Fluorescence optical imaging in anticancer drug delivery Tom´asˇ Etrych, Henrike Lucas, Olga Janouˇskov´a, Petr Chytil, Thomas Mueller, Karsten M¨ader PII: DOI: Reference:

S0168-3659(16)30075-X doi: 10.1016/j.jconrel.2016.02.022 COREL 8135

To appear in:

Journal of Controlled Release

Received date: Revised date: Accepted date:

24 September 2015 10 February 2016 11 February 2016

Please cite this article as: Tom´aˇs Etrych, Henrike Lucas, Olga Janouˇskov´a, Petr Chytil, Thomas Mueller, Karsten M¨ader, Fluorescence optical imaging in anticancer drug delivery, Journal of Controlled Release (2016), doi: 10.1016/j.jconrel.2016.02.022

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ACCEPTED MANUSCRIPT Fluorescence Optical Imaging in Anticancer Drug Delivery

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Tomáš Etrycha,*, Henrike Lucas b, c, Olga Janouškováa, Petr Chytila, Thomas Muellerc and Karsten Mäderb Institute of Macromolecular Chemistry AS CR, v.v.i., Heyrovský Sq. 2, 162 06 Prague 6,

Martin-Luther-University Halle-Wittenberg, Dept. of Pharmacy, Pharmaceutical Technology

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Czech Republic

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and Biopharmacy, 06120 Halle, Germany Martin-Luther-University Halle-Wittenberg, Dept. of Internal Medicine IV, Oncology /

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Hematology, 06120 Halle, Germany *

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Corresponding author: Tomáš Etrych, [email protected]

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Abstract

In the past several decades, nanosized drug delivery systems with various targeting functions and controlled drug release capabilities inside targeted tissues or cells have been intensively studied. Understanding their pharmacokinetic properties is crucial for the successful transition of this research into clinical practice. Among others, fluorescence imaging has become one of the most commonly used imaging tools in pre-clinical research. The development of increasing numbers of suitable fluorescent dyes excitable in the visible to near-infrared wavelengths of the spectrum has significantly expanded the applicability of fluorescence imaging. This paper focuses on the potential applications and limitations of non-invasive imaging techniques in the field of drug delivery, especially in anticancer therapy. Fluorescent imaging at both the cellular and systemic levels is discussed in detail. Additionally, we explore the possibility for simultaneous treatment and imaging using theranostics and combinations of different imaging techniques, e.g., fluorescence imaging with computed tomography.

Keywords: Fluorescence imaging, drug delivery, theranostics

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Introduction

The visualization of processes in biological systems is crucial for the understanding of diverse basic mechanisms on various levels ranging from cells to the whole body. Visualization is

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also very important for the investigation of novel drug delivery systems (DDSs), enabling specific targeting and controlled release in diseased cells or tissue. In particular, a detailed

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knowledge of drug biodistribution and intracellular trafficking is essential for the successful

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transition of research to clinical practice. This review attempts to cover available techniques and approaches based on fluorescent optical imaging (FI), one of the most important tools for

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visualization in preclinical DDS research. Drug delivery systems

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For several decades, nanosized DDSs have been intensively studied. Nanosized DDSs are macromolecules or self-assembly structures 1 to 100 nm in at least one dimension that bear drugs either attached or loaded and are capable of controlled release and targeted delivery of

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the drugs. Nanosized DDSs can substantially reduce the systemic toxicity of drugs, especially

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chemotherapeutics, and allow drugs to accumulate in target tissue, thereby improving the pharmacokinetics of the drugs [1]. Once the DDS has reached the target tissue, the drugs

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would be released either in the targeted cells or in their extracellular space so that the drug concentration would be maintained at sufficient levels to eliminate all diseased cells. Controlled drug release can be achieved using a DDS with a stimuli-based release mechanism, where a specific stimulus from either internal, e.g., the pH gradient between blood and the tumor environment, or external sources, e.g., a magnetic field or light, would trigger the release of the attached or loaded drugs [2]. A number of studies have been published demonstrating the advantages of DDSs over freely soluble drugs, both at the preclinical and clinical levels [3, 4]. Nanosized DDSs exhibit prolonged blood circulation, leading to their accumulation in solid tumors or at sites of inflammation. This phenomenon was first described by H. Maeda 30 years ago and was termed the Enhanced Permeability and Retention (EPR) effect [5, 6]. The EPR effect is a vascular phenomenon involving the extravasation of macromolecules. The EPR effect is caused by defective tumor blood vessels and an impaired lymphatic clearance due to the rapid and unorganized proliferation of tumor cells (Figure 1). It is also influenced by various vascular effectors, e.g., nitric oxide, bradykinin, and vascular endothelial growth factor (VEGF). Based on the EPR effect, high molecular weight DDSs preferentially accumulate in solid tumors. However, their nanosized

ACCEPTED MANUSCRIPT dimensions lead to reduced accumulation in healthy organs and tissues. Thus, nanosized DDSs are sometimes called passively targeted systems because of the EPR-based increased drug level at pathological sites and reduced severe side effect of drugs in healthy tissues [7,

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8]. Moreover, the accumulation of a nanosized DDS within a target tissue can be further enhanced using specific targeting moieties, e.g., antibodies, fragments of antibodies,

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oligopeptides, or lectins [9].

Figure 1. Schematic description of the EPR effect.

Although nanosized DDSs have several common characteristics, their architecture and inner structure vary widely. Nanocarriers of anticancer drugs may have the form of, e.g., liposomes, micelles, nanoparticles, dendrimers or polymer-drug conjugates, and schematic structure examples are shown in Figure 2. Some of these DDSs are in clinical trials or even in clinical practice [3].

