European Journal of Pharmaceutics and Biopharmaceutics 152 (2020) 123–143

Contents lists available at ScienceDirect

European Journal of Pharmaceutics and Biopharmaceutics

journal homepage: www.elsevier.com/locate/ejpb

Quicker, deeper and stronger imaging: A review of tumor-targeted, near- T

infrared fluorescent dyes for fluorescence guided surgery in the preclinical
and clinical stages
⁎
Jianhua Jiaoa,1, Jingliang Zhanga,b,1, Fa Yanga,1, Wei Songa, Donghui Hana, Weihong Wenc, ,
⁎
Weijun Qina,
a
Department of Urology, Xijing Hospital, Fourth Military Medical University, 127 West Changle Road, Xi’an 710032, China
b
Department of Health Services, Health Service Training Base, Fourth Military Medical University, 169 West Changle Road, Xi’an 710032, China
c
Institute of Medical Research, Northwestern Polytechnical University, 127 West Youyi Road, Xi’an 710072, China

A R T I C LE I N FO A B S T R A C T

Keywords: Cancer is a public health problem and the main cause of human mortality and morbidity worldwide. Complete
Targeted removal of tumors and metastatic lymph nodes in surgery is significantly beneficial for the prognosis of patients.
Tumor Tumor-targeted, near-infrared fluorescent (NIRF) imaging is an emerging field of real-time intraoperative cancer
Fluorescent imaging based on tumor-targeted NIRF dyes. Targeted NIRF dyes contain NIRF fluorophores and specific binding
Dye
ligands such as antibodies, peptides and small molecules. The present article reviews recently updated tumor-
Intraoperative fluorescence imaging (IFI)
targeted NIRF dyes for the molecular imaging of malignant tumors in the preclinical stage and clinical trials. The
Fluorescence-guided surgery (FGS)
Near infrared (NIR) strengths and challenges of NIRF agents with tumor-targeting ability are also summarized. Smaller ligands, near
infrared II dyes, dual-modality dyes and activatable dyes may contribute to quicker, deeper, stronger imaging in
the nearest future. In this review, we highlighted tumor-targeted NIRF dyes for fluorescence-guided surgery and
their potential clinical translation.

1. Introduction surgical interventions to resect all neoplasms—this requires resection of

all micro tumor nodules and tumor involved lymph nodes to obtain
According to the American Cancer Society, an estimation of 18.1 negative margins on the final pathology specimen. Positive surgical
million new cancer cases and 9.6 million cancer deaths occurred margins (PSMs) after surgical resections dominate the long-term clin-
worldwide in 2018 [1,2]. Most cancer patients still rely heavily on ical outcomes in almost all cancer types, such as rectal cancer, breast

Abbreviations: IMI, intraoperative molecular imaging; NIR, Near-infrared; NIRF, Near-infrared Fluorescent; IFI, Intraoperative fluorescence imaging; PSMs, Positive
surgical margins; NSCLC, non-small cell lung cancer; UA, ultrasound; CT, computed tomography; MRI, magnetic resonance imaging; PET, positron emission to-
mography; FGS, fluorescence guided surgery; SBR, signal-to-background ratio; ICG, Indocyanine green; EPR, enhanced permeability and retention; SPECT, single
photon emission computed tomography; ScFv, single chain antibody fragment; FRα, Folate receptor-α; FRβ, Folate receptor-β; RA, rheumatoid arthritis; IBD,
inflammatory bowel disease; PSMA, Prostate-specific membrane antigen; EGFR/ErbB-1/HER1, Human epidermal growth factor receptor; VEGF, human vascular
endothelial growth factor; FME, fluorescence molecular endoscopy; EAC, esophageal adenocarcinoma; AFTN, Afatinib; ERα, Estrogen receptor-alpha; CAIX, Carbonic
anhydrase IX; CCK2R, Cholecystokinin 2 receptor; CEA, Carcinoembryonic antigen; ssScFv, stabilized single-chain antibody fragment; PDAC, pancreatic ductal
cancer; HNSCC, head and neck squamous cancer; LPA, lysophosphatidic acid; RGD, arginine-glycine-aspartic acid; CTX, Chlorotoxin; MMP, Metalloproteinase; GRPR,
Gastrin-releasing peptide receptor; ER, endoplasmic reticulum; ROS, reactive oxygen species; LDLR, Low-density lipoprotein receptor; FGFR, fibroblast growth factor
receptor; uPAR, Urokinase-type plasminogen activator receptor; δOR, delta-opioid receptor; HGC, hydrophobically modified glycol chitosan; CA, glycyrrhetinic acid;
MBs, micro-bubbles; ANNA, N-carboxyhexyl derivative of 3-amino-1,2,4-triazole-fused 1,8-naphthalimide; GGT, Gamma-glutamyl transpeptidase; cPLA2, Cytosolic
phospholipase A2; pHLIPs, pH low insertion peptides; HCC, hepatocellular carcinoma; IHC, immunohistochemistry; QDs, quantum dots; In, indium; TROP-2, epi-
thelial glycoprotein-1; HSG, histamine–succinyl–glycine; PD-1, programmed cell death protein 1; PSCA, prostate stem cell antigen; MT1-MMP, membrane type 1
metalloproteinase; 800CW, IRDye800CW; λex/em, excitation wavelength/emission wavelength; EOC, epithelial ovarian cancer; NR, not reported; TER, tumor-
specific excretion retarded; TF, Thomsen-Friedenreich; TAG, Tumor-associated glycoprotein
⁎
Corresponding authors.
E-mail addresses: wenweih@fmmu.edu.cn (W. Wen), qinwj@fmmu.edu.cn (W. Qin).
1
These authors contributed equally to this work.

