Article Cite This: Acc. Chem. Res. 2018, 51, 3226−3235
pubs.acs.org/accounts
Harnessing Cyanine Reactivity for Optical Imaging and Drug Delivery Alexander P. Gorka,‡ Roger R. Nani,§ and Martin J. Schnermann*
Acc. Chem. Res. 2018.51:3226-3235. Downloaded from pubs.acs.org by OPEN UNIV OF HONG KONG on 01/21/19. For personal use only.
Chemical Biology Laboratory, Center for Cancer Research, National Cancer Institute, Frederick, Maryland 20850, United States
CONSPECTUS: Optical approaches that visualize and manipulate biological processes have transformed modern biomedical research. An enduring challenge is to translate these powerful methods into increasingly complex physiological settings. Longer wavelengths, typically in the near-infrared (NIR) range (∼650−900 nm), can enable advances in both fundamental and clinical settings; however, suitable probe molecules are needed. The pentamethine and heptamethine cyanines, led by prototypes Cy5 and Cy7, are among the most useful compounds for fluorescence-based applications, finding broad use in a range of contexts. The defining chemical feature of these molecules, and the key chromophoric element, is an odd-numbered polymethine that links two nitrogen atoms. Not only a light-harvesting functional group, the cyanine chromophore is subject to thermal and photochemical reactions that dramatically alter many properties of these molecules. This Account describes our recent studies to define and use intrinsic cyanine chromophore reactivity. The hypothesis driving this research is that novel chemistries that manipulate the cyanine chromophore can be used to address challenging problems in the areas of imaging and drug delivery. We first review reaction discovery efforts that seek to address two limitations of longwavelength fluorophores: undesired thiol reactivity and modest fluorescence quantum yield. Heptamethine cyanines with an Oalkyl substituent at the central C4′ carbon were prepared through a novel N- to O-transposition reaction. Unlike commonly used C4′-phenol variants, this new class of fluorophores is resistant to thiol modification and exhibits improved in vivo imaging properties when used as antibody tags. We have also developed a chemical strategy to enhance the quantum yield of far-red pentamethine cyanines. Using a synthetic strategy involving a cross metathesis/tetracyclization sequence, this approach conformationally restrains the pentamethine cyanine scaffold. The resulting molecules exhibit enhanced quantum yield (ΦF = 0.69 vs ΦF = 0.15). Furthermore, conformational restraint improves interconversion between reduced hydrocyanine and intact cyanine forms, which enables super resolution microscopy. This Account then highlights efforts to use cyanine photochemical reactivity for NIR photocaging. Our approach involves the deliberate use of cyanine photooxidation, a reaction previously only associated with photodegradation. The uncaging reaction sequence is initiated by photooxidative chromophore cleavage (using wavelengths of up to 780 nm), which prompts a C−N bond hydrolysis/cyclization sequence resulting in phenol liberation. This approach has been applied to generate the first NIRactivated antibody-drug conjugates. Tumor uptake can be monitored in vivo using NIR fluorescence, prior to uncaging with an external irradiation source. This NIR uncaging strategy can slow tumor progression and increase survival in a MDA-MB-468-luc mouse model. Broadly, the vantage point of cyanine reactivity is providing novel probe molecules with auspicious features for use in complex imaging and drug delivery settings.
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INTRODUCTION Experiments involving optical stimuli to visualize and/or alter biological processes are pillars of biomedical research.1 Tissue absorption and light scattering impose limitations on the scope of these experiments. Far-red and near-infrared (NIR) wavelengths are less subject to these effects, expanding the range of biological questions suitable for optical interrogation.2 This article not subject to U.S. Copyright. Published 2018 by the American Chemical Society
Over the past two decades, the benefits of using these wavelengths have been established in several contexts. Longer wavelengths provide orthogonal channels and low intrinsic toxicity, which has proven useful for in vitro and cellular Received: July 31, 2018 Published: November 12, 2018 3226
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Figure 1. Cyanine background: (A) key examples, (B) important photophysical features, and (C) chromophore reactivity.