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Figure 2. Schematic structures of most studied nanosized DDSs. Imaging techniques

To better understand and improve active and passive drug targeting, it is important to know

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the DDS pharmacokinetics. Blood, urine, tumor or tissue samples can be collected and

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examined ex vivo. However, modern techniques, such as positron emission tomography (PET), single photon emission computed tomography (SPECT), magnetic resonance imaging

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(MRI), and FI, have enabled non-invasive determination of DDS biodistribution, accumulation or elimination from the body [10-13]. Nevertheless, in many cases, SPECT or PET cannot be used for long-term studies due to the short half-life of the radioactive tracers involved [14-16]. Among the above mentioned techniques, FI is one of the most established imaging modalities with a wide spectrum of applications and future prospects for noninvasive visualization of DDSs in vivo. 1.3

Optical imaging

Generally, optical imaging (OI) is probably the most universal visualization technique employed in many research areas. While some other imaging techniques are based on ionizing radiation associated with potentially negative side effects to patient health, OI uses nonionizing radiation ranging from ultraviolet to infrared light. The lower risk for patients and typically faster analysis process enables long-term or repetitive observations of disease progression [17, 18]. In cancer research, OI is primarily used for the localization of tumors and metastases as well as the monitoring of disease progression or regression. One of the fundamental advantages of OI use in biomedical research is the accessibility to interactions

ACCEPTED MANUSCRIPT between light and tissue and the corresponding photophysical and photochemical processes at the molecular level (e.g., fluorescence, luminescence, multiphoton absorption, and secondharmonic generation). Progress in the development of OI technologies, especially in the

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quality of optical components and the enhancement of detection sensitivity, has resulted in commercially achievable instruments at affordable prices. In recent decades, FI has started to

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be widely used as a visualization modality of in vitro and in vivo DDS characteristics. FI

costs even for very long-term experimental designs.

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enables the easy following of in vitro trafficking and in vivo pharmacokinetics at moderate

Thus, this review attempts to describe in detail available techniques and approaches based on

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FI and utilized in DDS research. Drug delivery research can be broadly categorized into three areas: (I) in vitro studies, (II) preclinical, in vivo animal models and (III) human clinical trials.

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This review will describe the application of FI for the first two research areas in detail. The description herein will focus on the utilization of FI for in vitro cell studies and in vivo pharmacokinetic studies of advanced DDSs with treatment and diagnostic capabilities.

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Finally, future prospects in DDS research will be mentioned.

Principles of fluorescence optical imaging

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Generally, in vivo FI is based on the illumination of a target tissue with a light source of a specific wavelength or wavelength range (e.g., from ultraviolet to infrared) that is able to excite fluorophores. The excitation light has to penetrate through several tissue layers to reach the fluorophores and is, therefore, partially reflected and scattered. Photons are also absorbed by various types of molecules and tissue components. The interaction between the photons and fluorophores results in excitation of the fluorophores. On their subsequent return to the basal energetic state, the fluorophores emit photons with specific wavelengths. Most FI techniques used for visualization of DDSs are based on a planar epi-illumination method, also known as 2D-fluorescence reflectance imaging (FRI) [19-21]. In this method, photographic techniques are applied in fluorescence mode to non-invasively determine fluorescence intensity. After illuminating an animal, highly sensitive charge-coupled device (CCD) chip cameras are used to collect emitted light on the same side of the animal. FRI methods have gained wide popularity because they are easy to operate and have high throughput [22-24]. However, FRI methods have some significant drawbacks because depth cannot be resolved; these methods are unable to determine nonlinear dependencies of propagated signals detected from surrounding tissue [25]. Therefore, although fluorescent intensity is linearly dependent

ACCEPTED MANUSCRIPT (below quenching concentration) on fluorochrome concentration, it is strongly affected by tissue depth and the optical properties of surrounding tissue. For example, two tumors with the same fluorochrome concentration but different vasculature will show different overall

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fluorochrome intensities. The more-vascularized tumor (i.e., the tumor with a higher hemoglobin concentration) will give a lower signal because of the increased absorption of

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photons by the surrounding tissue. Similarly, two similar tumors at two different depths will show different fluorescent intensities in FRI.

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Although the quantification and interpretation of FI data are non-trivial, the advantages of FI prevail over those of other techniques mentioned above. The main advantages of FI are based

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on its relatively easy setup, lack of ionizing radiation, capability for long-term observations up to several months, and the ability to simultaneously use several fluorescent probes [26, 27].

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For example, the combination of far red and near-infrared (NIR) fluorescent dyes bound to the same polymer carrier, where the far red dye is bound by a biodegradable spacer, with multispectral FI can serve as a suitable platform for the simultaneous observation of the

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biodistribution and tumor accumulation of polymer carriers (NIR-labeling) as well as the

DDS labeling

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model drugs in solid tumors [10, 28].

Although some drugs, e.g., doxorubicin (Dox) or ellipticine, and some biological compounds, such as fluorescent proteins, can be directly observed using FI, the majority of DDSs require fluorescent labeling. Recently, several comprehensive reviews focusing on labeling approaches and dyes used for DDS labeling were published, e.g., the review published by [29, 30]. In general, dyes can be loaded, i.e., physically entrapped, or covalently bound to the investigated DDSs. For example, lipophilic NIR dye DiR can be entrapped into hydrophobic parts of nanoparticles [31]. Covalent linkages between DDSs and fluorescent dyes can be either non-biodegradable, e.g., amides, thioethers, or 1,2,3-triazole, enabling the visualization of polymer carrier fate, or biodegradable, e.g., hydrazone bonds, disulfide linkages, or enzymatically degradable oligopeptides, which can provide information about drug release. For example, Dyomics 676 dye can be bound by pH-sensitive hydrazone bonds or reductively degradable disulfide linkages [10, 28, 32]. Recently, an enzymatically degradable GFLG spacer was used to attach epirubicin (EPI) or Cy3 to a Cy5-labeled polymer carrier based on N-(2-hydroxypropyl)methacrylamide copolymers (pHPMA)[33]. The rate of spacer cleavage by lysosomal enzymes was

ACCEPTED MANUSCRIPT determined using fluorescence resonance energy transfer (FRET) in vitro. The loss of FRET signal indicated release of the Cy3 dye or EPI from the carrier (Figure 3). Nevertheless, these

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FRET pairs are not suitable for in vivo evaluation, as will be described later in the text.

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Figure 3. A) Fluorescence spectra of FRET conjugate P-EPI-Cy5 compared with those of PEPI, P-Cy5 and their mixture P-Cy5 + P-EPI. B) The change in FRET ratio of P-EPI-Cy5 conjugate revealed effective EPI release in A2780 ovarian cancer cells. A2780 cells

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overexpressing cathepsin B were incubated with P-EPI-Cy5 at 37 °C for 4 h and then were

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cultured in fresh medium for another 20 h. Then, at different time intervals, cell lysates were measured by fluorescence spectrometry. FRET ratio = IEPI/IFRET, where IEPI and IFRET are the

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fluorescence intensity at 590 nm and 664 nm, respectively (excitation 490 nm). The data are presented as the mean ± standard deviation (n = 3). *, p < 0.01. Reprinted from [33].