https://doi.org/10.1016/j.ejpb.2020.05.002
Received 13 October 2019; Received in revised form 3 May 2020; Accepted 3 May 2020
Available online 08 May 2020
0939-6411/ © 2020 Elsevier B.V. All rights reserved.
J. Jiao, et al. European Journal of Pharmaceutics and Biopharmaceutics 152 (2020) 123–143

cancer, non-small cell lung cancer (NSCLC), extrahepatic bile duct review: antibody conjugates, peptide conjugates and small molecule
cancer, prostate cancer, bladder cancer, cervical cancer, and pancreatic conjugates (Fig. 2E–G). Three strategies were shown to improve the
cancer [3–13]. Identification of sentinel lymph nodes and removal of all performance of targeted dyes (Fig. 2B–D). The first idea was to replace
lymph node metastases also greatly benefit the survival of the patients. the NIR I dye with a NIR II dye for deeper penetration ability and less
In hence, according to various surgical guidelines, surgeons have to light scattering. The second way was to add another probe such as
perform a histopathological examination by frozen section in- radiotracers to the structure to build a dual-modality dye for both NIR
traoperatively to determine the extent of surgical resection [14–16]. fluorescent imaging and ultrasound/CT/SPECT (single photon emission
However, the biopsies, which are low efficient and time consuming in computed tomography)/PET examination. The last strategy was “turn
the operating rooms, can interrupt the normal procedures of the sur- on/off” (activatable) method. The quenched targeted dyes can only be
geries and prolong the whole time of surgeries. Although imaging activated by specific enzymes expressed on cancer cells or low pH in the
techniques, such as X-ray, ultrasound(UA), computed tomography (CT), microenvironment of malignancies. As a result, signal-to-background
magnetic resonance imaging (MRI), and positron emission tomography ratio (SBR) can largely increase because of less auto-fluorescence in
(PET) have made rapid progress to locate tumors, intraoperative mo- background of blood or muscles.
lecular imaging offers a much more intuitive feeling to locate tumors in We summarized the structures of the commercialized dyes applied
fluorescence guided surgery (FGS) [17]. in tumor-targeted NIRF dyes (Fig. 3A). The structure-functional con-
Molecular imaging, first named by Weissleder R in 1999, offered an nections of the dyes vary a lot in different targeted dyes and we listed
excellent method of FGS which can provide real-time visualization of some interesting structures (Fig. 3B–D). An amide bond is the most
tumors in intraoperative imaging-guided surgery [18]. Near infrared commonly used connection in tumor-targeted NIRF dyes. The EDC/NHS
fluorescent (NIRF) light provides the most promising method of real- reaction is the most widely applied reaction to build this bond in tar-
time surgical navigation of FGS. Compared with traditional molecular geted dyes (Fig. 3B). Carboxylates (eCOOH) can be reacted to NHS or
imaging, optical imaging in the NIRF light spectrum (wavelength, Sulfo-NHS in the presence of a carbodiimide such as EDC, leading to a
λ greater than 650 nm) has four advantages: increased tissue penetra- semi-stable NHS or Sulfo-NHS easter, which may then be reacted with
tion of light, less scattering, increased signal-to-background ratio (SBR) primary amines (eNH2) to form amide crosslinks by a two-step reac-
of contrast agents and no radioactivity [19].With the development of tion. The commercial NIR dyes such as IRDye800CW NHS Ester from LI-
intraoperative molecular imaging (IMI), malignancies that were once COR Biosciences were reacted with a primary aliphatic amine such as
difficult to identify by visualization or palpation can be easily revealed lysine to create an amide bond. The condition of NHS reaction was in
by FGS. Moreover, the detection rate of lymph node metastases even phosphate-buffered saline (PBS) and room temperature. Hydrophilic
doubled with the help of IMI of FGS [20]. ability and hydrophobic ability should also be balanced in the targeted
Indocyanine green (ICG) was the first approved near-infrared (NIR) dyes. High hydrophilic dyes such as ICG can easily be excreted out and
fluorophore by the FDA for FGS in human [21,22]. However, due to the fail to provide long time for molecular imaging of FGS. High hydro-
relatively low specificity of ‘enhanced permeability and retention’(EPR) phobic dyes can hardly be dissolved in blood and circulated to targeted
effect, nontargeted NIR dyes such as ICG cannot locate the malignant tumor in human body. In addition, the example structures of a pH ac-
lesions accurately [23,24]. Beyond EPR effect, tumor-targeted NIRF tivated NIRF dye and a small molecular integrin αvβ3 targeted NIRF
probes delineate the margins of malignances by unique recognition of dye were shown in Fig. 3C-D.
specific biomarkers highly expressed on tumor cells. Therefore, tumor- In an ideal process of FGS, the targeted dyes for specific cancer need
targeted NIRF probes, namely conjugations of specific ligands such as incubation time to be circulated and bind biomarkers on cancer cells.
antibodies, peptides or small molecular analogs with NIR fluorophores, The surgeons resect all the tissues with fluorescence and confirmed no
proved to be a pivotal factor in FGS. NIR light spectrum can normally be fluorescence was left. The resected tissues were then confirmed by the
divided into NIR I (650 nm ~ 900 nm) spectrum and NIR II spectrum pathologists by hematoxylin and eosin staining (HE), im-
(1000 nm ~ 1700 nm). Most studies of NIR II dyes were still in their munohistochemistry (IHC) or directly under fluorescence microscope.
infancy and focused on material synthesis and reducing in-vivo toxicity. An ideal process of FGS was shown in Fig. S1.
As a result, the targeted dyes concluded in this review was mainly in
NIR I spectrum. 3. Quicker:Smaller ligands contribute to quicker imaging
In recent decade, many tumor-targeted NIRF dyes has been in
clinical trials and more tumor-targeted NIRF dyes show great potential Tumor-targeted NIRF dyes with smaller-size ligands contribute to
in clinical translation. In this review, we focused on tumor-targeted quicker imaging (Fig. 4). Quicker imaging of tumors and quicker
NIRF probes for molecular imaging in clinical trials and preclinical clearance of free NIRF dyes which largely depend on the size of ligands
stage, strategies to ameliorate them, their latest advances and potentials are both wanted to improve SBR of a targeted NIRF probe. According to
for clinical translation. As shown in Fig. 1, the imaging processes with the sizes of ligands in tumor-targeted NIRF agents, we normally divided
tumor-targeted NIRF dyes are becoming quicker, deeper and stronger: them into three different groups: antibody conjugates, peptide con-
smaller ligands contribute to quicker imaging; NIR II dyes and dual- jugates and small molecule conjugates. Antibody conjugates include full
modality dyes contribute to deeper imaging; “turn on/off (activatable) antibody and antibody fragments such as minibody, centyrin, affibody,
method” contributes to stronger imaging with higher SBR. Besides nanobody and single chain antibody fragment (ScFv). Theoretically, the
molecular imaging, NIR dyes can also be used for NIR photothermal ligands can be randomly conjugated to NIRF dyes, as long as the
therapy and drug release system. The tumor-targeted NIRF dyes re- structures of compounds is stable. Therefore, once mature commercial
viewed in the paper are mainly for molecular imaging so targeted dyes NIR fluorescent dyes such as IRDye800CW and Alexa dyes were in-
for photothermal therapy and drug release system are not included in troduced, tumor-targeted NIRF dyes explosively appeared. In hence,
the scope of the review. based on various cancer biomarkers, three groups of conjugates (anti-
body conjugates, peptide conjugates, small molecule conjugates) are
2. The structures and interesting linkers of tumor-targeted NIRF concluded as follows and the example tumor-targeted NIRF dyes are
dyes shown in Fig. 4.

