Detection of a fluorescent-labeled avidin-nucleic acid nanoassembly by confocal laser endomicroscopy in the microvasculature of chronically inflamed intestinal mucosa

Abstract

Inflammatory bowel diseases are chronic gastrointestinal pathologies causing great discomfort in both children and adults. The pathogenesis of inflammatory bowel diseases is not yet fully understood and their diagnosis and treatment are often challenging. Nanoparticle-based strategies have been tested in local drug delivery to the inflamed colon. Here, we have investigated the use of the novel avidin-nucleic acid nanoassembly (ANANAS) platform as a potential diagnostic carrier in an experimental model of inflammatory bowel diseases. Fluorescent- labeled ANANAS nanoparticles were administered to mice with chemically induced chronic inflammation of the large intestine. Localization of mucosal nanoparticles was assessed in vivo by dual-band confocal laser endomicroscopy. This technique enables characterization of the mucosal microvasculature and crypt architecture at subcellular resolution. Intravascular nanoparticle distribution was observed in the inflamed mucosa but not in healthy controls, demonstrating the utility of the combination of ANANAS and confocal laser endomicroscopy for highlighting intestinal inflammatory conditions. The specific localization of ANANAS in inflamed tissues supports the potential of this platform as a targeted carrier for bioactive moieties in the treatment of inflammatory bowel disease.

Keywords:

• confocal laser endomicroscopy
• inflammatory bowel disease
• diagnostics
• dextran sodium sulfate
• avidin-nucleic acid nanoassembly
• fluorescent nanoparticles
• ulcerative colitis

Supplementary material

Figure S1 shows the gel permeation chromatography analysis of biotin-C₆-red⁶⁸¹ (compound 3) before and after addition of avidin. Figure S2 shows the fluorescence intensity calibration curves of the same compound in phosphate-buffered saline, alone and in the presence of avidin. Figure S3 shows the results of dynamic light scattering analysis of the avidin-nucleic acid nanoassembly (ANANAS)-red⁶⁸¹ nanoparticles. Figure S4 shows histological images of colonic mucosa in dextran sodium sulfate (DSS)-treated and healthy mice.

Characterization of biotin-C₆-red⁶⁸¹

For analysis, a small aliquot of the coupling reaction solution (0.5 μL) was diluted to 218 μL with phosphate-buffered saline alone or phosphate-buffered saline containing avidin 1 mg/mL (Belovo Chemicals, Bastogne, Belgium), and analyzed by fast protein liquid chromatography (Superdex peptide column with phosphate-buffered saline as the eluent, 0.5 mL/min, ultraviolet absorption 681 nm). Figure S1 shows the overlapped chromatograms of the starting red⁶⁸¹-NHS, the reaction product as before and after the addition of avidin, with peak assignments. The hydrolyzed red⁶⁸¹-NHS (Red⁶⁸¹-COOH) and its biotin derivative were tested for fluorescence intensity. The biotin derivative was also tested upon addition of avidin. Fluorescence intensity calibration curves were generated on serially diluted phosphate-buffered saline solutions, starting from 1 μM red⁶⁸¹, with λ_exc 681 nm and λ_em 708 nm. The slope of the fluorescence intensity versus concentration curve was used to calculate the relative fluorescence intensity with respect to the starting red⁶⁸¹-COOH. The fluorescence intensity of avidin-linked biotin-C₆-red⁶⁸¹ was 43% that of the same reagent when analyzed free in solution. The same result was obtained using ANANAS instead of avidin (not shown).

Size distribution of ANANAS nanoformulation

Dynamic light scattering measurements were performed using a Malvern Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, UK) and the results are shown in Figure S3.

Induction of DSS chronic colitis

Chronic colitis was induced in Balb/c female mice^1,2 (aged 8–10 weeks) by using multiple cycles of dextran sodium sulfate (DSS, 40 kDa, Sigma-Aldrich, St Louis, MO, USA). We performed preliminary optimization experiments to identify the optimal DSS dose and administration schedule yielding chronic inflammation. During treatment, animal body weight, rectal bleeding, stool consistency, and the presence of blood in stools were assessed daily. Assessment of chronic inflammation was confirmed by histological analysis. The final induction protocol identified is reported below: initially, we administered two cycles comprising 7 days of 3% DSS in drinking water and 3 days of untreated drinking water. Next, 5% DSS was administered for 3–4 days until a 10% weight loss was achieved, then substituted with untreated drinking water for 3 days or until full body weight recovery. The 5% DSS/untreated drinking water cycle was repeated four times. Inflammatory changes³ (crypt architectural disarray, transmural inflammation with lymphoid follicles) were confirmed histologically in colonic sections from DSS-treated mice compared with control mice (Figure S4).

Figure S1 Chromatographic analysis of red⁶⁸¹ derivatives. (A) Starting NHS reagent, (B) biotin-C -red⁶⁸¹ derivative (compound 3), and (C) compound 3 in the presence of avidin.

Notes: Analyses were carried out using fast protein liquid chromatography apparatus (Akta Purifier, GE Healthcare, Little Chalfont, UK) equipped with a Superdex peptide column and eluted with 10 mM phosphate, 150 mM NaCl, pH7.4 (phosphate-buffered saline) at 0.5 mL/min. Chromatograms were registered at 681 nm.

Abbreviations: ANANAS, avidin-nucleic acid nanoassembly; NHS, N-hydroxysuccinimide.

Figure S2 Fluorescence intensity versus concentration of red⁶⁸¹-COOH (•) and biotin-C₆-red⁶⁸¹ as before (▲), and in the presence of avidin (□).

Abbreviation: RIU, relative intensity units.

Figure S3 (A) Intensity and (B) volume weighted size distribution of ANANAS assembly prepared for this work, as determined by dynamic light scattering.

Abbreviation: ANANAS, avidin-nucleic acid nanoassembly.

Figure S4 Histology images of dextran sodium sulfate-treated (A, B) and healthy (C) mouse colonic mucosa.

Notes: Colonic segments, fixed in 4% formaldehyde, were stained with hematoxylin and eosin and reviewed by a pathologist in a blinded fashion. Original magnification for histology, 40×.

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Acknowledgments

This research was funded by the Roberto Farini Association, Progetti di Ateneo, and Ex 60% at the University of Padova and National PRIN 2010–2011. We are grateful to ANANAS Nanotech for assistance with preparation and characterization of the nanoparticles, to Dr G Battaglia for promoting the acquisition and availability of the Mauna Kea system for CLE at the Venetian Institute of Molecular Medicine, Padova, and to Dr C Giacometti for advice on histology analysis.

Disclosure

MM is among the inventors of the ANANAS technology, and is a shareholder in ANANAS Nanotech s.r.l., a spinoff company that holds the intellectual property related to the ANANAS formulation. The other authors report no conflicts of interest in this work.