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Imaging on the cellular level

The methods of FI commonly performed today are fluorescence microscopy techniques useful for the investigation of pathogenesis at the tissue level. The prevalence of these techniques is due to the increasing accessibility of a broad spectrum of fluorescent dyes, probes and fluorescent proteins that offer the ability for the detection of gene expression, proteins, protein-protein interactions and cellular processes closely related to cancer progression, such as protease activity, apoptosis, autophagy and necrosis [34, 35]. Notably, In vitro studies present the possibility of studying molecular processes of diseases in detail. To prepare a viable diagnostic or therapeutic procedure, the initial steps require: a) identifying specific markers to be targeted for diagnostic or therapeutic purposes, b) the evaluation of drug targeting/localization/efficacy of action at the cellular level, and

ACCEPTED MANUSCRIPT c) the pre-evaluation of appropriate carriers of drugs or diagnostic agents, which also have to be screened for their in vivo delivery efficacy. The broad spectrum of available cancer cell lines is useful for initial screening. Immortalized

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cancer cell lines are ordinarily used to investigate unique molecular aspects of cancer biology

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and to explore the potential efficacy of anticancer drugs or functions of diagnostic agents. Fluorescent probes and proteins.

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The availability of fluorescent dyes [36], quantum dots [35], metallic nanoparticles [37] and fluorescent proteins [38, 39] has facilitated the development and applicability of FI in recent

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years. In general, there are plenty of small fluorescent dyes that can be used for in vitro FI as well as in combination with DDSs. The most representative and widely used fluorophores are

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coumarin, fluorescein, Alexa Fluor and cyanine dyes [40-43]. In addition, various types of quantum dots formed from CdSe in the core and ZnS in the shell or dots with CdSxSe1-x/ZnS compositions have been successfully used [44]. Another group of fluorescent probes are

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fluorescent proteins, and those commonly used include green fluorescent protein (GFP) or red

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fluorescent protein (RFP) [45, 46].

One FI option on the cellular level is based on the application of a pre-designed fluorescent

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probe; its target is a specific receptor or enzyme. These probes are constitutively active or activatable. Active probes that are selective for a target-specific ligand are essentially fluorochromes, such as monoclonal antibodies [47], antibody fragments [48], peptides [49, 50] or aptamers [51]. Activatable probes are based on quenched fluorochromes. Initially, fluorochromes are either self-quenched or quenched by a quencher placed via enzymespecific peptide sequences to the fluorochromes [52, 53]. This sequence can be cleaved by a specific enzyme and, subsequently, light is emitted from fluorochromes upon excitation. Smart probes or beacons belong in this category of probes[54]. Alternatively, the introduction of a fluorescent protein (FP)-encoding transgene into cells is another option. After expression, the FP serves as an intrinsic reporter probe that can be detected by FI [39, 55]. Stably transfected or transduced cells expressing a fluorescent protein under a promoter of interest are available for studying gene regulation. The fusion of FPencoding genes with the genes of interest offers the possibility of localizing and quantifying specific proteins in vitro and in vivo [56, 57]. These approaches are more useful in basic research because the fusion may change the structure and/or functionality of the original protein.

ACCEPTED MANUSCRIPT There are many fluorochromes applicable for in vitro studies because one can operate in broad spectrum of optical wavelengths. This advantage is lost in in vivo studies, in which it is preferable to work in the spectral NIR range because of the high absorption in tissues of all

In vitro fluorescence imaging

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other light wavelengths in the electromagnetic spectrum.

Generally, the in vitro evaluation of drug trafficking and accumulation in intracellular targets

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(organelles or compartments) at adequate concentrations has potential therapeutic use. At the cellular level, particle size, shape, charge, and even flexibility [58-60] and morphology [61]

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determine the rate of internalization, the intracellular localization, the anticancer drug release profile and the degradation of the DDS. For example, Qiu et al. studied the time-dependent intracellular localization of a fluorescent star copolymer-Dox conjugate [62]. Along with

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conjugate localization, they were able to study the increasing fluorescence of Dox during its release from the polymer carrier. The efficacy of active targeting using monoclonal antibodies

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or antibody fragments, selected oligopeptides or saccharides could be readily evaluated to

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confirm the primary concept and design of a targeted DDS [63]. A similar methodology was used to demonstrate the in vitro efficacy of an pHPMA conjugate with Dox targeted by antiCD20 monoclonal antibody rituximab to B cell lymphoma cells [64]. Moreover, most DDSs

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based on polymer carriers, micelles, nanoparticles and liposomes can be easily labeled with different fluorochromes; hence, various biological characteristics can be studied in detail by fluorescence microscopy [10, 65, 66]. FI using in vitro models is a powerful utility for studying the characteristics of novel DDSs at the cellular level and for comparing DDS properties, e.g., uptake, kinetics of cellular transport, efflux rates, and drug localization, with those of the parent free drug or other types of DDSs [67]. It is generally accepted that parent free drugs internalize by passive diffusion more rapidly than do DDSs, which have to enter cells by the slower processes of receptormediated or fluid-phase endocytosis. In vitro FI can distinguish the process by which a proposed DDS is able to enter cells, the rate at which it enters, and whether the drugs or drug model could be released within the cells and re-distribute to the organelles [68, 69]. The different time-dependent intracellular localization of Dox after a treatment of colorectal carcinoma cells with pHPMA conjugate-bound Dox or free Dox is shown in Figure 4 together with a visualization of pHPMA copolymer, which was labeled by the fluorescent dye Dyomics 676 [70]. Direct evaluation of the effect of anticancer drugs in vitro using cells can be suitably combined with the evaluation of specific processes, e.g., cell-death pathways,

ACCEPTED MANUSCRIPT proteolytic processes, protein expression, and colocalization with organelles and specific proteins [71]. In vitro cancer models can also determine the fate of drug carriers, which can facilitate the identification of DDSs suitable for the delivery of diagnostic agents or drugs. For

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example, the time-dependent intracellular localization of thermoresponsive diblock copolymers labeled with fluorescent dye and bound with cancerostatic drug pirarubicin via a

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pH-sensitive spacer was observed together with an unusual drug distribution. The cells showed morphological changes indicating oncotic or necrotic processes, and pirarubicin was

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mainly localized in the cytoplasm and colocalized with the nuclear membrane instead of the typical nuclear localization [72, 73]. A few anticancer drugs are known to have fluorescent

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properties, e.g., Dox and ellipticine, and can serve as extrinsic fluorescent dyes. Thus, realtime drug distribution factors, such as localization, accumulation, and time- and dose-

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dependent cell death, can be directly studied. The simultaneous observation of both drugs and DDS carriers can be achieved using two different fluorescent dyes: drug model dyes (attached via biodegradable spacers) and polymer labels (attached via non-degradable covalent bonds)

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[28]. In some cases, fluorescent intensity may change after preparing a DDS or during a