Traditional tumor-targeted NIRF dye contains a ligand which tar- 3.1. Antibody conjugates
gets biomarkers on cancer cells, a NIRF dye which emits near infrared
light and a linker to connect two parts above (Fig. 2A). The targeted 3.1.1. Antibody conjugates in clinical trial stage- VEGF
dyes were divided into three groups according to the ligands in this Bevacizumab, an therapeutic antibody of human vascular

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Fig. 1. The current and future developing trend of tumor-targeted NIRF dyes: quicker, deeper and stronger imaging. Smaller-size ligands facilitate quicker imaging
and quicker clearance of free targeted dyes through urine excretion. The dyes under 40KDa can be cleared through kidney while most probes whose size larger than
40KDa were cleared by liver [106]. NIR II dyes (λem = 1000–1500 nm) such as CH1055 provide deeper penetration by longer wavelength [106] . ER3 + -doped was
developed as a NIR II b dye (λem = 1500–1700 nm) [146]. The“Turn on/off (Activatable) method” contributes to higher stronger imaging with higher SBR.

endothelial growth factor (VEGF) approved by FDA in 2004, has been esophageal cancer, pancreatic cancer, sarcoma inverted papilloma,
widely applied to the treatment of cancer patients for colorectal cancer, Barrett esophagus, endometriosis were unfinished and results were still
NSCLC, breast cancer, renal cancer, and glioblastoma [25–29]. Bev- undeclared (Table 2, NCT02113202, NCT03620292, NCT03558724,
acizumab-IDRye800CW, with its TBR of 1.93 ± 0.40, was used for NCT02743975, NCT03913806, NCT03925285, NCT03877601,
intraoperative NIR fluorescence imaging at the submillimeter level in a NCT02975219). The NIRF endoscopy platform was reported to be used
tumor-bearing mouse model of A2780 ovarian cancer cells [30] as devices in four of these clinical trials (Table 2, NCT01972373,
(Table 1). University of Groningen leaded the clinical trials of Bev- NCT02113202, NCT03558724, NCT03877601).
acizumab–IDRye800CW of FGS. Laetitia E. Lamberts and colleagues
applied Bevacizumab–IDRye800CW in a phase I clinical study to con-
firm its safety for breast cancer surgical guidance using a systematic 3.1.2. Potential antibody conjugates in preclinical stage
validation methodology (Table 2, NCT01508572)[31]. Furthermore, More antibody conjugates were invented and tested in tumor-
Bevacizumab–IDRye800CW was also used for the FGS of colorectal bearing animal models and some of them may have the potential to
peritoneal metastases in 7 patients when 53% fluorescent tissues were enter clinical trials. Anti-CEA-IRDye800CW, a humanized antibody-dye
found to be tumor (Netherlands trial registry, NTR4632)[32]. The conjugate for Carcinoembryonic antigen (CEA), was synthesized to lo-
phrase I clinical trial in rectal cancer showed that Bevacizumab- cate tumors by laparotomy in tumor-bearing nude mice [34]. Two
IDRye800CW enabled a clear differentiation between tumor and tumor-specific NIRF dyes, Carbonic anhydrase IX (CAIX)-specific
normal tissues with a tumor-to-background ratio of 4.7 (Table 2, CA9Ab-680 and CA12Ab-680 (Table 1), synthesized for the noninvasive
NCT01972373)[33]. The phrase I clinical trials in esophageal cancer of detection of breast cancer lymph node metastasis [35] may has the
dysplasia, adenomatous polyposis coli, hilar cholangiocarcinoma, potential for clinical translation because another CAIX-targeted dual-
modality dye has already been in clinical trial (Table 2, NCT02497599).

Fig. 2. Structures of tumor-targeted NIRF dyes.

(A) Basic structural model of tumor-targeted
near-infrared fluorescent dye. (B) Tumor-tar-
geted NIR II fluorescent dye: NIR I dye was re-
placed by NIR II dye. (C) Model of dual-mod-
ality, tumor-targeted NIR fluorescent probes:
another probe such as 111In or nanogold was
attached to traditional structure. (D) Model of
“turn on/turn off” (activatable) dyes: the quen-
ched dyes can be activated by low pH or en-
zymes in the microenvironment of tumors. (E-G):
the tumor-targeted NIRF dyes can be divided
into antibody conjugates, peptide conjugates,
small molecule conjugates according to the type
of ligands.

