Dry eye disease (DED) is the most common ocular surface disease that affects about one third of the global population [1]. Its clinical manifestations are characterized by tear film deficiencies and persistent ocular surface inflammation, which result in ocular discomfort and even visual impairment. The pathogenic mechanism of DED is complicated and involves both intrinsic and extrinsic factors, such as tear film instability, ocular surface damage, and ocular surface inflammation [2,3]. At present, the first-line drugs for DED mainly include artificial tears, immunosuppressants, and others. However, these drugs usually need long-term administration (> 4 weeks). Even worse, most of them require a high frequency of dosing, e.g., six doses per day, which significantly reduces patient compliance [4,5]. Therefore, it is of great clinical significance to develop ocular drug carriers to enhance drug bioavailability and efficacy, thus reducing treatment cycles and frequency of administration.

Last decades have witnessed the rapid progress in the development of carriers for ocular drug delivery [6,7]. Among them, peptide-based platforms receive tremendous attention in terms of facile synthesis, good biocompatibility, and low immunogenicity [8,9]. More importantly, unlike other types of materials, peptides can be easily tailored into different morphologies (e.g., nanofibers, nanoribbons, crescents) by regulating sequences, e.g., simply switching amino acids, or controlling self-assembly pathways [[10], [11], [12]]. Notably, these morphologies determine the fate of nanocarriers in the body, i.e., nanofibers especially when formation of gels possess long retention time whereas spherical nanostructures have fast tissue or cellular internalization [13,14]. However, the structure-property relationships among the molecular composition of carriers, the assembled morphologies, and the capability to modulate the therapeutic efficacy of the drug within carriers remain elusive.

In recent years, we and others reported that constitutionally isomeric peptides that self-assemble into different morphologies by changing amino acid residue positions can be utilized for drug delivery [10,11,15,16]. Strikingly, the morphologies have direct influence on resultant bioactivities of these peptides or the therapeutic efficacy of drugs within assemblies. For instance, the Matson group found that variation in the position of amino acid residues led to different morphological formation and distinct hydrogen sulfide-releasing behaviors, ultimately impacting the cytoprotective capacities of hydrogen sulfide [10]. Zhou and coworkers found that alteration of the alanine residue in glycopeptide mimetics resulted in different biofilm elimination activities [16]. Although these results were appealing, almost all these studies were done in vitro and no such investigation has been performed in vivo. Consequently, how subtle changes in amino acid sequence influencing the in vivo bioactivity of peptides or peptide-based nanocarriers, further impacting the efficacy of the drug within carriers is still unclear up to now.

Herein, we report on a class of constitutionally isomeric pentapeptides. Each contains three isoleucine (I) residues, one histidine (H) residue, and one lysine (K) residue, with the N-terminus being capped with the naphthaleneacetictyl group (NP) to facilitate assembly. By regulating the positions of amino acid residues in the sequence, one-dimensionally discrete nanostructures with different morphologies were obtained. We hypothesized that these distinctly assembled morphologies would affect the viscosity when the concentration was elevated and further influence the retention time of drugs within assemblies as well. Furthermore, through molecular dynamics simulations, we aimed to map how these pentapeptides pack to form nanostructures and impact the retention time. Based on these results, in vivo investigation was carried out to assess the influence of these pentapeptides on the retention and the therapeutic efficacy of Diquafosol tetrasodium (DQS), a classical drug against DED. We envisioned that this combined experimental/computational approach could give insight into how molecular-level engineering can regulate the therapeutic efficiency of peptide-based materials in vivo.