Background: Porous silicon (pSi) nanoparticles (NPs) functionalized with suitable targeting ligands are now established cancer bioimaging agents and drug-delivery platforms. With growing interest in peptides as tumor-targeting ligands, much work has focused on the use of various peptides in combination with pSi NPs for cancer theranostics. Here, the authors investigated the targeting potential of pSi NPs functionalized with two types of peptide, a linear 10-mer peptide and its branched (Y-shaped) equivalent, that respond to legumain activity in tumor cells. Results: In vitro experiments established that the linear peptide-pSi NP conjugate had better aqueous stability under tumor conditions and higher binding efficiency (p < 0.001) toward legumain-expressing cells such as RAW 264.7 cells compared with that of its branched equivalent. In vivo studies (analyzed using ex vivo fluorescence) with the linear peptide-pSi NP formulation using a syngeneic mouse model of breast cancer showed a higher accumulation (p > 0.05) of linear peptide-conjugated pSi NPs in the tumor site within 4 h compared with nonconjugated pSi NPs. These results suggest that the linear peptide-pSi NP formulation is a nontoxic, stable and efficient fluorescence bioimaging agent and potential drug-delivery platform.

Porous silicon (PSi) with a suite of most desirable biomaterial properties has attracted great attention as a multifunctional nanoplatform for bioimaging and drug delivery. Various surface functionalization treatments have been reported for PSi to use as an active tumor cell targeting nanovector. In this study, we investigated surface functionalization treatments using a peptide that is specific to the emerging biomarker legumain. The PSi nanoparticles were coated with dextran and subsequently two types of legumain targeting peptide, Y-shaped and linear chain, were conjugated to produce the functionalized PSi. The functionalized (ligand-conjugated) PSi materials were characterized for morphology, size, functional groups, and fluorescence response using electron and fluorescence microscopy and vibrational spectroscopy techniques. Fluorescence microscopy imaging with two excitation wavelengths (450 nm and 600 nm) suggests comparable fluorescence response of the conjugated PSi to “bare” PSi and the suitability of the PSi functionalized with peptide for bioimaging.

The relative legumain‐targeting efficiency of a Y‐shaped peptide and a linear peptide with the same amino acid sequence was investigated using in vitro experiments and computer simulations. Flow cytometry and fluorescence microscopy of cell types with varying levels of legumain expression (RAW 264.7, 4T1, MCF 10 A, MCF 7 and MDA MB 231) revealed that the linear peptide has a higher legumain binding efficiency in legumain‐active cells compared to the Y‐shaped peptide. Peptide‐protein docking simulations showed that the more stable linear peptide binds at the active site of legumain, whereas the Y‐shaped peptide binds at a different site. The Y‐shaped peptide has its asparaginyl binding site (‐Asn‐) coiled into an α‐helix form, and this reduces access to legumain binding. The linear peptide conjugated to fluorescent carbon dots (CDs) displayed enhanced binding efficiency towards legumain. The peptide‐CDs conjugate nanoparticles are stable under pH, temperature, and medium composition conditions similar to that may be expected in tumor environments, suggesting their high potential as a bioimaging agent.

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