analyses.3 In Vivo Imaging Systems (IVIS) provide a convenient means to noninvasively track biological processes in live animals.4 Finally, optical imaging is emerging in clinical settings through the rapid development of fluorescence-guided surgery (FGS) procedures.5,6 In the context of altering biological systems, photodynamic therapy (PDT) methods were the first to recognize the benefits of NIR light for biological applications.7 While these methods provide a proven and versatile means to locally deliver reactive oxygen species, an enduring goal is to create NIR-activated photocaging platforms that can precisely deliver chemically diverse biological stimuli.8 While a range of nanomaterials and genetically encoded proteins has been explored, small molecule fluorophores and their bioconjugates have been central to the development of far-red and NIR imaging. Broadly speaking, these approaches benefit from a direct analogy to small molecule therapeutic agents−a theme that will be revisited in this Account. The principles and experience of medicinal chemistry offer critical insights regarding chemical discovery and optimization, as well as a clearly defined path to clinical translation. A unique feature of these molecules is the extended π-system, which is needed for a long-wavelength electronic transition. This conjugated system makes optimizing the in vivo pharmacodynamic/ pharmacokinetic (PD/PK) properties of these molecules a significant challenge. Furthermore, the requirement of a relatively high lying HOMO and low lying LUMO renders long wavelength fluorophores thermodynamically activated for further chemistry. As one of the few molecular scaffolds with suitable absorbance and emission maxima, cyanine fluorophores have played a particularly prominent role in the development of long wavelength imaging. The pentamethine and heptamethine cyanines, such as Cy5 and Cy7 (Figure 1A), are workhorse reagents for a range of applications.9 One NIR cyanine, ICG, has been used clinically for several decades, and, another, IR800CW is being explored for several FGS applications.10−12 The defining chemical feature of the cyanines is a chromophore comprising an odd number of unsaturated carbons connecting two nitrogen-containing heterocycles. While kinetically stable in biological media, this chromophore unit can undergo a range of thermal and photochemical
processes. The chemical reactivity of the cyanine chromophore has played a key role in the development of biomedical methods ranging from super resolution microscopy to biosensor molecules (as reviewed by our group in 2015).13 In many of these cases, the role of chromophore modification was discovered serendipitously and then defined through retrospective mechanistic analysis. This Account summarizes our recent studies to rationally employ the diverse chemistry of the cyanine chromophore. Cyanine reactivity has been applied toward two goals−the creation of novel imaging probes and the identification of NIR photocaging reactions. Reaction discovery drives our probe studies as we seek to develop synthetic transformations that modify the cyanine chromophore in novel ways. The resulting molecules have been applied toward live animal imaging and super resolution microscopy. To convert the cyanine scaffold into a NIR “photocage”, we have taken advantage of a photooxidative cleavage reaction previously only associated with cyanine photodegradation or photobleaching. The cyanine-based photocages have been used to create NIRactivated antibody drug conjugates (ADCs). Overall, this Account seeks to highlight how a focus on cyanine chromophore chemistry is leading to novel probe molecules with promising features for use in complex applications.