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Fig. 3. Structures of dyes applied in tumor-targeted near-infrared fluorescent dyes. (A) Structures of near-infrared fluorescent dyes that were reported. (B) A sketch
map of NHS reaction. (C) The structure of a pH activated NIRF dye: indole intermediate 2[57]. (D) VivoTag S680: a small molecular integrin αvβ3 targeted NIRF dye
[54,55]. The red arrowhead points out the NHS Ester in IRDye800CW to react with an aliphatic amine such as lysine in antibodies or antibody fragments. (For
interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

FGS with PD-1-800CW was first displayed in NIRF-guided tumor re- (tumor paint/ tozuleristide) was the first CTX-targeted NIRF probe in
section (Table 1)[36]. Anti-huCC49-800CW for tumor-associated gly- clinical trials. After testing its efficiency in human glioma-bearing mice
coprotein (TAG)-72 was used to illuminate colon tumor with a mean [42] (Table 3), BLZ-100 was then administered before surgery to dogs
tumor-to-liver ratio of 7.39 metastatic mouse models [37] (Table 1). with various naturally occurring spontaneous tumors and achieved its
Most antibody conjugates were based on therapeutic monoclonal full highest TBR in soft tissue sarcomas [43] (Table 3/4). BLZ-100 was then
antibody and may need more incubation time for imaging. evaluated in models of HNSCC and oral dysplasia in NSG mice with a
Besides full antibody, antibody fragments were also introduced to mean SBR of 2.51[44] (Table 3/4). After evaluation of its imaging,
synthesize antibody conjugates. SsSM3E, a disulfide-stabilized single- safety pharmacology, PK, and potential toxicology in human [45], BLZ-
chain antibody fragment (ssScFv), was conjugated to IRDye800CW to 100 was then evaluated in a phase I clinical trial of 23 breast cancer
display colorectal and pancreatic tumors with a TBR of 5.1 ± 0.6 at patients for visual real-time distinction between pathologically con-
72 h post injection (Table 1)[38]. A11 Mb-800CW, a minibody con- firmed breast cancer and normal tissues(Table 4, NCT02496065) [46]
jugate for prostate stem antigen (PSCA), was developed for FGS and (Table 3/4). Phase I study of BLZ-100 in glioma showed the safety of
validated in human PSCA transgenic mice [39] (Table 1). EpCAM-F800 BLZ-100 can be used for FGS of glioma with a safe dose up to 30 mg
including a Fab fragment from antibody MOC31 was invented for in- (Fig. 4C,D, Table 4, NCT02234297) [47]. BLZ-100 was also used for
traoperative imaging for colorectal cancer [40] (Table 1). Compared FGS of soft tissue sarcoma, skin neoplasm and pediatric central nervous
with full antibody conjugates, the smaller dyes including antibody system tumors in clinical trials (Table 4, NCT02464332, NCT02462629,
fragments can circulate to the targets in the shorter time. However, NCT02097875, NCT03579602). The results of other clinical trials have
more full antibody conjugates were under the evaluation of clinical not been declared.
trials because of relatively mature application of therapeutic antibodies Gastrin-releasing peptide is a regulatory peptide that functions
(Table 2). through the gastrin-releasing peptide receptor (GRPR) that is over-
expressed in various cancers [48]. Annie A. et al reported that they
designed, synthesized and tested a GRPR targeted peptide, TM1, and
3.2. Peptide conjugates
tested a targeted NIRF dye, TM1-IR680, in a murine orthotopic model
of oral cancer [49] (Table 3). Another GRPR targeted NIRF dye Bom-
3.2.1. Peptide conjugates in clinical trial stage- CTX/ GRPR
besin-like peptide (BBN)-Cy5.5 was also tested to resect prostate cancer
Chlorotoxin (CTX), isolated from scorpion venom, is a 36-amino
at 24 h after injection [50] (Table 3). The same ligand, BBN, was also
acid peptide with four disulfide bridges binds to Matrix metalloprotei-
used as a ligand in a dual-modality dye, 68Ga-BBN-IRDye800CW, to
nase-2 (MMP-2) and membrane type-I MMP [41]. In mouse models of
illuminate glioblastoma (Table 4, NCT02910804).
malignant glioma, medulloblastoma, prostate cancer, intestinal cancer,
and sarcoma, CTX-Cy5.5 bioconjugate was shown to delineate the
malignant tumors from adjacent nonneoplastic tissues [41]. BLZ-100

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Fig. 4. Representative figures from the imaging

process with the presented antibody conjugates,
peptide conjugates and small molecule con-
jugates in clinical trial stage. (A, B) In Situ NIRF
imaging with an antibody conjugate, panitu-
mumab-IRDye800CW, in FGS for head and neck
cancer. (A) Brightfield and (B) NIRF images
[124]. This research was originally published in
JNM. Stan van Keulen et al. The Clinical Appli-
cation of Fluorescence-Guided Surgery in Head
and Neck Cancer. J Nucl Med. 2019;
60:758–763. © SNMMI. (C, D) In Situ NIRF
imaging with a peptide conjugate, Tozuleristide
(BLZ-100), in FGS for gliomas. (C) Brightfield
and (D) NIRF merge images [47]. The figures are
used under the permission of Copyright © 2019,
Oxford University Press. (E, F) In Situ NIRF
imaging with a small molecule conjugate,
OTL38, in FGS for pulmonary adenocarcinomas.
(E) Brightfield and (F) NIRF merge images [81].
The figures are used under the permission of
Copyright © 2018 The Society of Thoracic Sur-
geons Published by Elsevier. Yellow circle marks
margins. Panitumumab-IRDye800CW was in-
travenously injected 1–5 days before surgery
[124]. Tozuleristide (BLZ-100) was administered
as a slow intravenous bolus injection 3 to 29 h
before surgery [47]. OTL38 was intravenously
injected 3 to 6 h before surgery [81]. Tumor
targeted NIRF dyes with smaller ligands can
contribute to quicker imaging in FGS. (For in-
terpretation of the references to color in this
figure legend, the reader is referred to the web
version of this article.)