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change in pH; as such, the results would have to be correlated. Fluorescent microscopy techniques for the study of DDSs in vitro

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FI remains a highly acceptable standard and flexible visualization tool, which can be continuously developed by increasing the number of new fluorescent imaging techniques. Laser scanning confocal microscopy (LSCM or CLSM), in comparison with fluorescent microscopy, is used to obtain high–resolution optical images at controllable depths and is suitable for in vitro DDS biodistribution studies. For example, in the human colorectal adenocarcinoma cell line DLD1, the difference in the in vitro intracellular trafficking of the anticancer drug Dox and a DDS based on a water-soluble pHPMA polymer conjugate with Dox (bound by a pH-sensitive hydrazone bond, pHPMA-Dox) was determined by LSCM and is shown in Figure 4A-C. After 6 h of incubation with pHPMA-Dox, a weak Dox fluorescent signal was detected inside the cells but outside the nuclei (Figure 4A). However, when treated with free Dox, a strong fluorescence signal was observed in the nuclei (Figure 4B). After 24 h of pHPMA-Dox treatment, Dox had localized to the cell nucleus; additionally, a diffuse fluorescence signal was observed in the cytoplasm. Dox bound to the pHPMA-based DDS required a longer time for uptake, intracellular trafficking and release from the carrier (Figure 4C). To determine the fate of the polymer carrier, pHPMA-Dox was labeled with the fluorescent dye Dyomics 676. The intracellular localization of Dox and the pHPMA labeled

ACCEPTED MANUSCRIPT with Dyomics 676 was evaluated after 24 h of incubation (Figure 4D). Dox (green) was observed to have localized in the cell nucleus. The polymer DDS (red) had localized to the cell membrane and cytoplasm but not inside the nucleus. The colocalization of both signals

polymer DDS at the same position/place. B)

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(yellow) corresponded to either Dox bound to the polymer or the localization of free Dox and

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Figure 4. LSCM (Olympus IX8) image of human colorectal adenocarcinoma cell line DLD1 incubated with free Dox or pHPMA-Dox conjugate: A) after 6 h of incubation with pHPMADox; B) after 6 h of incubation with free Dox; C) after 24 h of incubation with pHPMA-Dox; D) after 24 h of incubation of pHPMA-Dox labeled with Dyomics 676 (colocalization (yellow) of Dox (green) and polymer (red)). The cells were incubated with a final concentration of Dox equivalent to 1 µg Dox/mL. Reprinted with permission from [70].

Advanced FI methods, such as fluorescence-lifetime imaging microscopy (FLIM), can also be used at the cellular level. This technique produces images based on differences in the exponential decay rate of a fluorescent sample. FLIM is based on changes in a fluorophore lifetime due to local environmental factors, e.g., pH. This method is highly applicable for evaluating the drug release profile and localization and trafficking of DDSs in vitro. FLIM can also be used to simultaneously study DDS trafficking and drug colocalization in specific organelles [67, 74].

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studied using FI in in vitro models. For example, the incorporation of Dox in the nuclei of cells overexpressing ABC transporters can be documented by LSCM within a few hours;

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compared with that of cells that did not overexpress MDR-responsible proteins, the total accumulation was lower. A longer incubation with Dox could lead to decreased signals in the

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nucleus and, subsequently, in the cytoplasm [69, 75].

In this section, we discussed the in vitro use of FI for the study of molecular processes on a

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cellular level in the field of anticancer drug delivery. The in vitro evaluation of DDSs on selected cancer cell lines of different origins can eliminate inefficient and unsuitable DDSs

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from in vivo applications.

Whole-body imaging

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Conventional chemotherapy regimens rely on small molecular weight cytotoxic agents and

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have considerable limitations. Their nonspecific actions typically result in severe side effects after systemic administration because both malignant tumor cells and healthy cells suffer from

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this treatment. Unfortunately, the intestinal tract, hair and skin, and the immune system are affected. The development of chemotherapy resistance, especially after tumor recurrence and in metastatic diseases, is another crucial issue that must be solved in the development of new therapies. Severe side effects and chemotherapy resistance are considered local dosage issues. For effective therapeutic regimens, high levels of the active substance for a desired period of time are required to treat the targeted cells. However, the accompanying severe side effects limit the administrable dose and chemotherapeutic agent available to treat the tumor. Therefore, the design of new DDSs to improve anticancer therapies using passive accumulation or cell-specific active tumor targeting is desirable. The malignant tissues of many solid tumors have modified vascular systems. Rapid and partially unregulated vascularization leads to gaps in the endothelial layer. Macromolecular DDSs, such as nanoparticles, liposomes, or even water-soluble polymers, can leave the blood stream through these gaps and enter the tumor tissue[3]. Once there, these systems are trapped because the lymphatic system of the tumors is underdeveloped. This passive accumulation of the DDS in the malignant tissue can result in a sufficient effective local concentration of the transported and released cytotoxic drug. Thus, the administered dose can be reduced to minimize severe

ACCEPTED MANUSCRIPT side effects. In addition to passive accumulation, it is also possible to use specific biomolecules, such as antibodies or receptor ligands, for cell-specific active tumor targeting [76]. All developed innovative DDSs with demonstrated in vitro efficacy and safety should be

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evaluated in preclinical in vivo animal models where whole-body FI can be used as a reliable monitoring method. Scope of whole-body FI

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Whole-body FI is similar to in vitro FI: the light absorption and scattering characteristics of the samples are fundamentally analogical and are even more relevant in the complex system

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of a living animal. For in vivo FI, autofluorescence (AF) is an important factor in addition to absorption and scattering. The degree of absorption and AF depends on the range of the excitation wavelength [77, 78]. In the ultraviolet to red spectral range (200 to 650 nm), both

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parameters are significant. Within this range, hemoglobin and myoglobin are the main sources of absorption. Furthermore, melanin (a skin pigment) and fur dramatically reduce incident

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intensities. AF is also based on certain biomolecules, such as collagen, elastin, tryptophan and

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the coenzyme nicotinamide adenine dinucleotide (NAD). The main AF peaks are also located at a shorter wavelength. These effects apply for both excitation and emission radiation. In the short-wavelength spectral range, high absorption and AF limit the sensitivity of in vivo FI to