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GENERAL FEATURES OF CYANINES The photophysical properties of the cyanines have been defined in significant detail through efforts driven by, first, laser dye and sensitizer applications and, later, the single molecule imaging community.14 These molecules are characterized by a high absorbance cross section (ε often >200,000 M−1 cm−1), which is among the largest known for organic small molecules. This is due to a dramatic electronic reorganization upon S0 to S1 excitation leading to a high transition dipole, a feature apparent upon inspection of the HOMO and LUMO orbitals (Figure 1B).15 In contrast, the fluorescence quantum yield (ΦF) of these molecules is typically lower than other important chromophores (such as the rhodamines), with values often below 0.1. This is due to complex excited-state chemistry, especially bond isomerization processes, that compromises fluorescence emission.14 3227
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utility, the reaction proceeds readily with cyclic anhydrides to provide a bioconjugatable carboxylate product, 2, which we refer to as Frederick Near IR-774 or FNIR-774 (Figure 2A). To investigate FNIR-774 for in vivo imaging purposes, conjugates of FNIR-774 and EGFR-targeting antibodies were compared to those bearing IR-800CW (Figure 1A) in MDAMB-468 xenograft models. FNIR-774 conjugates exhibit dramatically decreased liver accumulation, along with some-
The chemical reactivity of the cyanines is complex and has been characterized in less detail than their optical properties.13,16 On first inspection, a randomly chosen organic chemist might guess that the cyanine chromophore is broadly reactive and, maybe, not even stable to a protic environment. The presence of embedded imine- and enamine-functional groups might be predicted to undergo a range of nucleophilic and electrophilic chemistry. However, cyanine reactivity is not readily defined by these chemical analogies. Most notably, these molecules are stable in aqueous buffer and, when coupled to targeting antibodies, have been shown to persist in vivo for days to weeks.17 This chemical stability arises from several features (Figure 1C). The delocalized cationic charge renders the nucleophilicity of the more electron-rich C1′ and C3′ positions modest. For example, direct protonation of the C1′ position is feasible but only under strongly acidic conditions (HClO4 in ethanol).18 The electrophilicity of the chromophore is also modest. Addition to the C2 indolenine position can occur but only with excess hydroxide or alkoxide at elevated temperature in alcohol solvents.19 While this twoelectron reactivity is largely irrelevant in biological settings, single-electron transfer chemistry can occur. Nucleophilic additions to C4′-chloro substituted cyanines are important preparative reactions, and the potential for these to occur with biological nucleophiles is described below. In addition, the light-promoted addition of thiol nucleophiles to the cyanine chromophore, first characterized in the context of single molecule imaging, involves photoinduced electron transfer (PET) processes.20 To ascribe another chemical analogy to these compounds, we suggest that the cyanine chromophore might be best thought of as an acyclic analog to nitrogen heterocycles, that is more “pyridinium-like”, rather than “enamine-like” or “imine-like”.
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DEVELOPMENT OF IMPROVED IMAGING PROBES Prompted by seminal studies on the “Cy” dyes by Waggoner and co-workers, there have been significant efforts to develop cyanines for biological use.9,21,22 Most of these studies were motivated by in vitro procedures (e.g., DNA sequencing), and these molecules were often subsequently applied in vivo with little further optimization. The limitations of existing probes for in vivo imaging use is outlined in this quote by Tsien and Nguyen:5 “longest possible wavelength...fluorophores are dim, chemically f ragile and more prone to nonspecific hydrophobic stickiness”. These issues have likely limited the broader implementation of NIR fluorescence techniques and suggest an ongoing need for probe development. To improve the chemical stability and physical properties of the heptamethine cyanine scaffold, we sought to identify new strategies that directly modify the C4′ position−a critical substituent of these molecules. Prior work had variably substituted this position with phenol, amine, and thiol substituents, which are accessed through direct addition from the C4′-chloro precursor. This reaction occurs through an electron-transfer-mediated SRN1 mechanism, which is not compatible with alkoxide nucleophiles.23 Enabling the preparation of otherwise inaccessible C4′-O-alkyl cyanines, we found that cyanines substituted with N-methylethanolamine (e.g., 1) undergo efficient N- to O-rearrangement with concurrent incorporation of an electrophile.24 Of particular
