Activatable Fluorescent Probes in Fluorescence-guided Surgery: Practical Considerations

Abstract

Fluorescence-guided imaging during surgery is a promising technique that is increasingly used to aid surgeons in identifying sites of tumor and surgical margins. Of the two types of fluorescent probes, always-on and activatable, activatable probes are preferred because they produce higher target-to-background ratios, thus improving sensitivity compared with always on probes that must contend with considerable background signal. There are two types of activatable probes: 1) enzyme-reactive probes that are normally quenched but can be activated after cleavage by cancer-specific enzymes (activity-based probes) and 2) molecular-binding probes which use cancer targeting moieties such as monoclonal antibodies to target receptors found in abundance on cancers and are activated after internalization and lysosomal processing (binding-based probes). For fluorescence-guided intraoperative surgery, enzyme-reactive probes are superior because they can react quickly, require smaller dosages especially for topical applications, have limited side effects, and have favorable pharmacokinetics. Enzyme-reactive probes are easier to use, fit better into existing work flows in the operating room and have minimal toxicity. Although difficult to prove, it is assumed that the guidance provided to surgeons by these probes results in more effective surgeries with better outcomes for patients. In this review, we compare these two types of activatable fluorescent probes for their ease of use and efficacy.

Graphical abstract

1. Introduction

In vivo medical imaging technologies have seen numerous developments due to advances both in imaging devices and imaging probes.¹ Optical imaging is a type of in vivo imaging that uses fluorescent probes that emit light at a range of wavelengths when excited by light of a lower wavelength. Optical imaging has attracted attention as a non-invasive tool with numerous preclinical and clinical applications for oncologic analysis, including tumor detection, biomarker visualization, and vascular/lymphatic mapping.² A particularly promising application of optical imaging is fluorescence imaging during surgery, which has become one of the most rapidly adopted optical imaging methods.³ Fluorescence imaging has the advantages of using low-cost, easy-to-use, portable equipment, with probes that have a high safety margin, and a high sensitivity for cancer in the picomolar range.^3–7

In cancer surgery, a major goal is to remove tumor as possible while preserving healthy tissues.⁸ Negative tumor margins or the complete resection of tumors is important for improving survival.^9,10 Surgery to resect tumors is largely based on the surgeon’s experience and ability to see anatomical features under the white light conditions of the operating theater.⁸ Due to the low contrast between cancerous and normal tissues, accurately identifying the border between cancer and normal tissues may be difficult with the unaided human eye.^9,11,12 In addition, tiny foci (< 2–3 mm) of cancer may be impossible to spot without the assistance of fluorescence imaging.^11–13

Currently, the gold standard for determining tumor margins is intraoperative frozen section analysis (IFSA). IFSA has several limitations including the requirement for skilled personnel over a prolonged time resulting in increased costs even while the method often is not accurate for positive margins.^{9, 15, 16} It is estimated that IFSA adds approximately 30 to 53 min, to surgical procedures, thus increasing anesthesia-related risks.^8,14 In addition to long processing times and insufficient sensitivity, only limited sampling of tissues is possible, which increases the possibility of false negative results leading to early recurrence.^15–17

A number of imaging methods have been proposed to aid surgery. For instance, intraoperative CT and MRI have played a significant role in the field of neurosurgical surgery.^18,19 However, intraoperative whole body systems are costly, complex, require space, and their use interrupts the normal workflow of the surgical procedure lengthening operative/anesthesia times.

A more promising alternative is intraoperative optical fluorescence imaging, which is a real-time imaging technology that is increasingly used to aid surgeons in identifying surgical margins for tumor resection.^6,12 Because white light cameras are used in many operating rooms and endoscopy suites already, optical fluorescence imaging is easily integrated into the workflow of intraoperative surgery and endoscopy.^6,20,21

Indocyanine green (ICG), methylene blue, and fluorescein are fluorescent probes approved by the United State (Scheme 1).

Scheme 1.

Chemical structures and molecular weight of clinically used fluorophores.