3.2.2. Potential peptide conjugates in preclinical stage applied for intraoperative NIRF imaging in a syngeneic rat model of
Integrin αvβ3, a biomarker that is strongly expressed in most bone colorectal metastases [54] (Table 6). IntegriSense680 was also applied
metastatic cancer cells, has been a therapeutic target in metastatic as intraoperative imaging in the FGS of ovarian cancer with a diagnostic
breast cancer patients [51]. In 2010, the first integrin αvβ3-targeted accuracy of 96.5% [55] (Table 6). Furthermore, IntegriSense750, which
NIRF probe, RAFTc (RGDfK)4-Alexa Fluor® 700, was designed for FGS has a shorter half-life period in tissues than IntegriSense680, was in-
[20] (Table 3). Another αvβ3-targeted NIRF probe containing two ar- vented to precisely identify in situ tumor nodes during FGS [56]. In the
ginine-glycine-aspartic acid units can exhibit increased fluorescence at same experiment, ProSense750EX and ProSense750FAST, which can be
720 nm when it binds to lysophosphatidic acid (LPA) [52] (Table 3). activated by cathepsin were also effective for optical imaging [56]
For metastases, RGD-ICG, was used to improve the quality of cytor- (Table 6). Besides, another integrin-targeted pH-sensitive NIR probe
eduction surgery for peritoneal carcinomatosis from gastric cancer [53] was also designed and synthesized by combining a pH-sensitive cyanine
(Table 7). Small molecule conjugates were also introduced. In- probe with an arginine-glycine-aspartic acid (cRGD) peptide-specific
tegriSense680 includes a small nonpeptide integrin αvβ3 antagonist and αvβ3 integrin [57] (Table 7). In addition to integrin αvβ3, a novel in-
an NIRF dye, VivoTag®-S680 (VisEn Medical). IntegriSense680 was tegrin αvβ6-targeted NIRF dye, R01-MG-IRDye800, was designed for

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Table 1
Tumor-targeted NIRF dyes in preclinical stage (antibody conjugates).
Contrast agent Ligand Target Fluorophore λex/em(nm) Cancer type Refs

Bevacizumab–800CW Bevacizumab VEGF IRDye800CW 778/796 Ovarian cancer [30]

Trastuzumab–800CW Trastuzumab HER2 IRDye800CW 778/796 Breast /Gastric cancer [30]
CA9Ab-680 CAIX mono-antibody (303123) HER2 VivoTag-S 680 673/691 Breast cancer [35]
CA12Ab-680 CAXII mono-antibody (315602) HER2 VivoTag-S 680 673/691 Breast cancer [35]
7D12-800CW nanobody 7D12 EGFR IRDye800CW 778/795 Tongue tumor [129]
ssSM3E-800CW ssSM3E (ssScFv) CEA IRDye800CW 773/792 Colorectal/pancreatic cancer [38]
cetuximab-800CW cetuximab EGFR IRDye800CW 778/796 HNSCC [120]
Cetux-Alexa647 cetuximab EGFR Alexa-647 635/670 Pancreatic ductal cancer [122]
Ag2Se–cetuximab cetuximab EGFR Ag2Se Qts 720/930 Orthotopic tongue cancer [123]
PD-1-800CW PD-1 monoclonal antibody PD-1 IRDye800CW 778/832 Breast cancer [36]
anti-CEA-800CW CEA antibody(hT84.55-M5A) CEA IRDye800CW 778/795 Colorectal cancer [154]
Centyrin-S0456 Centyrin(83v2Cys) EGFR S0456 770/790 NSCLC [130]
A11 Mb-800CW A11 Mb PSCA IRDye800CW 778/789 Prostate Cancer [39]
EpCAM-F800 Fab from antibody MOC31 EpCAM IRDye800CW 778/795 Colorectal/breast cancer [40]
anti-huCC49-800CW anti-huCC49 TAG-72 IRDye800CW 778/796 Colorectal cancer [37]
anti-HER2-MnCuInS/ZnS anti-HER2 HER2 MnCuInS/ZnS 561/741 Breast cancer [136]