Figure 2. Development of N-to-O rearrangement reactions on the cyanine scaffold. (A) Synthesis of FNIR-774 (2) from 1. (B) In vivo imaging of IR-800CW (IR800) and FNIR-774 in an MDA-MB-468 xenograft tumor model. (C) Reactivity of C4′-O-alkyl and O-aryl cyanines to thiol nucleophiles. Lef t: Fraction remaining following exposure to 1 mM glutathione in pH 7.0 PBS. Right: SDS-PAGE following incubation of IR-800CW and FNIR-774 with cell lysate (24 h, rt). 3228
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what improved tumor targeting (Figure 2B).25,26 Motivated by uncovering fluorescent labels of the ureter for use in abdominal surgery applications, we have also used this chemistry to develop a class of untargeted C4′-O-alkyl cyanines that undergo nearly exclusive renal clearance.27 In probing the basis for these improved in vivo properties, we examined the thiol reactivity of various C4′-substituted cyanines. Phenol-modified cyanines (e.g., IR-800CW) react readily with glutathione to provide C4′-thiol substituted products.24 By contrast, alkyl ethers, such as FNIR-774, are immune to this reaction (Figure 2C). To test if analogous reactivity occurs with cellular proteins, these molecules were incubated in cell lysate (24 h, rt, HEK-239 cells). IR-800CW covalently labels an array of proteomic components, whereas FNIR-774 and other C4′-O-alkyl cyanines are unreactive.27 Of note, Usama et al. has recently reported a related set of observations with C4′-chloro cyanines, looking particularly at the role of albumin as an in vitro labeling partner.28 Broadly speaking, studies to determine if, and in what context, cyanine protein modification occurs in vivo would be of significant interest. In total, C4′-O-alkyl substitution represents an excellent approach for preparing bioconjugatable heptamethine cyanines. These studies also suggest that covalent reactivity with biomolecules, commonly evaluated for therapeutic agents, also be considered for imaging probes. As pointed out in the quote above, another limitation of long-wavelength probes is modest ΦF. The major nonemissive pathway for cyanine excited state deactivation involves chromophore isomerization.13 Using the trimethine scaffold, Cy3, this pathway was blocked by installing 3 fused 6membered rings along the polymethine linker to form Cy3B. This modification leads to a dramatic improvement in ΦF (0.09 for Cy3 to 0.85 for Cy3B).29 A clear extension of this work - assembling restrained penta- and heptamethine cyanines - requires the attachment of 4 and 5 fused rings, respectively, directly onto the chromophore, which increases the synthetic complexity of these targets. Our studies were the first to assemble conformationally restrained polycyclic pentamethine cyanines. The approach, shown retrosynthetically in Figure 3A, involves a tetracyclization reaction from protected dialdehyde, 4. This reaction sequence involves an intramolecular Michael addition followed by a dihydropyran ring-forming cascade. This reaction engages the more nucleophilic positions of the cyanine chromophore but still requires a strong Lewis acid (BBr3) to proceed. To our delight, the resulting compounds exhibit dramatically improved ΦF relative to the unconstrained cyanines (ΦF = 0.69 for 6 vs ΦF = 0.15 for Cy5 5) and extended fluorescence lifetimes (Figure 3B).30 Pentamethine cyanines are the chemical component of extensive single molecule localization microscopy (SMLM) efforts (i.e., in dSTORM). The central chemical challenge in SMLM is the controlled generation of nonfluorescent and fluorescent states. The most extensively used strategy has been to subject cyanines to thiol and phosphine nucleophiles in deoxygenated buffer. Mechanistic studies have shown the resulting blinking processes involve nucleophilic modification of the C2′ position.31 Our chemical studies suggested these reactions are disfavored following ring installation. Another approach has been to chemically reduce (with NaBH4) the cyanine chromophore and restore it using UV irradiation in a process likely involving ROS (Figure 3C).32 Advantageously, conformationally restricted cyanines undergo the reduction/
Figure 3. Synthesis and characterization of conformationally restrained pentamethine cyanines. (A) Retrosynthetic approach to 3. (B) Key spectroscopic properties of conventional pentamethine cyanine 5 and 6 (in MeOH). (C) Left: Recovery from NaBH4 reduction (UV-irradiation of reduced 5 and 6 in pH 7.4 PBS). Middle: SMLM of a phalloidin-conjugate of 7. Right: Photon count using 7 with NaBH4 in PBS relative to Alexa 647 in a conventional STORM buffer.