ICG is one of the most frequently employed near-infrared fluorescent probe. Throughout its history, ICG has maintained a high safety index. ²² ICG is routinely used to evaluate hepatic function and clears from tumors such as gliomas at a slightly lower rate than normal tissue.^22–24 When injected into the body, ICG increases fluorescence signal after albumin binding. However, it is difficult to design a targeted fluorescent probe with ICG because ICG loses fluorescence after covalent conjugation with proteins.²²

In addition to ICG, a number of other dyes have been used for intraoperative guidance. Methylene blue is a near-infrared fluorescent agent but it has low quantum yield which hampers its clinical application.²⁵ Fluorescein has been shown to significantly improve resection of gliomas, yet like the others, it is not tumor-specific and can give false-negative or false-positive signals.^26,27 Moreover, its light has minimal tissue penetration in vivo. In Europe, 5-aminolevulinic acid (5-ALA) is an approved probe used to assist in tumor resection and has been shown to improve 6-month progression free survival in patients with malignant gliomas.^28,29 However, low specificity of fluorescent probes at tumor margins introduces error as the agents cannot differentiate tumors from reactive vascularity.^28–30 As the strategy of removing cancerous tissues during surgery faces limitations, it is becoming increasing important to further develop the next generation of fluorescent imaging probes.^2,9,31

There are two types of fluorescent probes used for fluorescent imaging: “always-on” and “smart” or “activatable” probes.¹² Always-on probes continuously emit signal regardless of their relative proximity to or binding with target cells and they, therefore, accumulate both at the target and in background tissue.^32,33 Therefore, using always-on probes produce relatively low target-to-background ratios (TBR), making it more difficult to visualize the tissue of interest. An adequate TBR is only reached after waiting a considerable time for probes in the background to clear, but at the same time, probes bound to the tumor will also begin to clear and produce a lower signal.^21,34 On the other hand, activatable probes remain undetected until they are turned on by specific enzymes or environmental conditions and emit signal, leading to increased contrast and sensitivity: a bright tumor against a dark background (Fig. 1) ^35.36

Fig. 1. Comparison of always on vs activatable imaging with ¹¹¹In/ICG-dual-labeled anti-HER2 antibody.

An always on radionuclide image (center) with ¹¹¹In shows biodistribution of anti-HER2 antibody that accumulates in both specific and non-specific tumors as well as normal tissues. In contrast, an activatable ICG image (right) only shows the HER2+ target tumor with minimum background.

Activatable probes can be widely categorized into 1) enzyme-reactive and 2) molecular-binding probes. Enzyme-reactive probes can be both systemically and topically applied and can remain quenched until cleaved by enzymes that are known to be upregulated in cancer cells.³⁷ Examples of cancer-associated enzymes include cathepsin-B, cathepsin-L,^38,39 matrix metalloproteinases-2 (MMP-2),^40,41 β-galactosidases,^42,43 and γ-glutamyltransferase (GGT) (Fig. 2).⁴⁴

Fig. 2. Serial fluorescent endoscopic images of peritoneal ovarian cancer in mouse using gGlu-HMRG.

gGlu-HMRG probe depicts disseminated SHIN3 ovarian tumors in the peritoneal cavity as early as 30 seconds after spraying. Fluorescence signal of tumors increased as time.

On the other hand, molecular-binding probes which are applied systematically via intravenous injection remain quenched until bound to specific receptors on the target cell, become internalized through endolysomal processing, and then activated by chemical processes within the lysosome.³² Within the lysosome, catabolism can occur under conditions such as low pH, protease activity, or oxidation, which can turn on the fluorescence signal.³²

As the objective of fluorescent imaging is to improve contrast or the TBR,⁴ activatable probes can be considered the future of optical imaging and fluorescence-guided surgery. However, when topically applying fluorescent probes to patients either in the surgical or endoscopic suite, rapid activation is also crucial for activatable probes to be practical. In this review, we will evaluate the translational potential of enzyme-reactive and molecular-binding activatable probes during intraoperative fluorescence-guided surgery.

