Elsevier

Biomaterials

Volume 35, Issue 10, March 2014, Pages 3356-3364
Biomaterials

Near-infrared fluorescence imaging using organic dye nanoparticles

https://doi.org/10.1016/j.biomaterials.2014.01.004Get rights and content

Abstract

Near-infrared (NIR) fluorescence imaging in the 700–1000 nm wavelength range has been very attractive for early detection of cancers. Conventional NIR dyes often suffer from limitation of low brightness due to self-quenching, insufficient photo- and bioenvironmental stability, and small Stokes shift. Herein, we present a strategy of using small-molecule organic dye nanoparticles (ONPs) to encapsulate NIR dyes to enable efficient fluorescence resonance energy transfer to obtain NIR probes with remarkably enhanced performance for in vitro and in vivo imaging. In our design, host ONPs are used as not only carriers to trap and stabilize NIR dyes, but also light-harvesting agent to transfer energy to NIR dyes to enhance their brightness. In comparison with pure NIR dyes, our organic dye nanoparticles possess almost 50-fold increased brightness, large Stokes shifts (∼250 nm) and dramatically enhanced photostability. With surface modification, these NIR-emissive organic nanoparticles have water-dispersity and size- and fluorescence- stability over pH values from 2 to 10 for almost 60 days. With these superior advantages, these NIR-emissive organic nanoparticles can be used for highly efficient folic-acid aided specific targeting in vivo and ex vivo cellular imaging. Finally, during in vivo imaging, the nanoparticles show negligible toxicity. Overall, the results clearly display a potential application of using the NIR-emissive organic nanoparticles for in vitro and in vivo imaging.

Introduction

In near-infrared (NIR) spectral range, organisms and tissues have low absorption of light and possess low intrinsic autofluorescence. Autofluorescence is the natural emission of light from biological structures. If biomarkers are fluorescent in NIR range, they can be better detected and identified from the surrounding biological environment. Therefore, NIR fluorescence imaging in wavelength range of 700–1000 nm is particularly attractive for early detection of cancers, which is expected to significantly contribute to improved cancer therapy and increased survival rates of patients [1], [2], [3], [4].

Currently, the most widely used NIR probes are still organic dyes, which are usually encapsulated in various nanoparticles (NPs) to overcome the intrinsic limitations of conventional NIR dyes including poor hydrophilicity, low photostability, small quantum yield (QY) and instability in bio-environment [5], [6], [7], [8]. However, most of these encapsulating nanoparticles including silica NPs, calcium phosphate NPs, and lipoprotein NPs, only act as inert carriers but do not contribute to brightness improvement of the NIR probes [9], [10], [11], [12]. Another concern is that Stokes shift of conventional nanoparticle-based dye probes is usually small and optical interferences (light scattering and autofluorescence) caused by biosubstrates often exist, which greatly reduces detection sensitivity [13]. Furthermore, the clearance of inert carriers from patients still remains a great concern. Conjugated polymer dots (Pdots) were then reported to be utilized as the matrix to load organic dyes for imaging, in which Pdots served as light-harvesting agents to transfer their energy to organic dyes to enhance brightness [14], [15].

Recently, small-molecule organic dye nanoparticles (SM ONPs) have also been developed as a new class of promising fluorescent probes [16], [17], [18], [19], where organic dyes themselves were directly assembled into pure dye nanoparticles without nanocarriers. Despite at early stage, SM ONPs have attracted much attention, because they possess large absorption cross-sections, non-blinking property and favorable biocompatibility. More importantly, compared to Pdots, there is great variety and flexibility in materials design and thus tunability in optical properties and functionalities [20], [21]. Previously, dye molecules with rigid structures or aggregation-induced enhanced emission (AIEE) properties were used for preparing SM ONPs to avoid concentration quenching of traditional dyes [22]. For example, recently, we reported a type of ultrabright and ultrastable NIR dye nanoparticles which were prepared from a NIR dye of bis(4-(N-(2-naphthyl)phenylamino) phenyl)-fumaronitrile (NPAPF) with AIEE effect [16]. However, if traditional NIR dyes are employed to make nanoparticles, severe quenching will happen.

To address the challenges of developing NIR fluorescence probes with high brightness, large Stokes shift and photo- and bio-environmental stability, herein, we encapsulated an NIR dye into red-emissive ONPs. We hypothesize that the efficient fluorescence resonance energy transfer (FRET) from ONPs to NIR dyes will enable the resultant NPs to possess superior properties for bioimaging. To confirm this, we systematically investigated the optical properties, water-dispersity, photo- and bio-environmental stability and in vivo toxicity of the NIR dye doped NPs and tested their application for in vitro and in vivo imaging.

Section snippets

Materials and characterization

NIR712 dye (2,9,16,23-tetra-tert-butyl-29H,31H-phthalocyanine) was purchased from Sigma Aldrich, Inc. Tetrahydrofuran (THF) was ordered from Shanghai LingFeng Chemical Reagent Co., Ltd. High-purity water (resistivity = 18.2 MΩ cm) was produced with a Milli-Q apparatus (Millipore). Fetal bovine serum (FBS), Roswell Park Memorial Institute-1640 (RPMI-1640) medium, folic acid (FA)-free RPMI-1640 and Penicillin-streptomycin solution were obtained from Invitrogen (San Diego, CA).

Synthesis and characterization of NIR712-doped NPs

NPAFN ONPs with characteristic of aggregation-induced enhanced emission were chosen as encapsulating red-emissive NPs, and a non-water soluble NIR712 was selected as doping NIR dye. The molecular structures of NPAFN and NIR712 are shown in Fig. S1. Reason of the choice is that the photoluminescence (PL) spectrum of NPAFN ONPs overlaps well with the absorption spectrum of NIR712 (Fig. S3), which ensures efficient energy transfer in the guest-host systems and thus leads to enhanced optical

Conclusions

We have demonstrated an approach of encapsulating NIR dyes into NPAFN NPs to enable efficient FRET to achieve NIR emission for in vitro and in vivo imaging. Energy transfer from the host NPAFN NPs to the guest NIR712 molecules render this nanoprobe with large Stokes shift and amplified fluorescence emission. Furthermore, surface modification of the NPs with amphipathic surfactant endows them with water dispersity, stability and biocompatibility in various bio-environments. Last but not least,

Acknowledgments

This work was supported by National Basic Research Program of China (973 Program, Grant Nos. 2013CB933500, 2012CB932400, 2011CB808400), Major Research Plan of the National Natural Science Foundation of China (No. 91027021, 91233110), and National Natural Science Foundation of China (Nos. 51173124, 51172151). We also thank Natural Science Foundation of Jiangsu Province (No. BK2010003) and a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions.

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