oxidation cycle with dramatically improved yield and efficiency, an unanticipated benefit of the conformational restriction strategy (Figure 3C). This observation has been used for SMLM with a phalloidin conjugate of 7. These studies provide photon count in oxygenated PBS that matches that obtained with a phalloidin conjugate of standard pentamethine cyanine, Alexa 647, in STORM buffer (Figure 3C). Applying these molecules in other contexts, as well as the extension of conformational restriction to the heptamethine scaffold, is a promising avenue for future study. 3229
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REPURPOSING CYANINE PHOTOBLEACHING FOR NIR PHOTOCAGING In addition to imaging, optical methods can also introduce biological stimuli with otherwise unattainable specificity. An important strategy has been to “cage” bioactive molecules with photoremovable protecting groups.33 In particular, caging neuromodulatory small molecules with UV-light responsive caging groups has proven to be a versatile and enduring method.34 Several strategies have been investigated to translate these approaches into more complex settings. Two-photon (2P) methods use pulsed NIR lasers that deliver sufficient local flux to initiate excitation of probe molecules with absorbance maxima in the visible range. In these methods, release is confined to small 2P focal volumes (nanoliters to picoliters), making uncaging in bulk tissue difficult.35 There have also been significant efforts to develop metallic nanoparticle-based strategies involving localized heating or upconversion.36,37 However, heavy metal toxicity concerns and difficult bioconjugation have made broad use a significant challenge.38 Single-photon reactions using organic scaffolds are needed to broadly implement caging strategies for various complex in vivo applications. Exciting recent progress, particularly using the BODIPY scaffold, has extended photocaging wavelengths well into the visible range, but general strategies in the 700−900 nm range are still needed.39−42 To develop NIR uncaging groups, we have sought to repurpose the intrinsic photochemistry of long-wavelength fluorophores. This tactic offers several advantages: (1) Fluorescence imaging allows compound accumulation to be assessed prior to uncaging. (2) Many foundational elements are already in place for deploying NIR fluorophores, including toxicity profiles and imaging infrastructure. (3) Prior reports of fluorophore photobleaching provide useful chemical starting points to design long-wavelength caging reactions. Below, we describe our efforts using the cyanine scaffold, but we have also applied this approach with silicon phthalocyanines. The latter led to a class of molecules where uncaging with 690 nm light is conditional on hypoxia and the presence of thiol reducing agents.43 Cyanine photodegradation had been shown to involve a photooxidative cleavage reaction, exemplified in Figure 4A with the conversion of ICG to carbonyl products 9 to 12.44,45 We carried out mechanistic studies that support a reaction sequence involving self-sensitized 1O2-generation, which reacts with high regioselectivity to form thermally labile dioxetane intermediates.46 To adapt photooxidation for photocaging, we developed the general scheme shown in Figure 4B. The sequence includes photochemical and thermal components. The photochemical process entails photooxidative cleavage of the cyanine polyene. The thermal phase entails C4′-N hydrolysis and then intramolecular cyclization to release phenol payloads. The central premise is that 13 is hydrolytically stable, while photooxidation products 14 and 15 undergo hydrolysis under physiological conditions. This altered reactivity can be rationalized by a change in the π-conjugation of 14 and 15 to increase the electrophilicity of the key C4′-N bond through increased iminium character. Initial testing was carried out with compound 16, which releases the absorbance reporter 4-nitrophenol.47 Cyanine photooxidation occurs with single-photon light power (1−200 mW/cm2), requiring 2−3 min for completion when using flux at the higher end of this range. Of note, prior work has
Figure 4. Development of cyanine-based caging group. (A) Photooxidation of ICG to provide 9 to 12. (B) Design of cyanine photocaging reaction. (C) Characterization of uncaging reaction with nitroaryl-releasing cyanine 16. Lef t: change in absorbance of 50 μM 16 (red) and uncaged 4-nitrophenolate (blue) in cell culture media (DMEM with 10% FBS) as a function of irradiation time (3 mW/cm2, 690 nm). Right: effect of intermittent irradiation (1 mW/cm2, 690 nm) on absorbance of 50 μM 16 and uncaged 4-nitrophenolate in 50 mM HEPES (pH 7.5).