2. Comparison of Enzyme-reactive and Molecular-binding Activatable Probes

Molecular imaging probes in the U.S. require the same Food and Drug Administration (FDA) approval process as therapeutic drugs, which can take over a decade and cost millions of dollars.⁸ When designing fluorescent activatable probes, their practicality and toxicity must be evaluated, if they are to be translated into actual clinical practice⁴⁵. Unless large amounts of a probe are systemically injected into a patient days ahead of surgery there may not be enough of the probe accumulated in the tumor to be useful during surgery. The probe, injected in the quenched state, must react with sufficient speed to become visible by the time of surgery without too narrow of a window. Needless to say, probes used in patients must not be toxic and should produce limited side effects to pass safety tests set by regulatory bodies.

2.1 Size

In general, size of activatable fluorescent probes distinguishes whether probes are applied topically or systemically. Some enzyme-reactive activatable probes can be designed as small molecules. They are often initially hydrophiliic molecules which are altered to become hydrophobic molecules after enzyme cleavage, which makes topical or local application possible.¹ At the same time, several other enzyme-reactive probes^38,39,46 are large molecular weight molecules, preventing them from being applied topically.⁴⁷ Molecular-binding activatable probes are often large molecular weight probes and must also be systemically injected. They typically consist of three parts: a signaling payload, a carrier, and a targeting moiety in order to effectively deliver to target tumors and specifically bind to target molecules.¹ Monoclonal antibodies are often used as the targeting moiety to specifically bind the probe to cell-surface receptors, which makes the molecular weight of the molecular-binding probes about 150kD.^9,47 Such molecular-binding activatable probes are too large to permeate to deep layers of cells and therefore must be intravenously injected into the body ahead of time of surgery or endoscopic procedure. In fact, large molecular weight enzyme-reactive probes may be similar in size or even larger than molecular-binding probes.

The drastic difference in size leads to different processing mechanisms, reaction speeds, and pharmacokinetics for the different types of activatable probes.

2.2 Speed of Reaction

In the “turned off” state, activatable probes are quenched by one of several mechanisms such as spirocyclization, H- or J-type dimer formation, homo- or hetero-Förster resonance energy transfer (FRET), or photon induced electron transfer (PeT).^8,32 Under environmental conditions unique to each probe, the quenched probe will become activated and fluorescent. For probes quenched with PeT, the transfer of an electron in the acidic lysosome will “turn on” the probe.¹ Probes quenched under spirocyclization only require an enzyme to cleave the quenching ”cage”, and the breakdown of the spirocyclic ring allowing the probe to become fluorescent.^8,33 For probes that have formed dimers or are quenched by FRET, digestive enzymes in the lysosome will “cut” the quenched probes apart and produce activated probes.

For enzyme-reactive activatable probes that are applied topically, only “a single cut” or one enzyme reaction is required to fully activate the signal, making the speed of the reaction is very rapid.¹³ For example, the activatable probe HMRef-βGal is quenched through a spirocyclization strategy and activated after cleavage by the enzyme β-Galactosidase.¹⁴ As the time for the enzyme reaction is on the order of milliseconds, the fluorescence can rapidly become visible. For γ-glutamyl hydroxymethyl rhodamine green (gGlu-HMRG), which is a fluorescent probe activated by the enzyme GGT, small ovarian implants even < 1 mm in size could be detected within just 2 min of the probe application (Fig. 2).⁴⁴ Enzyme-reactive activatable probes require only a single catabolic process after reaching the activation site, resulting in a rapid re-activation.

For probes that are applied systemically, the first step of probe activation is a biological process. In this process, probes circulate through the body after intravenous administration and extravasate into target tissues due to the leaky tumor vasculature, which is known as the enhanced permeability and retention (EPR) effect.^48,49 This biological process requires at least days before adequate amounts of the probe leak out of permeable vasculature, and bind to the target tissue.⁴⁹ After the biological process, large molecular weight enzyme-reactive probes only require the “single cut” or enzyme reaction for activation. However, for molecular-binding probes, a catabolic cascade must follow. This process involves internalization of the antigen-antibody pair and subsequent endolysosomal processing that activates the probe, a process that can take at least 1 hour to achieve an adequate TBR.^32,49 Therefore, combining the biological and catabolic process, sufficient activation of both large molecular weight enzyme-reactive fluorescent probes and molecular-binding activatable fluorescent probes requires days, which impedes the practicality of the probe for real-time imaging on demand.^49,50 Depending on the probe, it may be necessary to inject the probe hours or several days in advance.⁹