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the point of not being detectable even at prolonged exposure times. Therefore, to obtain reliable data from preclinical animal experiments using FI, an appropriate animal model and a complementarily labeled DDS are required. First, the animals should be relatively small. Thereby, the depth through which incident light penetrates is minimized. Considering this, mice are the best choice. Second, albino and hairless phenotypes are preferred so that light absorption, scattering and AF are minimized. For pharmacokinetic studies, for example, the healthy but hairless SKH-1 Elite Mouse (Crl:SKH1-Hrhr) strain from Charles River Laboratories International, Inc., can be useful. Additionally, for research on human xenografted tumors, an animal model with an immune deficiency is essential (e.g., the athymic nude mouse). A DDS formulation can be oral or parenteral; however, oral applications may require a special non-fluorescent diet to avoid high background signals from the animal feed. Independent from the administration route of a DDS and its carried model drugs, or active substances as in theranostics (see below), the working wavelength range should ideally be in the near-infrared spectral region, i.e., the optical range between 650 and 900 nm where tissue absorption and AF are at the lowest [77, 78]. Therefore, the preferred dyes for labeling DDSs or for use as small-molecule model drugs are near-infrared dyes, such

ACCEPTED MANUSCRIPT as IRDye®800CW (LI-COR, Inc.), XenoLight CF 750 (PerkinElmer, Inc.), Dyomics 782 (Dyomics, Inc.), and DiR or Alexa Fluor® 680 and 750 (Life Technologies from Thermo Fisher Scientific, Inc.).

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The analysis of in vivo FI data is critical because the quantification of fluorescence intensities is difficult. Background fluorescence in an animal should be detected. After the

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administration of a (covalently) labeled DDS, one can follow its distribution by subsequent imaging at defined time points. Figure 5A shows the typical results of a DDS pharmacokinetic

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study performed in healthy, hairless SKH-1 mice with a MaestroTM in vivo fluorescence imaging system (CRi, Inc.; now PerkinElmer, Inc.). All these tested DDSs were based on

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natural sugars and were labeled with IRDye®800CW. Pseudo-colored images of different animals at certain time points are displayed. It is obvious that the biodistribution of the

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hydroxyethyl starches (HES 200 and 450) differs from that of the dextran (DEX 500). DEX 500 showed a significant accumulation in the liver. Regarding the influence of molecular weight, the larger HES 450 showed a longer circulation time compared with that of the

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smaller HES 200. This is clearly visible in the images 1 day post HES 450 injection. The

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yellow to orange color indicates higher fluorescence intensities of HES 450 compared with the red images of HES 200. The tumor accumulation of the polymer detectable for the longest

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time, HES 450, was tested in athymic nude mice with two different human colorectal carcinoma xenografts. A passive tumor accumulation was apparent (Figure 5B). In addition to in vivo pharmacokinetics, subsequent ex vivo analyses of necropsied organs/tumors can provide information about DDS biodistribution in the body (Figure 5C) [79]. 4.2

Relative quantification of FI data

All the results mentioned were based on the detection of fluorescent intensities. No relative or absolute quantification was performed. However, quantification is fundamental for comparing different DDSs with varying doses in different animals at various time points. So, how can we compare data obtained under different conditions? A relative quantification could be a useful method. Data obtained from identical individuals and experimental settings can be applied to a defined, fixed point. Thus, it is possible to compare the mean of one group with that of another (Figure 5D). Relative total fluorescence intensities over time are shown; the graphs display the different elimination characteristics of the polymers. HES 450 could be detected in the mice for over three weeks. Another relative approach, the tumor accumulation value (TAV), can be useful for the determination of tumor accumulation [79]. Here, fluorescence from tumor areas was compared with the signals from the remaining healthy areas of the

ACCEPTED MANUSCRIPT mice. For HES 450, maximum tumor enrichment was reached after 24 h (Figure 5D, right). Further widely studied DDSs using FI include nanoparticles and nanocapsules, among others. Many groups are working on the application of nanoparticles, especially for cancer

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diagnostics and therapy [80]. For example, the application of indocyanine green (ICG) as an NIR label of diagnostic proteinoid-poly(L-lactic acid) nanoparticles was investigated by

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Kolitz-Domb et al. [81]. Their data showed that the particles were readily eliminated from the body over the first 24 h after i.v. administration. The LS174T colon tumor model showed

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good ex vivo fluorescence after a contrast enema; therefore, these nanoparticles may be useful in fluorescent endoscopy [81]. Benoît and colleagues published data about the biodistribution

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of lipid nanocapsules and compared their FI results with those of nuclear counting and imaging. They demonstrated that both techniques revealed the same findings [82].

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Conclusions such as “the fluorescence intensity showed that 1 mg of the injected dose has accumulated in the tumor after 24 h” are impossible to determine solely from in vivo images. For such statements, absolute quantification is necessary. Of course, one can determine

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absolute intensities from images using imaging software. However, these values have too

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many factors to account for and may require additional studies. First, there are the properties of the DDS to consider. The batch-to-batch fluorescent intensities of a DDS can differ. The

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dye load and fluorescent intensity have to be verified for each batch to prevent selfquenching. Second, in vivo data are unique; each animal has its own pharmacokinetic conditions that affect the DDS and the fluorescent intensity. Some parameters include sex, age, body size and weight, skin and fur status, and fatty tissue content (e.g., subcutaneous, visceral, brown adipose tissue). For tumor-bearing animals, the parameters could also include the localization, size, shape, vascularization, and amount of necrosis of the tumor. Some of these parameters can be controlled by the experimental setup, such as sex and age, while others are unavoidable, such as body size and weight or tumor shape and vascularization. Third, FI comes with technique-independent parameters that affect the measured fluorescence intensity. Instrument settings are important: the strength of the excitation light, the distance from the light source to sample and from the sample to the light detector, the type and quality of the filters used and the other optical components (e.g., objective lenses), the sensitivity and size of the detector (often a CCD camera chip), internal camera gain and the method of signal projection (2D vs. 3D). Some instrument variables can be controlled via hardware and software. However, the inherent sample characteristics influencing the optical path are difficult to control, especially the local concentrations of a DDS and its fluorophores. Tissue

ACCEPTED MANUSCRIPT thickness and optical quality above the fluorescent source can dramatically decrease the intensity of excitation light, resulting in reduced emission signals. Additionally, these properties are not constant over the whole experimental period. Tumors are very dynamic and

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complex systems. They continue to grow over time, and their mass and tissue density are in constant flux. Additionally, the blood and connective tissue content of tumors is constantly

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changing. Under therapy, the overall tumor structure can change. In addition to remission, hemorrhages can occur. Finally, the characteristics of tumors from different individuals with

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different genetic backgrounds and diverse genetic mutations are not comparable. These factors affect the optical issues of tumors and the accumulation of DDSs.