estimated the quantum yields of cyanine photooxidation (Φpd) to be between ∼5 × 10−3 and ∼5 × 10−4.44 The reaction is reasonably high-yielding (60−75%) and only moderately sensitive to pH. Intermittent irradiation experiments (Figure 4C) show that the cyanine absorption decrease is directly linked to irradiation, while the reporter signal increases in interim dark periods. These results are consistent with the proposed mechanism - photooxidation of the cyanine initiates the uncaging process, and subsequent steps, C4′-N hydrolysis and cyclization, are light-independent. Detailed mass spectral analysis observed each of the proposed intermediates, and suggested that the hydrolysis of 14 and 15 is the rate-limiting, postphotolysis step.47 Guided by an interest in developing molecules suitable for use as NIR-activated ADCs (described below), we carried out a significant set of optimization studies.48 These efforts were geared toward improving protein labeling, stability to biological media, and extending the wavelength of excitation. 3230
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ability to initiate uncaging with 780 nm light (see below). These studies highlight the dramatic role that single atom substitutions can have on photocaging molecules−another notable similarity with the optimization of small molecule therapeutics.49
Analogs were tested for photooxidation/release efficiency, background release, and optical properties. Key observations are summarized in Figure 5. Modifications to the linker
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NIR-ACTIVATED ADCS The treatment of locally disseminated solid tumors with NIR light, applied either from an external source or intraoperatively, is a longstanding goal. The principal existing strategy, PDT, relies on photosensitizers that locally generate ROS upon irradiation.50,51 The past decade has seen significant progress in the development of improved variants of this general strategy. Particularly promising are photoimmunotherapy (PIT) strategies, which use photosensitizers conjugated to tumor-targeting antibodies.52,53 For example, a conjugate of cetuximab and a silicon phthalocyanine photosensitizer is in clinical trials for recurrent, inoperable head and neck cancer.54 These ongoing efforts, as well as the progress of FGS methods, suggest that the conventional view of novel probe-based optical methods being difficult to translate into clinical use may be changing. In fact, the examples of both FGS and PIT indicate there is a real potential for a new generation of optical methods to have dramatic translational impact. The NIR light-targeted release of potent bioactive molecules could serve as a useful alternative, or complement, to methods that rely on traditional photosensitizers alone. Locally targeted pharmacological agents can offer advantages over ROSmediated effects alone, including the ability to engage specific biological mechanisms and to improve potency. With regard to the latter, the duocarmycin-releasing constructs described below require in vitro concentrations that are 3−4 orders of magnitude lower than PDT methods (which are typically used in 10−500 nM range). Our efforts to date have focused on the development of a light-activated ADC strategy. Extensive clinical work with ADCs has illustrated the benefits of antibody-based approaches, including excellent biodistribution (even with complex payloads) and antigen targeting.55
Figure 5. Summary of structure−function relationship studies of the cyanine caging group.
domain were predicted to reduce the rate of background hydrolysis (through steric effects), while also extending the λabs further into the NIR range (a prediction informed by computational studies). In practice, we found that a small change to the nitrogen substituent of the ethylenediamine linker−N,N′-dimethyl to N,N′-diethyl−is coupled with a nearly 2-fold improvement in stability and a 40 nm bathochromic shift in λabs. These chemical observations translate to a biological setting through improved therapeutic index and the
Figure 6. Development of cyanine-based NIR ADC strategy. (A) Modifications leading from Cy-Pan-CA4 to CyEt-Pan-Duo. (B) In vivo imaging (Cy-Pan-CA4) pre- and postirradiation (100 J, 690 nm hν) showing selective depletion of intratumoral fluorescent signal of one of two A431 tumors implanted in the dorsum. (C) Impact on tumor progression (CyEt-Pan-Duo) in an MDA-MB-468-luc xenograft. 3231
