Intravitreal Delivery of Cationic DMP Micelles Suppresses Choroidal Neovascularization

Abstract

Neovascular age-related macular degeneration (AMD) is characterized by choroidal neovascularization (CNV), which remains a major cause of irreversible vision loss worldwide [1]. Although intravitreal anti-vascular endothelial growth factor (VEGF) therapy has improved clinical outcomes, repeated injections, incomplete responsiveness, and long-term treatment burden remain important limitations [2]. Nanomaterial-based ocular delivery systems have attracted increasing interest because of their favorable biocompatibility and sustained delivery potential. However, whether cationic micelles possess intrinsic therapeutic activity independent of drug loading remains largely unexplored.

Here, we investigated DMP, a cationic micelle assembled from 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) and methoxy poly(ethylene glycol)-block-poly(ε-caprolactone) (mPEG-PCL), as a stand-alone therapeutic strategy for CNV (Figure 1A). DMP micelles were prepared by thin-film hydration and had a mean hydrodynamic diameter of 86.48 ± 3.78 nm, a polydispersity index of 0.101, and a zeta potential of +46.40 ± 3.87 mV. Transmission electron microscopy confirmed their spherical morphology (Figure 1A).

We evaluated DMP uptake and cytocompatibility in rat Müller rMC-1 cells, consistent with the species used for the in vivo experiments. Rhodamine-labeled DMP was readily taken up by rMC-1 cells. MTT assays showed that cell viability remained above 80% at DMP concentrations up to 600 μg/mL and decreased at 800 μg/mL, while remaining substantially higher than that following PEI25K treatment (Figure 1B). Among the inhibitors tested, M-β-CD significantly reduced DMP uptake, whereas CPZ and filipin III had no significant effects. Latrunculin A increased DMP-associated fluorescence, which may reflect altered cortical actin constraints or vesicular trafficking rather than actin-dependent uptake. These findings support a cholesterol-sensitive component of DMP uptake but do not establish a single internalization pathway (Figure 1B). Following intravitreal injection, rhodamine-labeled DMP was detected across multiple retinal layers, supporting its distribution within the retina (Figure 1C).

To evaluate the anti-neovascular effect of DMP, a laser-induced CNV model was established in Brown Norway rats, followed by intravitreal administration of DMP 1 day after laser photocoagulation. Fundus fluorescein angiography (FFA) on Day 7 showed lower CNV leakage grades in DMP-treated eyes than in saline-treated controls (p < 0.0001; Figure 1D). IB4 staining on Day 14 likewise showed a smaller CNV area after DMP treatment (79,700 ± 18,083 vs. 169,537 ± 23,220 μm²; p = 0.0140; Figure 1D).

To preliminarily explore biological pathways associated with DMP treatment, we performed RNA-seq analysis on retinal tissues after intravitreal administration. Transcriptomic analysis revealed upregulation of immune- and antigen presentation-related genes, including RT1-Da, RT1-Ba, RT1-Db1, RT1-Bb, and Slpi (Figure 1E). GO and gene set enrichment analyses identified transcriptional enrichment of MHC class II- and immune response-related terms (Figure 1F,G). These results indicate an association between DMP treatment and retinal immune-related transcription.

Ocular safety was further assessed using electroretinography (ERG) and optical coherence tomography (OCT). No significant differences in ERG b-wave amplitudes, retinal thickness, or retinal architecture were detected among the DMP-treated, saline-treated, and untreated groups at baseline, 2 weeks, or 1 month (Figure 1H). These findings provide preliminary evidence of preserved retinal structure and function following a single DMP injection. Histological examination of the heart, liver, spleen, lung, and kidney revealed no obvious treatment-related abnormalities, providing preliminary histological evidence of systemic tolerability (Figure 1I).

Unlike previous studies that primarily utilized cationic micelles as delivery vehicles for nucleic acids or therapeutic agents [3], cargo-free DMP reduced vascular leakage and CNV area in vivo. Most nanomaterial-based ocular systems are designed to enhance tissue penetration or drug retention [4]. Our findings extend their potential role to therapeutic activity independent of loaded cargo. Nevertheless, because endothelial migration and tube formation were not examined, a direct endothelial mechanism cannot be inferred. Similarly, the RNA-seq results should be considered hypothesis-generating until supported by protein-level and cell-specific validation. The findings from the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, ERG, OCT, and organ histology findings suggest preliminary biocompatibility but do not establish long-term safety, particularly because cationic nanomaterials may cause membrane or inflammatory toxicity [5].

Several limitations should be acknowledged. Laser photocoagulation induces acute disruption of Bruch's membrane and a rapid inflammatory and angiogenic response, whereas human neovascular AMD develops chronically in an aged retinal environment involving RPE dysfunction, oxidative stress, complement dysregulation, recurrent exudation, fibrosis, and retinal atrophy. DMP was administered 1 day after injury and evaluated after a single dose; thus, the results primarily demonstrate suppression of early injury-induced neovascularization and cannot be directly extrapolated to established or recurrent neovascular AMD. Immune-related transcriptional changes in this acute setting may also differ under chronic conditions. Although a transient immune response may accompany CNV suppression, persistent immune stimulation in the aged retina could promote chronic inflammation, retinal toxicity, or fibrotic scarring. The duration, cellular source, and long-term consequences of this response remain unknown. Moreover, systemic safety assessment was limited to short-term organ histology. Future studies should evaluate pharmacokinetics, dose optimization, repeated-dose safety, and long-term efficacy in chronic models, together with cell-specific protein validation and endothelial functional assays.

In summary, we demonstrate that intravitreal administration of cationic DMP micelles effectively suppresses CNV development in vivo. DMP combines antiangiogenic activity with modulation of retinal immune-related pathways while maintaining favorable retinal biocompatibility. These findings expand the therapeutic application of cationic micelles beyond conventional delivery systems and support the further development of DMP as a potential nanomedicine strategy for neovascular AMD.

Yingying Chen: investigation, formal analysis, funding acquisition, and writing – original draft. Jieping Wu: investigation, formal analysis, and resources. Ke Men: writing – review and editing, supervision, and funding acquisition. Jiamei Yang: investigation and formal analysis. Mohan Liu: conceptualization, methodology, formal analysis, writing – review and editing, and supervision. Meixia Zhang: conceptualization, methodology, supervision, and funding acquisition. All authors have read and approved the final manuscript.

During the preparation of this manuscript, the authors used the AI-powered language-editing service provided by American Journal Experts (AJE) to improve the clarity and readability of the text. All authors reviewed and edited the revised text as needed and take full responsibility for the final content of the publication. No AI-generated images were included in the manuscript. This work was supported by the National Natural Science Foundation of China (82473289), the Sichuan Provincial Science and Technology Support Project (2024YFFK0303), and the Sichuan Provincial Natural Science Foundation for Youth (2025ZNSFSC1712).

All animal experiments were approved by the Animal Ethics Committee of West China Hospital, Sichuan University (approval no. 20230213004).

The authors declare no conflicts of interest.

The RNA-seq data generated in this study have been deposited in the GEO database under accession number GSE297943.