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To solve these issues with absolute quantification or to approximate true values, significant efforts are required. Extensive in vitro and ex vivo tests can provide data for comparison with

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in vivo images. As many parameters as possible should be constant. For example, ex vivo FI of defined tumor masses (0.5 g) from differently treated animals were homogenized in a defined buffer volume (0.5 mL of physiological sodium chloride solution) and measured in

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instrument settings. This reduces data variability and minimizes random errors. In this manner, correlation analyses are more feasible. One should keep in mind that all these

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procedures need to be re-verified for each new fluorophore. The detection of in vivo fluorescent signals for DDSs in animal models for cancer therapy is quite easy using readily available instruments for fluorescence imaging if some basic conditions are considered. Regarding quantification, relative comparisons are possible. However, great care must be taken to achieve reliable absolute quantification.

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Figure 5. Representative in vivo images for DDS monitoring using FI performed on a MaestroTM in vivo fluorescence imaging system (CRi, Inc.; now PerkinElmer, Inc.). Inc.). Taken and adapted from Hoffmann et al. [79] All mice received different NIR-labeled NIR sugar-based polymers as indicated.

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Fluorescent imaging as a tool in theranostics

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HES 200, HES 450 and DEX 500 were labeled with IRDye®800CW (LI-COR, Inc.) via amide bonds. As a control, DEX was also labeled with the same dye using a rapidly cleavable ester bond. The dosage was 1.5 mg of polymer per mouse via tail vein injection. Images are intensity-weighted, pseudo-color images of the NIR fluorescence signal. (A) Display of the pharmacokinetics of different possible macromolecular DDSs in healthy but hairless SKH1-Elite mice (Crl:SKH1-Hrhr from Charles River Laboratories International, Inc.). Bladder signals indicate renal excretion. The longest circulation time was observed for HES 450. DEX 500 showed a strong liver accumulation. DEX 500 (ester) was rapidly de-labeled. Thus, the small dye molecules were quickly eliminated via renal excretion (strong bladder signal after 5 h). (B) Monitoring tumor accumulation of the HES 450 polymer as a long circulating DDS in two subcutaneous tumor xenograft models of colorectal carcinoma using the human cancer cell lines DLD-1 and HT-29 grown in athymic nude mice. Already, at 4 h post injection, the fluorescence signal had concentrated in the tumor area. (C) Ex vivo images of organs (left) and tumors (right) 24 h and 48 h after DDS administration, respectively. For each organ image, from left to right – top: fat, liver, heart; middle: spleen, gallbladder, lung; bottom: intestine, kidneys, uterus and ovaries. All DDSs showed liver accumulation, but the highest liver intensity was measured for DEX 500. For HES 450, some signal was detectable in the spleen as well as in the uterus. In both tumor xenografts, the HES 450 signal showed a rim enhancement and a relatively homogeneous distribution over the tumor cross-sections, with only a few hot spots. (D) Left: plot of relative fluorescence intensity decreasing over time, indicating the degradation and elimination of the DDS (data represents the mean ± min/max, n = 3). HES 450 was traceable for up to three weeks in the mice. Right: TAVs in DLD-1 and HT-29 human colorectal cancer xenografts. In both tumor types, the TAVs for the injected HES 450 rapidly increased during the first few hours. A plateau was reached within the first day after injection (data represents the mean ± min/max, n = 3).

As a portmanteau, theranostics links the words therapy and diagnostics. Under this heading, disease treatment and the generation of diagnostic information are merged using one multifunctional system. Theranostics is an essential part of personalized medicine. The aim of personalized medicine is to find the right therapy for the right patient at the right time. One well-known example established in the late 1990s is the improved personalized breast cancer treatment using the specific monoclonal antibody trastuzumab (trade name Herceptin®, F. Hoffmann-La Roche Ltd.). The application of this therapeutic agent is only indicated and reasonable if the tumor is positive for HER2/neu receptor overexpression [83]. Here, the genetic diagnosis of an individual patient tissue sample leads to a targeted treatment and a better therapy outcome. In addition to individual diagnostics, active and direct monitoring of a therapy and its efficacy has become more relevant in academic and industrial research and in clinical practice. FI can be a perfect tool for the observation of pharmacokinetics (see previous section) and for the visualization of a therapy and its effects on the whole animal body in preclinical models. The following paragraphs illustrate the potential of whole-body FI in cancer theranostics.

ACCEPTED MANUSCRIPT However, the main principles can be extended to address other clinical questions, such as the understanding and treatment of inflammatory processes or targeted drug delivery into the brain because FI allows the simultaneous examination of different aspects using one set-up at

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one definite time point. This characteristic is based on the inherent technique flexibility rendered by a wide variety of useful fluorophores, ranging from dyes to fluorescent proteins. Tumor labeling strategies

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FI permits tumor burden monitoring in preclinical rodent cancer models. This can be realized by different strategies: (a) endogenous labeling, (b) exogenous staining or (c) the use of in

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vivo contrast agents (CAs). Endogenous labeling requires the genetic modification of tumor cells. A standard method for stably marking tumor cell lines is lentiviral transduction with a DNA-encoding FP [84]. This viral-based technique ensures that the DNA is integrated into

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the cellular genome. The marker gene is expressed by the tumor cells at the highest levels. However, a disadvantage of this method is in the selection of an appropriate clone. The

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selected FP-expressing clones may not represent all the specific characteristics of the tumor

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cell line from which they were derived. The transient labeling of tumor cells with a marker gene using, e.g., plasmid transfection, is possible. However, the marker gene can be lost through dilution from mitosis and/or degradative processes. These limitations also apply to

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the second strategy, exogenous staining, where the tumor cells are marked with nanocrystals, such as Qdot® probes, or very lipophilic dyes, such as DiR (Life Technologies; Thermo Fisher Scientific, Inc.), that are integrated into the cell membrane. This method is only usable in slowly or non-replicating cells and is not optimal for the long-term determination of cancer animal models. The usage of CAs in vivo permits tumor detection without prior cell modifications. That is why CAs are perfect for the imaging of spontaneous or induced cancer models. Examples of CAs in FI include injectable dyes and dyes in complexes. The dyes can also be coupled to long-circulating macromolecules or specific, tumor-binding antibodies. Of course, one could also use these CAs in cell line-derived tumors. However, two factors have to be considered. Each injected CA has its own pharmacokinetic properties, and a perfect CA that enhances the contrast between all healthy tissues and all possible malignancies has yet to be created. One has to evaluate the best CA for each model. 5.2

Tracking DDSs

In addition to tumor monitoring, the FI technique allows the tracking of DDSs and therapeutic agents (e.g., Dox). The double coupling of a dye as a DDS tracer and a (autofluorescent) drug