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However, both “on-target” (undesirable target expression in benign tissue) and “off-target” effects (premature release in circulation or nonspecific uptake in benign tissue through Fcγreceptors and other mechanisms) are associated with significant adverse outcomes. This approach seeks to combine the potency and in vivo properties of ADCs with the highly targeted nature of NIR photoactivation. Prior to pursuing ADCs, we examined unconjugated cyanine photocages in several cellular experiments. We first investigated the inherent toxicity of C4′-N-dialkyl heptamethine cyanines. These efforts were informed by previous studies suggesting that other heptamethine cyanine fluorophores are generally only weakly cytotoxic, even upon protracted irradiation.56 A sulfonated cyanine modified to release only phenol, a nontoxic payload, had minimal impact on cell viability (IC50 of >200 μM in both HeLa and MCF-7 cells) either in the presence or absence of NIR irradiation.57 A major reason for this modest toxicity is likely that cyanine photooxidation at least partially consumes 1O2 and, concurrently, destroys the chromophoric element. These results also suggest that the byproducts of the photooxidation process are well tolerated. Of note, there have been some efforts to use certain hydrophobic cyanines as conventional PDT type reagents.58,59 However, the sensitizer concentrations required in these studies (μM) exceed that required with other more efficient 1O2-sensitizing chromophores (e.g., porphyrins or silicon phthalocyanines), which often function in the nanomolar range. These encouraging results led us to pursue ADC constructs. Our efforts centered on conjugates of panitumumab (Pan), a clinically used anti-EGFR antibody. Head and neck, ovarian, and bladder cancer are NIR accessible and often EGFR+ and thus feasible candidate tumor classes for these approaches. Using our first-generation construct, Cy-Pan-CA4 (Figure 6A) and the analog that releases the fluorescence reporter 4-methyl umbelliferone, we demonstrated the following: (1) Release proceeds efficiently, including in the acidic environment (pH 5.0) characteristic of the lysosome. (2) Background release is modest (1−2% after a 24 h incubation in human serum). (3) Biologically relevant hypoxia has minimal effect on release. (4) Cellular studies revealed similar efficacy to free drug upon irradiation and a ∼70X therapeutic window (defined here as the ratio between unirradiated and irradiated viability IC50’s). (5) Imaging studies demonstrated that cyanine fluorescence could be used to track the conjugate in vivo (Figure 6B). Moreover, external tumor irradiation with a 690 nm laser led to the disappearance of the cyanine signal−a useful marker to measure the light dose needed for drug release (Figure 6B).57 While Cy-Pan-CA4 was useful in developing these key components, we quickly recognized that improved small molecule potency was a likely prerequisite for in vivo efficacy, particularly in a single-dose administration format. Duocarmycin-class payloads, developed through detailed mechanistic and SAR studies by Boger and co-workers, exhibit picomolar potency and are finding clinical use in conventional ADC applications.60 Furthermore, these molecules can be modified through the phenol of the seco-form, providing a convenient handle for conjugation that also traps the compound in an initially inactive form. Guided by the optimization studies described above, we prepared and characterized two fully elaborated cyanineantibody constructs.48 These compounds, CyMe-Pan-Duo and CyEt-Pan-Duo (Figure 6A), differ only in the ethylene diamine
N-alkyl substituent (N,N′-dimethyl to N,N′-diethyl). The CyEt-Pan-Duo construct exhibits a 45 nm bathochromic shift relative to CyMe-Pan-Duo and is almost 2-fold brighter due to a higher molar absorptivity. Cellular studies demonstrate that both compounds exhibit excellent potency upon irradiation (20−30 pM) but that the CyEt-Pan-Duo construct exhibits a roughly 2-fold improved therapeutic index (∼580X for antigen positive cells). We also find that CyEt-Pan-Duo could be activated efficiently with a low dose of 780 nm light (5 J/cm2) in cellular studies. Of note, CyEt-Pan-Duo exhibits a nearly 8fold improved therapeutic index relative to Cy-Pan-CA4 and an over 400-fold improvement in potency. Building on these cellular studies, we examined the impact of CyEt-Pan-Duo on in vivo tumor progression. Effects of irradiation (80 J, 690 nm) on tumor proliferation were assessed through luminescence imaging of luciferase activity (short-term) and caliper measurement of tumor size (longterm) in a MDA-MB-468-luc xenograft. Immediate decreases in luciferase activity were observed for two doses (Figure 6C), and these effects on tumor growth correlate with increased survival (p < 0.01). Notably, the treated mice exhibited no statistically significant differences in body weight relative to untreated animals. These studies provide the first evidence that cyanine photocaging can be applied to modulate biological outcomes in live animals and provide proof-of-concept demonstration for our drug-delivery efforts. Enduring goals in this area include improving the tumor uptake of these constructs and extending this approach to more advanced preclinical models.61 