Topical application of small molecular weight enzyme-reactive activatable fluorescent probes is straightforward and reduces the logistical issues of intravenous injections that arise when using molecular-binding activatable enzymes. In an intraoperative environment with limited time to determine tumor margins, the more practical probe is the topically-applied enzyme-reactive activatable fluorescent probe with its rapid activation.^49,50

2.3 Dosage

Although dosages for imaging probes are less than the traditional dose used for therapeutic drugs, any potential toxicities in patients must be determined when attempting to bring a new imaging agent into clinical trials and later commercialization.^9,20 Required doses are vastly different between topically (~20 μg/kg) and systemically-applied probes (~4 mg/kg) that are relevant or a little greater than small and large molecular nuclear medicine imaging probes, respectively. ³³ Due to the large volume of distribution, systemically-applied probes require a larger dose in order for an adequate amount of probe to accumulate in the target tissue.⁹ For small molecular weight enzyme-reactive probes, a topical or local application can minimize the overall dose and, therefore, any adverse side effects.⁹ Topical application of enzyme-reactive probes is an appealing choice to limit exposure to the fluorescent probe.

2.4 Toxicity and Clearance

An important issue is the fate of any fluorescent probe that is not bound to the tumor or probes bound to cancerous tissue that are not removed by the operating surgeon.⁵¹ Predicting the elimination pathways of the probes will allow better assessments of potential toxicities, interactions between probes, and pharmacokinetics of the probe.³⁶

Drugs and probes alike are eliminated through liver or renal clearance, or otherwise remain in circulation.³⁶ The small molecular weight enzyme-reactive probes is under the 6 nm diameter renal excretion limit and will be rapidly excreted by the kidney through glomerular filtration.^1,33 Excretion into the urine will occur rapidly in the course of minutes, which will prevent the excess buildup of enzyme-reactive activatable probes in the body.³³ Therefore, even if these probes are not cleaved by the target enzyme and absorbed into the body, they will diffuse into the vasculature, be filtered and excreted through the kidney. While these probes will be excreted rapidly thereby increasing TBR, it is also important to keep in mind that rapid clearance may decrease the absolute accumulation of the probe in the tumor when it is systemically injected.

Compared to small molecules, the clearance of monoclonal antibodies or large molecular weight probes is much slower.⁵² Monoclonal antibodies that are used as moieties for large molecular-binding activatable probes can be are quite large (> 10 nm), easily exceeding the renal filtration limit (~ 6 nm), and therefore will be excreted by alternate routes.^53,54 In fact, the pharmacokinetics and pharmacodynamics of monoclonal antibodies are complex because they involve factors such as antibody structure, size, and charge.^52,55 Monoclonal antibodies and large molecular-weight probes have long circulating half-lives ranging from days to weeks until they become trapped by one of several systems and metabolized into peptides and amino acids.³³ Such systems include slow hepatic excretion in the reticuloendothelial system (RES) in the liver or spleen, target-mediated elimination, and nonspecific endocytosis.^56,57 These large molecular weight probes are more likely to induce allergic reactions during catabolism in the liver compared to small molecular weight probes.

To reduce the amount of time that the imaging probe will remain in the body and limit any toxicity associated with the presence of the probe, enzyme-reactive fluorescent probes should be used topically for intraoperative surgical guidance.

3. Challenges

3.1 Regulatory and Market Issues

The translation of imaging agents into the clinic faces many obstacles including regulations regarding safety and efficacy, market forces, lower profit margins than therapeutic drugs, and lack of reimbursement strategies.³ Imaging agents will likely be required to exhibit the same or higher safety profiles than therapeutic drugs.⁹

For imaging agents that are used only once or twice during surgery compared to therapeutic drugs that are dosed repeatedly, developing a probe which can be used to target multiple cancer types during multiple different procedures can reduce financial risk.^8,9 Each patient may only receive one or two doses of the agent during a lifetime as opposed to a drug that is used every day. Probes that target a general feature found in many types of cancers and probes that target a specific feature on more than one type of cancer cell are both important to develop. However, improving specificity for a specific cancer can significantly reduce market size.⁸ Enzyme-reactive fluorescent probes that are activated by enzymes present in many cancer types may be more likely to succeed in the market.