ACCEPTED MANUSCRIPT to a DDS (a soluble polymer or a complex nanoparticle) allows diagnostic and therapeutic applications from a single system. In principle, it is possible to combine specific probes for the pH-dependent release of an active substance. There are many avenues open for research

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and evaluation. An example of tumor therapy using a polymer polymer-based, Dox-loaded loaded DDS is shown in Figure 4. Using the NIR NIR-labeling labeling of the DDS, the biodistribution and the tumor

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accumulation of the DDS can be visualized in vivo (Figure 6A). A). Because Dox is aan autofluorescent fluorescent drug, it can also be visualized; visualized however, due to its spectral properties (Ex/Em

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of 480/590 nm), only ex vivo imaging can be achieved (Figure 6A). A). The therapeutic efficacy of this Dox-loaded loaded DDS in a Dox resistant model was monitored by caliper measurement measurements

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and tumor volume calculations (Figure 6B).

Figure 6. (A) Top: lateral mouse images of a subcutaneous, subcutaneous growing xenograft tumor 24, 72 and 120 h after DDS administration. The in vivo tumor accumulation of the NIR fluorescently labeled DDS (containing Dox attached via pH-sensitive sensitive hydrazone bonds) bond ) is clearly visible (yellow to white area). Beneath, a tumor cross section shows a central area with a high Dox accumulation. (B) The plot displays data from mice bearing a Dox Dox-resistant, subcutaneous xenograft tumor treated either by free Dox (blue curve) or by the Dox-loaded loaded DDS (red curve). Therapeutic administrations are indicated by the grey arrows. It is obvious that free Dox could not initiate tumor regression, whereas DDS DDSdelivered Dox could significantly reduce tumor volume after the third injection. injection. This remission is induced by therapeutic intratumoral Dox levels caused by EPR effect effect-based based DDS accumulation. The

plot is based on data published by [85].

In addition to the targeted release of classical chemotherapeutic compounds, such as Dox or irinotecan, into a tumor, DDSs can also transport other classes of therapeutic agents agents, such as siRNA. Liu et al.. worked on self-assembling self assembling nanoparticles for intratumoral siRNA delivery to effectively knockdown the VEGFR2 gene. They combined two OI techniques: bioluminescence imaging (BLI), LI), to monitor the tumor burden of the animals, and FI, to

ACCEPTED MANUSCRIPT localize the nanoparticles carrying the therapeutic siRNA [86]. This work is exemplary for the possibilities of theranostics. Liposomes loaded with quantum dots emitting in the NIR spectral range for FI and with the chemotherapeutic agents camptothecin and irinotecan were

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studied by Fang and colleagues. They were able to demonstrate that the cationic liposomes were detectable by FI and could increase the concentration of both therapeutic agents after

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intratumoral injection [87].

Because FI utilizes a huge range of fluorophores, several questions can be addressed in a

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single subject at a given time point. This simultaneous monitoring is one major advantage of FI compared with other imaging techniques, such as MRI, computed tomography (CT) or

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even BLI. The localization of tumor cells, the measurement of tumor burden, the grade of vascularization, the accumulation of macromolecular DDSs in a solid tumor, and the drug

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release can all be observed with an adequate system design. Additionally, tumor reaction to the therapy is detectable after therapeutic administration. The use of fluorescent proteins as endogenous markers and as expression indicators driven by specific promoters can reveal

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biological relationships in tumor research. Because many FI studies are available and still

are provided [81, 88-90].

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forthcoming, a complete, exhaustive literature summary is omitted here. Only a few examples

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In addition to visualization in anticancer drug delivery and tumor biology research, the treatment of malignancies or other neoplasms is possible using photodynamic therapy (PDT) in combination with DDSs [91]. A per se nontoxic photosensitizer-DDS conjugate accumulates at a target site after administration. Irradiation of the target location with harmless light activates the photosensitizer and, subsequently, induces the generation of toxic substances (often reactive oxygen species). These molecules often initiate cell death and lead to the therapeutic treatment of disease. For example, porphyrin-based nanoparticles can act as a singlet oxygen converter. Additionally, they can be used in photothermal therapy (PTT), where light is converted into destructive heat inside tumor tissue [92]. In summary, the flexibility of FI is based on the large number of available fluorophores. The specific characteristics of each fluorophore enable a wide range of in vivo theranostics. Fluorescence-based techniques have been used in clinical practice in surgery, phototherapy, and the detection of inflammatory events [91].

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3D fluorescence imaging

Generally, FRI is recognized as an outstanding tool for imaging nano-scaled DDS biodistribution in superficial tissues, for instance, their accumulation in subcutaneously

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inoculated tumors. Nevertheless, FRI fails in absolute quantification of results and cannot be used to determine accumulation in deep tissues [25]. However, FRI enables the comparison of

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kinetics of various DDS systems, for example, the comparison of kinetics of DDSs for cell-

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specific or passive tumor targeting. Unfortunately, FRI results cannot be quantified. The determination of absolute amounts of accumulation within tumors or in physiologically relevant healthy organs by FRI is complicated, perhaps even impossible, at this time. Efforts

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have been made to overcome this limitation, e.g., by combining in vivo signaling and ex vivo analysis of tissue to determine the dose percentage reaching the targeted tissues.

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During the past decade, the progress in OI methods led to the improvement of standard 2D methods. The use of short light pulses in a raster-scan design has been used as a suitable OI

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method for retrieving signal depth to enable three dimensional imaging [93]. Additionally, the depth-dependent attenuation of different wavelengths has been proposed as another property

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to exploit for measuring the depth of fluorophores in the body [94]. FI has also been

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combined with high-resolution microcomputed tomography (µCT) [21].

Fluorescence molecular tomography

Fluorescence molecular tomography (FMT) is based on 3D reconstruction of the distribution of fluorophores in the body based on multiple projections and measurements of light around the boundary of the investigated tissues. The collected emitted light is combined with a mathematical equation that describes photon transport through the tissues. A number of planar detectors, such as CCD chip cameras, record the excitation and emission images of NIR fluorophores in small animals. Advanced algorithms volumetrically reconstruct the concentration of optical imaging agents. FMT may be capable of overcoming the drawbacks of FRI; FMT also facilitates quantitative analyses, even in deep tissues. In addition to small animal imaging, some early achievements toward clinical imaging have also been published. However, because some fluorescent dyes have been approved for human use, it can be expected that the number of reports describing studies on human patients will grow significantly. For example, exogenous fluorescence contrast will likely play a major role in endoscopic methods for the detection of even very small disease foci or micrometastases [95]. A very interesting and promising application of FMT is optical mammography because

ACCEPTED MANUSCRIPT of the relative transparency of human breast tissue to NIR light. Thus, breast cancer [96-98] can be optically detected with high sensitivity [99, 100]. FMT can offer an interesting alternative to X-ray mammography by improving detection specificity and enabling long-term

Hybrid methods

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observation of treatment efficacy.