NIRF-guided surgery of pancreatic ductal cancer (PDAC) [58] (Table 3). may not be a good target for targeted NIRF dyes.
The TBR of R01-MG-IRDye800 in the tumors of transgenic mice was
3.6 ± 0.94[58]. Meanwhile, another integrin αvβ6 targeted NIRF 3.3. Small molecule conjugates
probe, IRDye800-PEG28-A20FMDV2-K16R-PEG28, was used for ima-
ging of orthotopic pancreatic tumor model [59] (Table 3). LS301, a 3.3.1. Small molecule conjugates in clinical trial stage- FRα
peptide-based NIRF probe that targeted unspecified integrins, was Folate receptor-α (FRα), a member of the folate receptor (FOLR)
conjugated with Cy7.5 for targeted NIR laser colonoscopy in early de- family, which has a high affinity for folate, is overexpressed in many
tection and endoscopic resection [60] (Table 3). Compared with other cancers such as ovarian cancer, lung cancer, breast cancer, colorectal
peptide conjugates in preclinical stage, more studies focused on in- cancer and renal cancer [73–76]. In 2011, conjugation of folate and
tegrins, which may greatly facilitate their clinical translation and trials. fluorescein isothiocyanate (FITC, λexcitation/λemission, λex/
Besides integrins, various receptors were also used to invent tumor- λem = 470/520 nm), termed folate-FITC (EC17), made a breakthrough
targeted NIRF dyes. The first receptor is urokinase-type plasminogen as a tumor-specific agent in the cytoreductive surgery of ovarian cancer
activator receptor (uPAR). For uPAR, ICG-Glu-GluAE105 was tested in patients [77]. However, due to fluorescence in the visible light spec-
human glioblastoma xenograft mice [61] (Table 3). One year later, trum, the major shortcomings of EC17 were its poor depth of penetra-
Karina Juhl and his colleagues demonstrated the feasibility of com- tion and autofluorescence from nearby tissues [78]. To overcome this,
bining ICG-Glu-Glu-AE105 (fluorescent agent) and 64Cu-DOTA-AE105 in OLT38, FITC was then replaced by S0456, an NIRF dye (λex/
(PET agent) together to localize their signal in an orthotopic xenograft em = 776/796 nm) [79]. In an experimental comparison of these two
model with a mean TBR of 2.5 in vivo [62] (Table 3). Another receptor fluorescent agents of EC17 and OTL38, OTL38 appeared to have better
is delta-opioid receptor (δOR), a member of the G-protein-coupled re- sensitivity and brightness, namely, superior clinical efficacy [78]. Due
ceptor family [63]. Huynh, A. S. et al designed a δOR-targeted fluor- to the high affinity for FRα + cancers and quick clearance from FRα–
escent imaging probe based on Dmt-Tic (a synthetic peptide antagonist) tissues, OTL38 was used to identify malignant lesions in a couple of
conjugated to a Cy5 fluorescent dye [64] (Table 3). Furthermore, the hours after intravenous injection [80]. As a result, OTL38 has already
same research team developed a new δOR-targeted fluorescent imaging been tested in intraoperative NIRF imaging-guided surgeries of pul-
probe based on Dmt-Tic conjugated IRDye800CW [65] (Table 3). The monary adenocarcinoma, renal carcinoma, and ovarian cancer [81–84].
third receptor is low-density lipoprotein receptor (LDLR), where pep- OTL38 was also practiced in phase I/II trials to investigate its efficacy,
tide-22 showed good potential to bind [66] (Table 3). Peptide-22-Cy7 safety, and clinical value for patients of lung cancer (Fig. 4 E-F)[81],
was evaluated to guide surgical navigation to achieve R0 resection of pituitary neoplasms [85], bladder cancer, gastric cancer and renal cell
orthotopic pancreatic cancer of the mice [67]. The fourth receptor is carcinoma (Table 5, NCT02872701, NCT02317705, NCT02629549,
fibroblast growth factor receptor 2 (FGFR2) which belongs to FGFR NCT02769156, NCT02769533, NCT02852252, NCT02645409). After
family that includes glycoproteins situated at the cell surface and a the test of optimal dosage and time window of IMI, OLT38 was used to
cytoplasmic domain [68]. A FGFR2-targeted NIRF agent comprising a remove an additional 29% more of the malignant lesions than only
synthesized peptide SRRPASFRTARE(SRR) and Cy5.5 were validated in inspection and palpation in 12 patients with ovarian cancer [83].
esophageal squamous cell carcinoma ex vivo [69] (Table 3). In addition Furthermore, Jarrod D. Predina and colleagues showed that up to 80%
to receptors, some other biomarkers were also used to invent targeted pulmonary adenocarcinoma and 70% squamous cell carcinomas (SCCs)
NIRF dyes. An NIRF light-up probe DBT-2EEGIHGHHIISVG was de- can be detected in FGS by OLT38 (Table 5, NCT02602119)[81,86].
signed for lysosomal protein transmembrane 4 beta (LAPTM4B) pro- They also showed that the sensitivity of OTL38(95.6%) was better than
teins in hepatocellular tumor-bearing mice [70]. A targeted agent, that of PET (73.5%) and utilization of OTL38 greatly improved the
Cy5.5-MT1-AF7p-H4R, including a nonsubstrate peptide (MT1-AF7p) detection rate of lung cancer nodules [87]. A larger, multicenter phase
was synthesized to bind to the “MT-Loop” region of MT1-MMP which is II study claimed that sensitivity of OTL38 was estimated at 97.97% in
an essential regulator of tumor invasion, growth and angiogenesis [71] 44 ovarian patients [88]. Now OTL38 has been in Phase 3 clinical trial
(Table 3). A38C-alexa647 was invented to recognize Thomsen-Frie- of FRα positive ovarian cancer patients (Table 5, NCT03180307) and
denreich (TF) antigen for intraoperative detection of submillimeter the result was not disclosed. In total, OTL38 has the potential to be the
nodules in an ovarian peritoneal carcinomatosis mouse model [72] first tumor-targeted NIRF dye to be widely applied in clinical surgeries.
(Table 3). Although many peptide conjugates were in their preclinical Nevertheless, inevitable OTL38 uptake of folate receptor-β (FRβ) in
stage, just a few of them may enter clinical trials. More studies still need cancer and stromal cells interference the signal from FRα while sa-
to focus on the biomarkers with excellent accuracy and reliability. The tisfying OTL38 signals can hardly be obtained from low FRα expressing
biomarkers such as MMP, which can be secreted to extracellular region, patients [89]. Based on the hypothesis that OTL38 may accumulate in

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Table 2
Tumor-targeted NIRF dyes in clinical trials (antibody conjugates).
Contrast agent Ligand Target Fluorophore ClinicalTrials.gov Condition or disease Start date Complete date Phase Actual Enrollment Locations
identifier (participants)

Bevacizumab-IRDye800CW/ Bevacizumab VEGF IRDye800CW NCT01508572 Breast Cancer 10/01/11 01/01/15 Phase 1 20 University Medical
Bevacizumab-800CW NCT02583568 Breast Cancer 10/01/15 02/01/17 Phase 2 26 Center Groningen
NCT01972373 Rectal Cancer 10/01/13 01/01/17 Phase 1 30
NCT02129933 Esophageal cancer or dysplasia 04/01/14 11/01/16 Phase 1 14
NCT02113202 Adenomatous Polyposis Coli 03/01/14 10/01/15 Phase 1 17
NCT03620292 Hilar Cholangiocarcinoma 10/01/18 01/01/20 Phase 1/2 12
NCT03558724 Esophageal Cancer 10/29/18 12/31/19 Phase 1 30
NCT02743975 Pancreatic Cancer 09/01/16 03/01/18 Phase 1/2 26
Cetuximab-IRDye800CW Cetuximab EGFR/ErbB-1/ IRDye800CW NCT03134846 Head and Neck Squamous Cell 12/16/17 01/01/21 Phase 1/2 79
HER1 Carcinoma
NCT02736578 Pancreatic Adenocarcinoma 07/01/16 05/22/17 Phase 2 8 Stanford University
NCT02855086 Brain Neoplasm /Malignant Glioma 10/01/16 11/22/16 Phase 1/2 3