3.2 Drawbacks of Enzyme-reactive Activatable Probes

While it may seem that topically-applied small molecular weight enzyme-reactive probes are superior to molecular-binding probes in signal amplification, sensitivity, rapid reaction, and lack of side effects compared with systemic injection, there are some drawbacks associated with them.^58,59 During surgery, topical application (i.e. via a spray) of enzyme-reactive probes may only penetrate a fraction of a millimeter into tissue and fail to visualize lesions below the surface.⁹ Probes that are injected systemically can highlight tumor foci deeper below the surface provided that the fluorophore used emits in the near infrared where tissue penetration of light is better. However, enough time must elapse for signal to develop in the target tissue and any nonspecific fluorescence must wash out so the surgeon can see tumors below the surface.^8,9,58 Enzyme-reactive probes may encounter issues with specificity: probes activated by an enzyme on the cell surface may diffuse away from the cell and contribute to background signal.³² Furthermore, probes that target enzymes may be able to activate a wide variety of physiological and pathological conditions including inflammation and neoplasia, leading to lower specificity for tumor compared to molecular-binding probes.³²

There are also weaknesses of fluorescence imaging probes in general, such as dilution encountered when body fluids or other liquids are present in the visual field and the difficulty of determining margins of tumor in organs involved in the excretion of the probes such as the kidney, liver, and the gastrointestinal tract.^9,12,33 Such probes are also temperature sensitive as the enzyme reaction kinetics are governed by temperature..

4. Summary

During surgical resections of tumors, it is crucial to avoid leaving residual tumor which can lead to recurrent disease.¹¹ Fluorescence-guided surgery with activatable fluorescent probes is a promising approach to improve the efficacy of surgical procedures. Two types of activatable fluorescent probes were reviewed: enzyme-reactive and molecular-binding probes. Small molecular weight enzyme-reactive probes rapidly activate and produce signals when they are topically applied, while large molecular weight enzyme-reactive probes and molecular-binding probes may take days to establish an adequate TBR. Topical application of enzyme-reactive probes requires a smaller dose thus, limiting side effects, and allowing for more rapid urinary excretion of the probe, while intravenously injected molecular-binding probes with their long circulating half-life require larger doses, increasing the possibility of systemic side effects. Considering practicality, patient outcomes, and the regulations set by the FDA, enzyme-reactive activatable probes would seem to be preferred to intravenously injected agents, except in specific instances.

Enzyme-reactive activatable fluorescent probes can aid the surgeon or endoscopist in identifying disease with high sensitivity and specificity if used correctly.⁵⁹ For instance, a surgeon or endoscopist might use an enzyme-reactive activatable fluorescent probe by spraying it on a suspicious area during surgery. This would allow him/her to more completely resect the tumor. In the case of ovarian cancer resections improved resections clearly improve the time to recurrence. The use of enzyme-reactive probes may reduce costs by decreasing the need for IFSA thus, improving the accuracy of margin determination.¹⁷ The use of fluorescence-guided surgery represents an important step towards improved cancer surgery, however, bringing such agents to market where they can be widely used will be challenging given the unfavorable economics. Hopefully, the argument can be made that the improvements in outcome more than justify the added costs associated with fluorescence guided surgery.

Abbreviations

ALA

aminolevulinic acid

CT

computerized tomography

EPR

enhanced permeability and retention

FDA

Food and Drug Administration

FRET

Föster (fluorescence) resonance energy transfer

gGlu

γ-glutamyl

GGT

γ-glutamyltransferase

HMRG

hydroxymethyl rhodamine green

ICG

Indocyanine green

ISFA

intraoperative frozen section analysis

MRI

magnetic resonance imaging

PeT

photon induced electron transfer

RES

reticuloendothelial system

TBR

target-to-background ratio