Inflammatory cell imaging

Implantation of a biomaterial elicits a dynamic foreign body reaction (FBR) at the implant site (Anderson et al., 2008). The recruitment of macrophages and neutrophils at the implant site is the hallmark of host inflammatory responses to implanted biomaterials (Anderson et al., 2008). Activated macrophages and neutrophils are widely implicated in the degradation of polymeric implants, which subsequently leads to increased fibrotic reactions, tissue damage, and delayed tissue regeneration (Hoemann et al., 2010). Evidently, the number of recruited macrophages and neutrophils at an implant site directly relates to the severity of implant-associated inflammation (Anderson et al., 2008). Consequently, direct imaging of these inflammatory cells around implanted biomaterials presents a clear and viable option to noninvasively assess the degree of biomaterial-associated inflammation. To this end, targeted cell imaging of activated macrophages and neutrophils near the vicinity of an implant via receptor-targeting NIR nanoprobes (NIR NPRs) has been successfully employed.

It is well established that activated monocytes/macrophages overexpress the folate receptor (FR) (Turk et al., 2004; Puig-Kroger et al., 2009). Interestingly, an FR-targeting fluorescein isothiocyanate (FITC)-conjugated macrophage antibody was used to evaluate the extent of FBR to implanted biomaterials in vivo (Bratlie et al., 2010). However, low tissue penetration and high autofluorescence limits the use of FITC-conjugated probes for optical imaging applications in vivo. Polymer nanoparticles encapsulated with NIR dye were synthesized and conjugated with folate peptides (Zhou et al.,

2011). IR750 dye encapsulated poly(N-isopropyl acrylamide-co-styrene) nanoparticles (PNIPAM-co-St NPs) with an average diameter of 100 nm, and emission wavelength of 760 nm was synthesized by emulsion polymerization. Folate peptides were conjugated to the dye entrapped PNIPAM-co-St NPs using carbodiimide-coupling protocols. The FR-targeting NIR PNIPAM-co-St NPs demonstrated a high affinity for lipopoly- saccharide (LPS)-activated macrophages with fluorescence intensities, correlating linearly with a number of activated macrophages in vitro (Fig. 3.1(a) and (b)). Furthermore, FR-targeting NIR PNIPAM-co-St NPs were able to quantify FBR around

Near-infrared fluorescence

Figure 3.1 Near-infrared fluorescence (NIRF) imaging of activated macrophages around injected biomaterials using poly(N-isopropyl acrylamide-co-styrene) nanoparticles. (a) Effect of incubation time on the accumulation of folate receptor (FR)-targeting and control nanoparticles (NPs) onto lipopolysaccharide (LPS)-activated macrophages in vitro. (b) Correlation between LPS-activated macrophage number and NP fluorescence intensity in vitro. (c) Representative image of the bioimaging data obtained from live animals following intravenous administration of FR-targeting NPs in poly(L-lactic acid) (PLA), saline, and polyethylene glycol (PEG)-injected groups at different time points. (d) Fluorescence intensities from tissues surrounding PLA, PEG, and saline injection sites at different time points (mean ± standard error (SE), n = 4). (e) NIRF signals (red) acquired from unstained and unfixed tissue sections at 20x and 40x magnification. (f) Representative histological tissue sections. (g) Quantification of CD11B+ inflammatory cells and monocytes/macrophages (MOMA+) in tissues surrounding PLA, PEG, and saline-injected sites (mean ± SE, n = 4).

subcutaneously injected poly(L-lactic acid) (PLA) and polyethylene glycol (PEG) particle implants in the dorsal regions of mice. Of note, PLA and PEG were chosen as model materials for their ability to elicit mild and weak inflammatory responses, respectively (Weng et al., 2004; Ruan and Feng, 2003). Bioimaging results showed that fluorescence intensities at PLA-injected sites were consistently higher than PEG sites, while saline-injected controls had low or no fluorescence during the same time points (Fig. 3.1(c) and (d)). In addition, fluorescent images of unstained and unfixed tissue sections confirmed significant accumulation of FR-targeting NIR PNIPAM-co-St NPs surrounding PLA sites compared to normal tissue (Fig. 3.1(e)). Immunohistological analysis also revealed that PLA microparticles recruited three times higher CD11B+ inflammatory cells and monocytes/macrophages (MOMA+) compared to PEG NPs (Fig. 3.1(f) and (g)). Overall, the synthesized FR-targeting NIR PNIPAM-co-St NPs demonstrated high affinity toward activated macrophages and shows great promise for detecting inflammatory responses to subcutaneously implanted biomaterials.

A similar strategy was employed to target neutrophils at the site of FBR (Zhou et al., 2012a). The peptide, cinnamoyl-Phe-(D)Leu-Phe-(D)Leu-Phe (cFLFLF), has been shown to have a high affinity to formyl peptide receptors (FPR) of activated neutrophils (Locke et al., 2009). This peptide was conjugated to an Oyster 800 (O800) dye-incorporated, eight-arm PEG amine through carbodiimide-coupling reaction. Characterization studies showed that the FPR-targeting PEG-O800 NPR had an absorbance peak at 280 nm, corresponding to that of cFLFLF peptide and a fluorescence spectral emission at 800nm, matching that of free Oyster 800 dye. In vitro studies showed that PEG-O800-cFLFLF NPRs were specific to activated neutrophils with fluorescence intensities in direct correlation with neutrophil numbers. Next, to evaluate the ability of PEG-O800-cFLFLF NPRs to detect localized inflammatory responses in vivo, FPR-targeting PEG-O800 NPRs were delivered intravenously in mice subcutaneously injected with PLA and PEG particle implants. Results demonstrated that fluorescence signals were significantly higher in PLA injected sites compared to PEG sites and saline-injected controls. Furthermore, fluorescent images of tissue sections around PLA and PEG implant sites revealed significantly higher concentrations of FPR-targeting PEG-O800 NPRs in PLA compared to PEG implants. This observation was further confirmed by histological analysis, which showed that PLA particles recruited neutrophils to a greater extent than PEG implants, while negligible neutrophil numbers were observed in saline-injected controls. In contrast, neutrophil-depleted mice demonstrated a substantial reduction in neutrophil recruitment (~75%) around PLA implant sites compared to controls. Cumulatively, these results prove that FPR-targetingPEG-O800-cFLFLF NIR NPRs are promising for quantitatively assessing inflammatory responses associated with activated neutrophils around implanted biomaterials in vivo.

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