However, FMT still has a major drawback. The reconstructed probe accumulation signal

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cannot be accurately assigned to treated tissue [101, 102]. Several pioneering studies combining FMT and µCT for molecular imaging purposes in small animals have recently

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been published [103-106]. These studies indicated the potential afforded by the combination of the very precise 3D determination of µCT and the high fluorophore sensitivity of FMT within animal organs. In these studies, imaging performance and accuracy were considerably

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better for the hybrid method than they were for FMT alone. Hybrid FMT-µCT imaging was recently employed to non-invasively evaluate the

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accumulation of nano-DDSs, even in deeper tissues [107]. The biodistribution of NIR dye-

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labeled pHPMA polymer carriers was visualized and quantified using the combination of FMT and anatomical µCT in tumor-bearing mice. Here, several physiologically relevant

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organs and tumor were presegmented, as shown in Figure 7. The long-circulating, NIRfluorophore-labeled DDS were preferentially accumulated in tumor tissue, while the accumulation in healthy organs was minimal. These results showed that the combination of anatomical µCT with molecular FMT could be an effective method for the non-invasive assessment of nanomedicine biodistribution.

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Figure 7. CT-based organ segmentation and hybrid CT-FMT imaging. (A) High-resolution

µCT scans of CT26 colon carcinoma-bearing nude mice, depicting highly electron-dense anatomical structures (i.e., bones), presegmented organs (cf. panel C), FMT-based biodistribution data overlaid on highly electron-dense anatomical structures, and FMT-based biodistribution data overlaid on presegmented organs. (B, C) Two-dimensional planes representing individual organs (B) and pHPMA-Dy750 accumulation in cross sections of these organs (C), analyzed by merging µCT and FMT data sets. Reprinted with permission from [107]. Copyright [2013] the American Chemical Society.

The pHPMA polymer carrier containing a moiety targeting (e.g., oligopeptide containing an RGD or NGR sequence) angiogenesis-related surface receptors was compared with a nontargeted polymer carrier using the hybrid FMT-µCT imaging method [108]. In this study, the biodistribution of a far red-labeled polymer DDS bearing RGD- or NGR-containing oligopeptides targeted to tumor blood vessels and an oligopeptide-free NIR-labeled DDS were compared after their co-injection into mice bearing both rapidly growing, highly leaky CT26

ACCEPTED MANUSCRIPT and slowly growing, poorly leaky BxPC3 tumors. Both DDSs were rapidly and efficiently found in tumor blood vessels due to the EPR effect. Passive targeting was significantly more efficient than the oligopeptide-mediated targeting. Although these conditions may be different

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for other types of tumors than for solid tumors or other DDSs based on micelles or liposomes, these insights indicate that the contribution of cell-specific active targeting should not be

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overestimated.

In addition to the water-soluble polymer DDS, the pharmacokinetic profiles of nanoparticles

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were determined using a FMT-CT modular imaging method [109]. Fluorescently labeled and targeted particles for siRNA delivery were administered in mice. As expected, the

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nanoparticles showed prolonged blood circulation time and had more profound gene silencing in tumor xenograft mice models than did the parent siRNA. Similarly, the biodistribution of

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lipid nanoparticles tailor-made for siRNA delivery was determined by an FMT-CT system using fluorescently labeled siRNA [110]. Interestingly, it was determined that a major portion of the nanoparticles accumulated in the spleen. The distribution data obtained by FMT-CT

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imaging were consistent with the amount of plasma/tissue siRNA determined by a PCR-based

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method.

In addition to CT, other techniques for “anatomical” imaging have been combined with FMT,

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e.g., MRI [111, 112] and ultrasound [113-115]. The development of contrast agents (typically containing Gd) has strengthened MRI for the determination of tumor characteristics, such as pH, vascularization, and metabolism [116, 117]. However, contrast agent sensitivity and acquisition time are limited. Nevertheless, tumor-targeted liposomes [118] and magnetic nanoparticles [119] with NIR-fluorescent and magnetic resonance probes containing siRNA have also been studied.

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Conclusions and future prospects

In recent years, FI has become an integral part of drug delivery research in part due to the adaptability of the method in obtaining highly relevant pharmacokinetic data with reasonable costs and instrumentation. FI is a highly evolving method, as indicated by the growing number of applications and technological opportunities. This paper has presented current, state-of-art and prospective technologies that can be used to perform in vitro, ex vivo and, above all, whole-body in vivo FI. Particular focus was placed on the potential uses and limitations of FI and data relevance with respect to employed imaging techniques. The majority of in vivo applications are still based on 2D FRI providing fine biodistribution

ACCEPTED MANUSCRIPT information in superficial tissue, but not in deeper tissue. However, combinations of advanced optical technologies with high-spatial-resolution structural modalities, such as MRI or CT, should overcome such drawbacks. A reliable method has yet to be developed for the absolute

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quantification of FI data either via the combination of easy-to-perform techniques based on in vivo planar FRI and ex vivo tissue analysis or by more demanding techniques combining 3D

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FMT and CT.

In summary, hybrid techniques that combine the highly efficient and practicable FMT

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tomography with other 3D tomography methods seem to be powerful quantitative tools for the 3D imaging of novel DDSs. Authors strongly believe that there are huge challenges for the

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development of FI, especially for long-term observations of human patients and treatments in the fields of diagnostics and theranostics. We think that 3D FI method will be incorporated

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into day-to-day practice within a decade, most likely in endoscopic methods for the detection of even small diseases, e.g., endoscopic tumor surgery, or in optical mammography because

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Acknowledgements

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observation of treatment efficacy.

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of the higher transparency of breast tissue, for improved detection specificity and long-term

This work was supported by the Grant Agency of the Czech Republic (grant No. 15-02986S), by the Ministry of Education, Youth and Sports of CR within the National Sustainability Program I, Project POLYMAT LO1507, and by the Deutsche Forschungsgemeinschaft (MA 1648/7-1 and MA1648/8-1).

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Graphical abstract

Fluorescence optical imaging in anticancer drug delivery.

In the past several decades, nanosized drug delivery systems with various targeting functions and controlled drug release capabilities inside targeted...
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