There are several features of this cyanine-based uncaging approach that merit additional comment. First, the kinetics of release are slower than with conventional UV/blue photocages (t1/2’s for release are in 10−30 min range, depending on substrate). This means that biological phenomena being perturbed should occur over the same time frame. While not an issue for drug-delivery approaches discussed here, these kinetic parameters preclude applications in certain other settings (e.g., local release of neuromodulatory small molecules). Of note, we have applied this approach for the intracellular release of an estrogen receptor antagonist to initiate spatially resolved Cre-LoxP-mediated recombination, and Mitra and co-workers used an initial cyanine photooxidation step to uncage a platinum(II) species.62,63 Finally, achieving sufficient light doses has not imposed a significant challenge in our studies (typically 2−3 min irradiations using a continuous wave laser in vivo). This may be in part because this cyanine-based approach differs from “conventional” uncaging reactions in that the chromophoric unit is destroyed by the photooxidative cleavage reaction, thus limiting the effects of optical shielding by uncaged but still light-absorbing chromophores. Nevertheless, the identification of long-wavelength photocaging reactions with more rapid kinetic parameters and lower light-dose requirements is still a critical goal.
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CONCLUSION Developing imaging probes and photocaging reactions for use in advanced biological applications is a challenging process. Rationally modifying the few existing long-wavelength chromophores provides an enduring path to develop promising new molecules.64,65 Cyanine fluorophores are unique because the polymethine chromophore is sterically accessible and not stabilized through aromaticity. This feature is sometimes 3232
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collaborators, listed on the associated publications, who contributed to many of these studies.
described as a liability of these molecules. However, the studies above, as well as the experiences of others, demonstrate that chromophore reactivity can play productive, and even central, roles in the use of these molecules. Incorporating this feature as an upfront “design” element provides the opportunity to create novel cyanines that target specific biological questions. Future uses of cyanine reactivity will continue to span the nano- to macroscales. Super resolution microscopy inherently pushes the limits of photon budget and labeling specificity. Bright, photostable NIR probes that can be incorporated through protein-based modern labeling strategies are still needed. The development of molecules for in vivo use imposes its own set of issues. Just as with small molecule therapeutics, PK/PD properties are critical for achieving efficient in vivo imaging and drug delivery. Thus, while photochemical parameters (ε, ΦF, etc.) are critical, so are water solubility, efficient bioconjugation, stability to biological media, and optimal biodistribution. New chemical design strategies have a critical role to play in the identification of probe molecules optimized for specific challenges in fundamental and applied settings.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Martin J. Schnermann: 0000-0002-0503-0116 Present Addresses ‡
Department of Chemistry, University of Connecticut, Storrs, CT 06269, USA. § 4Catalyzer, Guilford, CT 06437, USA. Author Contributions
The manuscript was written through contributions of all three authors. All authors have given approval to the final version of the manuscript. Notes
The authors declare no competing financial interest. Biographies Alexander P. Gorka obtained his Ph.D. at Georgetown University with Prof. Paul Roepe in 2013. He was a postdoctoral scholar at the NCI with Martin Schnermann until 2017 before joining the University of Connecticut Chemistry Department as an Assistant Professor. Roger R. Nani obtained his Ph.D. at the California Institute of Technology with Prof. Sarah Reisman in 2013. He was a postdoctoral scholar at the NCI with Martin Schnermann until 2017 before joining 4Catalyzer. Martin J. Schnermann is an investigator in the Chemical Biology Laboratory of the Center for Cancer Research, National Cancer Institute. He obtained his Ph.D. in Chemistry at the Scripps Research Institute with Prof. Dale Boger. He conducted postdoctoral research with Prof. Larry Overman at the University of California, Irvine before joining the NCI in 2012.
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ACKNOWLEDGMENTS This work was supported by the Intramural Research Program of the National Institutes of Health, National Cancer Institute, Center for Cancer Research. We gratefully acknowledge our 3233
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