A robust cornea constitutes a transparent and avascular tissue situated on the ocular surface, maintaining its transparency is crucial for normal visual function [1]. Under physiological conditions, the cornea exists in a state of “angiogenic privilege,” in which anti- and pro-angiogenic factors intricately uphold a dynamic equilibrium [2,3]. However, in various pathological conditions such as infections, trauma, post-corneal transplantation, and prolonged contact lens use, the balance of avascularity can break down, leading to the development of corneal neovascularization (CoNV) [4]. CoNV represents a serious condition that poses a threat to vision. It is reported to have an estimated annual incidence of 1.4 million individuals, with 12 % experiencing subsequent vision loss [5]. Corticosteroids have become a mainstay of CoNV treatment because of their low cost and effectiveness [4]. However, the long-term application of such drugs can cause repeated infections, elevate intraocular pressure, and accelerate cataract development [6]. Vascular endothelial growth factor (VEGF) and its receptors play vital roles in the angiogenic process [7]. Additionally, anti-VEGF drugs such as ranibizumab, bevacizumab, and aflibercept have been used in CoNV treatment to improve the vision of patients with abnormal ocular angiogenesis [8]. Moreover, both animal models and clinical trials have demonstrated their therapeutic effects against CoNV [9]. Nonetheless, some patients present an insufficient response to such treatments or experience CoNV recurrence because of drug resistance or injury [10,11]. Nucleic acid drugs, such as small interfering RNA (siRNA) and antisense oligonucleotides, are promising approaches for preventing angiogenesis by selectively inhibiting angiogenesis-related gene expression [12], and may represent a new strategy for CoNV treatment.

Antisense oligonucleotide drugs are a class of biologic entities that have had a fast-growing pace of development in recent years. Some, such as fomivirsen (an antiviral agent) and nusinersen (a therapeutic agent for spinal muscular atrophy), have already been approved for clinical use by the United States Food and Drug Administration (FDA) [13,14]. For the treatment of ocular neovascular diseases, aganirsen—an antisense oligonucleotide drug targeting insulin receptor substrate-1—has been demonstrated as safe and efficacious in multi-center clinical trials, and no treatment-related adverse events were reported [15,16]. In the present study, Itgb1 was revealed to play a crucial role in CoNV. Single-cell sequencing demonstrated that Itgb1 expression was significantly increased in mouse corneas with CoNV, and Itgb1 knockdown significantly reduced CoNV. We therefore investigated the feasibility of Itgb1-siRNA for CoNV treatment.

The delivery of oligonucleotides to target cells remains challenging in vivo [17]. Lipid nanoparticles (LNPs) are currently the most effective delivery carriers for nucleic acids. They are also considered as a promising carrier for ocular drug delivery because of their ability to enhance drug solubility, improve bioavailability, and provide sustained release [18]. Ionizable cationic LNPs (icLNPs), which adopt a net neutral surface charge, avoid the rapid clearance and toxicity that are associated with permanently cationic LNPs in vivo [19]. They also have excellent performance in nucleic acid delivery, with high encapsulation, good cellular uptake, and effective endosomal escape [[20], [21], [22]]. Because of these positive properties, icLNPs have obtained FDA approval as Onpattro (the therapeutic siRNA molecule developed by Alnylam Pharmaceuticals) and COVID-19 mRNA vaccines (developed by Moderna and Pfizer-BioNTech) [[23], [24], [25], [26]]. Although icLNPs have robust delivery efficacy for nucleic acids, inflammatory side effects have raised concerns. Ionizable lipids can induce interleukin-1 (IL-1) expression and inflammatory reactions [27], which may promote the formation of CoNV. This limits the use of icLNPs for CoNV therapy [12]. Triptolide (TP) is one of the main active ingredients of Tripterygium wilfordii, a widely used traditional Chinese medicine with a long history. It showed strong immunosuppressive and anti-inflammatory effects in the treatment of a variety of immune diseases [28,29], and has also been reported to possess anti-tumor angiogenic activity [[30], [31], [32]]. Moreover, TP significantly inhibits IL-1β-induced collagen degradation by corneal fibroblasts [33], and TP-modified LNPs exhibit good anti-inflammatory activity [34]. Therefore, TP-modified icLNPs have great potential for the delivery of oligonucleotide drugs to treat CoNV.

Targeted drug delivery systems can ensure that a drug effectively binds to a specific tissue microenvironment or specific receptor on the cell surface, to achieve more effective treatment outcomes [35]. Anti-Flt1 peptide is an FLT1 (the gene name of VEGFR1)-specific antagonist with a sequence of Gly-Asn-Gln-Trp-Phe-Ile. Anti-Flt1 peptide specifically binds to VEGFR1 and blocks the interaction of VEGFR1 with various ligands (e.g., VEGFA, VEGFB, and placental growth factor), thereby inhibiting VEGF-induced vascular endothelial cell (VEC) migration and tube formation [36]. In the current study, single-cell sequencing revealed that FLT1 expression was significantly increased in VECs and pericytes of CoNV in mice. Anti-Flt1 peptide may therefore be a viable choice for targeting CoNV and enhancing the therapeutic efficacy of the drug.

In the present study, we developed the TP-modified icLNPs that targeted VECs and pericytes of CoNV by coupling them with anti-Flt1 peptide. These TP-modified icLNPs, loaded with Itgb1-siRNA, had good therapeutic effects on CoNV.