Elsevier

Journal of Controlled Release

Volume 262, 28 September 2017, Pages 118-126
Journal of Controlled Release

Gastroresistant oral peptide for fluorescence imaging of colonic inflammation

https://doi.org/10.1016/j.jconrel.2017.07.024Get rights and content

Abstract

The use of molecular markers for inflammation in the gastrointestinal tract could empower optical imaging modalities for early diagnosis and eventually personalized timely treatments. A major hurdle to the widespread use of functional fluorescence imaging is the absence of suitable contrast agents, in particular to be administered via the oral route due to the usual proteolytic susceptibility of the biomarkers. By designing a retro-inverso peptide, starting from a previously described sequence specific for N-cadherin, we achieved resistance to gastrointestinal degradation and even slightly improved specificity towards the target, both in ex vivo and in vivo experimental colitis. Simulations at fundamental molecular level suggested that the introduced retro-inverso modifications did not affect the folding of the peptide, leaving its ability to interact with the binding pocket of the monomeric N-cadherin unaltered, even when fluorescently labeled. Possible further derivatization of this sequence could be envisaged to further extend the potential of the designed retro-inverso peptide as diagnostic or theranostic agent for the oral route.

Introduction

The first, crucial tile in the multifaceted process of chasing and defeating a disease is an accurate and specific diagnosis. The majority of the diagnostic modalities enables to gather mainly anatomical and macrofunctional information. However, with the aid of biomarkers binding specifically to therapeutic targets and thanks to a restless technological improvement, a deeper understanding of molecular functions increases the possibility of timely therapeutic treatments and personalized therapies [1].

The most common route of administration of contrast media is either intravenous or topical [2], [3]. Though, for some pathologies such as inflammatory diseases in the gastrointestinal (GI) tract enteral administration would be more convenient. Albeit topical application to the GI mucosa reduces the risks connected to the systemic administration of a diagnostic agent [4], it seldom enables an exhaustive screening of the disease area. An a priori knowledge of the region to be imaged becomes in this case a requirement [5]. Compared to topical application, the oral route represents a simpler approach to stain the full intestinal tube in a minimally invasive way. A GI contrast agent should be not (i) absorbable to minimize the risk of systemic side effects; (ii) resist digestion; (iii) bind with high affinity and specificity to the affected tissue; (iv) be retained for some time at the binding site; and (v) be easily manufactured.

The main reason underlying the scarce success of oral targeted contrast media relies, in part, on their poor ability of biomarkers in resisting the harsh environment of the GI tract [6]. Small ligands are generally more resistant to acid and enzymatic degradation than biologics (e.g. antibodies) but often lack specificity and affinity; better target-to-background ratios are generally achieved with biologics, but their permeability through the mucus and stability in the GI tract is limited [7]. Peptides have attracted increasing attention as possible molecular imaging probe owing to their intrinsic advantages over larger biomacromolecules [8], [9]. Basic and sophisticated synthetic strategies allow to tune the chemical properties of the amino acid sequences while preserving the binding ability of the peptide to the target [10], [11]. An elegant modification strategy to tackle the peptide susceptibility to proteolysis has been introduced in the late 70's by Goodman and Chorev [12]. While retaining the spatial arrangement of the side chains, the amino acid sequence can be made resistant to enzymatic degradation by inverting the orientation of the peptide bond. So, while by simply reverting the sequence (i.e. generating the retro all-L sequence) the structural equivalence is not necessarily retained, by also inverting the chirality (retro all-D or retro-inverso, RI, sequence) the side-chain topology is maintained [13], [14].

In the present study, we aimed to develop an oral contrast agent for fluorescence-based detection of inflammation of the GI tract. Starting from three sequences previously identified for luminal targets [15], [16], [17], we investigated their susceptibility to pH and to proteolytic degradation in simulated GI fluids following and modified their structure to improve stability. The ex vivo binding affinity of the original and modified peptide to colon tissues harvested from mice with colitis was assessed. The most stable sequence showing the highest ex vivo target-to-control fluorescence intensity ratio was found to be the one selected against N-cadherin and modified to be a retro-inverso sequence. The peptide was then orally administered to validate the in vitro gastroresistance and the specificity towards inflamed tissue resulted from the ex vivo investigations. Computational simulations shed light on the impact of the chemical modifications on the peptide folding and on the energy profiles of the interaction between ligand and target.

In view of the function that N-cadherin has been revealed to hold in chronic conditions in the GI tract, such as Crohn's disease [18], the identification of a gastroresistant disease-specific biomarker could empower the diagnostic potential of emerging high-definition fluorescence imaging modalities, such as confocal endomicroscopy and image-guided surgery, of the inflamed, fibrotic or dysplastic GI lumen [4].

Section snippets

Materials

Peptides were purchased from Peptide 2.0 Inc. (Chantilly, VA). Capital letters stand for l-amino acids, while lower-case letters stand for d-amino acids. Peptides of at least 95% purity were conjugated to FITC at their C-terminus for their further detection in the animal experiments (Table 1). The quality of the peptides was controlled by HPLC analysis, using an autosampler and pump system (Ultimate 3000, Dionex, Thermo Fisher Scientific, Reinach, Switzerland) equipped with a reversed-phase

In vitro stability of the selected peptides

An ideal contrast agent should resist digestion in the GI tract, bind with high affinity and specificity to the disease site, while at the same time guiding the clinician in his/her timely assessment of the severity of the disease to help him/her devise the best and most cost-effective treatment. Peptide sequences that are reported to bind selectively the intestinal mucosa could theoretically serve as optimal biomarkers, but their translational usage is hindered by their short life in vivo.

Conclusions

Starting from three sequences previously identified for luminal targets, we found that the susceptibility to pH and to proteolytic degradation of one sequence described as specific for N-cadherin can be dramatically decreased using a retro-inverso sequence. Notably, the ex vivo and in vivo binding affinity of the retro-inverso peptide for the target in murine experimental colitis model also increased. The interactions of the retro-inverso sequence with N-cadherin needs to be further

Author contributions

PL and JCL designed research, analyzed the data and wrote the paper; AEHM performed research and analyzed the data; TC, SL, MRS, AP, and JS performed research, analyzed the data and wrote the paper; KA performed research; IWF designed research; GR designed research and wrote the paper.

Conflict of interests

None.

Acknowledgements

JCL and AEHM gratefully acknowledge the financial support from Novartis Foundation (formerly Ciba – Geigy – Jubilee - Foundation). Lipoid GmbH is acknowledged for the endowment to the University of Jena (PL). Steven T. Proulx is acknowledged for his technical support with the fluorescence imaging. Julia Geissmann is acknowledged for her technical help with the sample preparation for the stability study. The authors acknowledge the computational resources provided with Brutus and Euler clusters

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  • Cited by (4)

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    P. L. and A.E.H.M. contributed equally to this work.

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