129
NCT01987375 Head and Neck Cancer 11/01/15 04/19/17 Phase 1 1
Panitumumab-IRDye800CW Panitumumab EGFR IRDye800CW NCT02415881 Head and Neck Cancer 11/01/15 06/01/20 Phase 1 23
NCT03405142 Head and Neck Squamous Cell 07/16/18 07/13/20 Phase 2 20
Carcinoma
NCT03582124 Lung Carcinoma /Metastatic Malignant 07/19/18 06/30/21 Phase 1/2 30
Neoplasm in the Lung
NCT03384238 Pancreatic Adenocarcinoma 02/07/18 02/07/22 Phase 1/2 24
NCT03510208 Malignant Brain Neoplasm/Malignant 05/14/18 05/14/22 Phase 1/2 22
Glioma
NCT03733210 Squamous Cell Carcinoma of the Head 01/07/19 02/07/22 Phase 1 14
and Neck/Carcinoma of the Head and
Neck
ProstaFluor HuJ591 PSMA IDRye800CW NCT01173146 Prostate Cancer 12/01/11 06/01/15 Phase 1/2 —— Spectros Corporation
anti-PSMA monoclonal MDX1201 PSMA A488 NCT02048150 Adenocarcinoma of the Prostate(Stage 03/05/15 01/01/19 Phase 1 20 City of Hope Medical
antibody MDX1201-A488 IIB/III /IV) Center
111In-DOTA-Girentuximab- Girentuximab carbonic IDRye800CW NCT02497599 renal cell carcinoma 06/01/15 04/01/19 Phase 1 30 Radboud University
IRDye800CW anhydrase IX
European Journal of Pharmaceutics and Biopharmaceutics 152 (2020) 123–143
J. Jiao, et al. European Journal of Pharmaceutics and Biopharmaceutics 152 (2020) 123–143

Table 3
Tumor-targeted NIRF dyes in preclinical stage (peptide conjugates).
Contrast agent Ligand Target Fluorophore λex/em Cancer type Refs
(nm)

RAFTc(RGDfK)4-Alexa Fluor® 700 RGDfK peptides Integrin αvβ3 Alexa Fluor 700 702/723 Peritoneal carcinomatosi [20]
PSMA-1–800CW PSMA-1 PSMA IRDye800CW 778/832 Prostate cancer [107]
PSMA-1–Cy5.5 PSMA-1 PSMA Cy5.5 678/695 Prostate cancer [107]
BLZ-100 chlorotoxin (CTX) peptide MMP-2 ICG 785/813 Giloma [42]
BLZ-100 CTX peptide MMP-2 ICG 785/813 Spontaneous Solid [43]
Tumors
BLZ-100 CTX peptide MMP-2 ICG 785/813 oral dysplasia [44]
BLZ-100 CTX peptide MMP-2 ICG 785/813 Breast cancer [46]
TM1-IR680 TM-1 GRPR IR680 680/694 Oral cancer [49]
ICG-Glu-GluAE105 uPAR agonist uPAR ICG 788/813 Glioblastoma [61]
Dmt-Tic-800CW Dmt-Tic δOR IRDye800CW 778/832 Lung cancer [65]
Dmt-Tic-Cy5 Dmt-Tic δOR Cy5 650/670 Colorectal cancer [64]
G4RGDSq2 arginine-glycine-aspartic acid units Integrin αvβ3 Squaraine 710/720 Ovarian cancer [52]
SRR*-Cy5.5 SRR(synthesized,SRRPASFRTARE) FGFR2 Cy5.5 678/695 Esophageal cancer [52]
ICG-Glu-GluAE105 uPAR agonist uPAR ICG 788/813 Tongue tumor [62]
KSPNPRF-800CW KSPNPRF(peptide) HER2 IRDye800CW 778/832 Breast cancer [134]
800CW-SCE succinimidyl-Cys-C(O)-Glu PSMA IRDye800CW 775/800 Prostate cancer [108]
R01-MG-IRDye800 R01(a cysteine knotti peptide) Integrin αvβ6 IRDye800CW 778/832 PDAC [58]
800CW-PEG28-A20FMDV2-K16R- PEG28-A20FMDV2-K16R-PEG28 Integrin αvβ6 IRDye800CW 778/833 Pancreatic cancer [59]
PEG28
A38C-alexa647 A38C TF antigen alexa647 635/670 ovarian peritoneal cancer [72]
Cy5.5-MT1-AF7p-H4R MT1-AF7p MT1-MMP) Cy5.5 678/695 Breast cancer [71]
peptide-22-Cy7 peptide-22 LDLR Cy7 759/788 Pancreatic cancer [66]
LS301-Cy7.5 LS301 Integrin Cy7.5 788/808 Colorectal cancer [60]
CDGM NPs CPLGVRGRGDS MMP-2 Ce6 660/– Lung cancer [161]
peptide-PGC GPKPYRS WMK (peptide) MMP-3 Cy5.5 630 /680 EOC [162]
SBP-M13 M13 SPARC protein single-walled carbon 808/970 Ovarian cancer [144]
nanotubes
ATTO665-EGF EGF EGFR ATTO665 600/684 Squamous cancer [164]
BBN-Cy5.5 Bombesin-like peptide (BBN) GRPR Cy5.5 673/691 Prostate cancer [50]

