between indicated two groups. ##P＜0.01 between indicated two groups. # means unpaired Student’s t-test (2 tailed) between indicated two groups. Each point shown in the bar charts indicates an individual data point. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 7

TPEN-loaded nanoparticles promoted remarkable more axon regeneration. (A) Images of optic nerve wholemounts showed the regenerated axon labeled with CTB (red) after 2 weeks of ONC. Scale bar, 200 μm. (B) Quantification of regenerated axons at 0.25 mm and 0.5mm to lesion site in different groups. (C) Quantification of regenerated axons at 0.75 mm and 1 mm to lesion site in different groups. All the data were analyzed by one-way ANOVA with Bonferroni’s post hoc test and unpaired Student’s t-test (2 tailed). ∗ P＜0.05, ∗∗P＜0.01, ∗∗∗∗P＜0.0001 between indicated two groups. #P＜0.05, ##P＜0.01 between indicated two groups. # means unpaired Student’s t-test (2 tailed) between indicated two groups. ns, no significance between indicated two groups. Each point shown in the bar charts indicates an individual data point. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 8

Multiple treatment of TPEN solution promoted significantly more RGCs survival and axon regeneration. (A) Detection time of TPEN in retinas by UPLC-ESI-MS/MS. (B) Change dynamic of concentration of TPEN in retinas after different days of ONC. (C) Representative images of flat-mounted retinas showing the both Rbpms and βⅢ tubulin positive RGCs in different groups. Scale bar, 20 μm. (D) Quantification of average number of RGCs/mm² after 2 weeks of ONC in different groups. (E) Representative images of optic nerve wholemounts showed the regeneration of axon labeled with CTB after 2 weeks of ONC. Scale bar, 200 μm. (F-G) Number of regenerated axons at different distances to lesion. TPEN (0,3) means vitreous injections of TPEN (100 μM) were applied immediately and 3 days after ONC. TPEN (0,7) means vitreous injections of TPEN (100 μM) were applied immediately and 7 days after ONC. Data were analyzed by one-way ANOVA with Bonferroni’s post hoc test and unpaired Student’s t-test (2 tailed). ns, no significance between indicated two groups. ∗P＜0.05, ∗∗P＜0.01, ∗∗∗P＜0.001, ∗∗∗∗P＜0.0001 between indicated two groups. ##P＜0.01 between indicated two groups. # means unpaired Student’s t-test (2 tailed) between indicated two groups. Each point indicates an individual data point.

Fig. 9

TPEN-loaded nanoparticle had no superiority over multiple treatment of TPEN in the same or half dosage on RGCs survival and axon regeneration. (A) Bar charts showed the average number of both Rbpms and βⅢ tubulin positive RGCs/mm² in the retinas. (B-C) Quantification of regeneration of fibers at different distances to lesion site in each group. (D) Representative images of flat-mounted retinas showing the both Rbpms and βⅢ tubulin positive RGCs in different groups. Scale bar, 20 μm. (E) Quantification of average number of RGCs/mm² after 2 weeks of ONC in different groups. (F) Representative images of optic nerve wholemounts showed the regeneration of axon labeled with CTB after 2 weeks of ONC. Scale bar, 200 μm.(G-H) Number of regenerated axons at different distances to lesion site. Data were analyzed by one-way ANOVA with Bonferroni’s post hoc test. ns, no significance between indicated two groups. Each point shown in the charts indicates an individual data point.

Fig. 10

TPEN-loaded nanoparticle had comparable therapeutic effect with TPEN in the same dosage in preserving visual function. (A) Representative pictures of pERG waveform performed 4 weeks after ONC injury of different groups. The representative points of N1, P1 and N2 of control group were showed in the picture. (B) Bar plots showed the quantitative analysis of pERG P1–N2 amplitudes before ONC (Baseline), 2 weeks and 4 weeks after ONC of different groups. (C) Representative images of waveform in different groups of f-VEP. The points of P1, N2 and P2 in the waveform were showed in control group. (D) Bar graph showed the quantitative analysis of N2-P2 amplitude of f-VEP in different groups before and after 2 weeks of ONC injury. All data were displayed as mean ± SEM and were analyzed using one-way ANOVA followed by Dunnett’s multiple comparisons test or unpaired Student’s t test (2 tailed). ∗ P < 0.05, ∗∗P < 0.01, ∗∗∗P＜0.001, ∗∗∗∗P＜0.0001 between indicated two groups. #P＜0.05, ##P＜0.01 between indicated two groups. # means unpaired Student’s t-test (2 tailed) between indicated two groups. ns, no significance between indicated two groups. Each point shown in the statistic charts indicates an individual data point.

Fig. 11

TPEN-loaded nanoparticles preserved significantly more RGCs and axon after AOH than solution. (A) A diagram of AOH injury modeling. (B) Representative images of retinal flat mounts labeled with Rbpms (red) and βⅢ tubulin (green) after 2 weeks of AOH injury in different groups. Scale bar, 20 μm. (C) Bar plots showed the quantitative analysis of RGCs/mm² in 2 weeks after AOH of different groups. (D) Representative images of SMI32⁺ axons in different groups. Scale bar, 40 μm. (E) Representative HE staining images of paraffin‐embedded retinal sections from different groups. Scale bar, 80 μm. (F) Bar graph showed the number of SMI32⁺ axons per micrograph in different groups after 2 weeks of AOH injury. (G-H) Quantification of the thickness of the RNFL and GCC in central, middle, and peripheral retina regions from different groups. All data were displayed as mean ± SEM and were analyzed using one-way ANOVA followed by Dunnett’s multiple comparisons test or unpaired Student’s t test (2 tailed). ∗P < 0.05, ∗∗P＜0.01, ∗∗∗P＜0.001, ∗∗∗∗P＜0.0001 between indicated two groups. #P＜0.05 between indicated two groups. # means unpaired Student’s t-test (2 tailed) between indicated two groups. Each point shown in the statistic charts indicates an individual data point. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)