inflamed tissues by binding to FRβ of activated macrophages, a phase I 3.4. Three examples: Smaller ligands are proper for intraoperative imaging
trial was conducted to visualize and monitor the inflammation in
rheumatoid arthritis (RA) and inflammatory bowel disease (IBD) 3.4.1. Prostate-specific membrane antigen (PSMA)
(Table 5, NCT03938701), showing the potential for disease monitoring. Prostate-specific membrane antigen (PSMA), a class II membrane
glycoprotein that encoded by the FOLH1 (folate hydrolase) gene, has
become a promising target for the molecular imaging of prostate cancer
3.3.2. Potential small molecule conjugates in preclinical stage [100,101]. Antibody was first introduced to illuminate PSMA. Takahito
Receptors were also frequently used as targets for small molecule et al synthesized a conjugate containing a humanized anti-prostate
conjugates. Estrogen receptor-alpha (ERα), a nuclear hormone receptor specific membrane antigen (PSMA) monoclonal antibody (J591) linked
which can be regulated by the hormone estrogen, such as 17β-estradiol to ICG derivatives [102]. However, due to the special quenching trait of
(E2), was overexpressed in nearly 70% of breast cancer patients [90]. ICG, the conjugate failed to illuminate prostate cancer in vivo by
An ERα-targeted NIRF agent, IRDye800CW − E2, including an E2 identifying the margins of prostate cancer and positive lymph nodes in
analogue ethinyllestradiolamine as a ligand was developed [91] real-time resection [102]. To address the problem, ICG was replaced by
(Table 6). For Cholecystokinin 2 receptor (CCK2R), CRL-LS288 was IRDye800CW in ProstaFluor (HuJ591-800CW), which was the first
designed to facilitate intraoperative identification of malignant tissues targeted dye tested in clinical trials of prostate cancer patients (Table 2,
in CCK2R-expressing HEK 293 murine tumor xenografts [92] (Table 6). NCT01173146). Quantum dot800-J591 was also used for optical
Suberoylanilide hydroxamic acid (SAHA), a FDA approved HDAC in- fluorescence imaging but no further studies were reported [103].
hibitor, was conjugated to IRDye800CW in orthotopic HCC fluores- Meanwhile, MDX1201, another novel anti-PSMA antibody, was con-
cence imaging and FGS at 6–12 h after injection [93] (Table 6). Dasa- jugated with the NIRF probe A488, to be tested in patients with stage
tinib, the inhibitor of Src Family Kinases, was introduced to conjugated IIB/III/IV prostate adenocarcinoma (Table 2, NCT02048150). Due to
with a novel cyanine based dye, MHI-148, to image subcutaneous the bulky size of full antibody, the imaging experiments had to be
glioblastoma tumor in vivo [94] (Table 6). MHI-148 was conjugated performed 4–5 days after intravenous injection. In hence, smaller an-
with the hydrophobically modified glycol chitosan (HGC) micelle by tibody fragments, ScFv and minibody, were then introduced to mini-
EDC/NHS chemistry to target 4 T1 breast cancer cells in vivo and mize the time between injection and imaging. A fully human ScFv
trigger drug release [95]. For cancer cell mitochondria, IR-DBI, a novel targeting PSMA called gy1-IRDye800CW was developed by our group
analog of ICG was developed to target albumin II in mitochondria via to recognize PSMA-positive tumor tissue [104]. Meanwhile, another
enhanced permeability and EPR effect [96]. One year later, the same ScFv, D2B, was also developed to delineate prostate tumors [105].
team synthesized IR-34 for tumor imaging and induced death of NSCLC Compared with antibody fragments, peptides with a lower molecular
cells by uncontrolled reactive oxygen species (ROS)[97] (Table 6). weight (less than40KD) can easily be excreted by kidney [106]. Wang
Besides orthotopic tumor, some probes were also designed for metas- Xinning and colleagues synthesized a peptide-based PSMA ligand,
tases. P800SO3, a bone tissue-specific NIR fluorophore to image carti- PSMA-1, labeled it with NIR dyes 800CW and Cy5.5, and demonstrated
lage and monitor metastatic bone tumors in FGS [98,99] (Table 6). that PSMA-1-800CW and PSMA-1-Cy5.5 had better binding affinities
With the development of small molecule inhibitors, more small mole- than their parent ligand Cys-CO-Glu [107] (Table 3). Urea scaffold, as
cule conjugates were designed but their binding ability and perfor- smaller molecule, is essential for synthesizing PSMA-based imaging
mance still need further investigation.

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Table 4
Tumor-targeted NIRF dyes in clinical trials (peptide conjugates).
Contrast agent Ligand Target Fluorophore ClinicalTrials.gov Condition or disease Start date Complete date Phase Actual Enrollment Locations
identifier (participants)

BLZ-100/tozuleristide /Tumor CTX/Chlorotoxin Annexin A2/ ICG NCT02464332 Sarcoma, Soft Tissue 06/08/15 05/26/16 Phase 1 —— Blaze Bioscience Inc.
Paint peptide MMP-2 NCT02234297 Glioma 09/09/14 04/06/16 Phase 1 17
NCT02496065 Tumors, Breast 07/14/15 07/13/17 Phase 1 30
NCT02462629 Central Nervous System Tumors 06/01/15 08/01/18 Phase 1 32
NCT02097875 Skin Neoplasms 03/27/14 04/21/15 Phase 1 21
NCT03579602 Pediatric Central Nervous System —— —— Phase 2 114
Tumor
EMI-137/NAP c-Met Cy5 NCT03205501 Barrett EsophagusEsophageal 02/09/17 12/12/18 Phase 1 25 University Medical
CancerDysplasia in Barrett Center Groningen
Esophagus
NCT03360461 Colonic Cancer/Metastasis to 02/14/18 03/14/19 Phase 2 10 University of Leeds
Lymph Node
NCT03470259 Papillary Thyroid Cancer/Lymph 06/20/18 04/01/20 Phase 1 32 University Medical
Node Metastases Center Groningen
NCT02676050 Lung Cancer 07/01/18 07/01/19 Phase 1 20 University of
Edinburgh

131
LS301 octapeptide —— Cy7.5 NCT02807597 Breast Cancer 05/31/19 05/31/21 Phase 1 22 Washington
University School of
Medicine
QRH-882260 heptapeptide EGFR Cy5 NCT02574858 Healthy Adults 10/14/15 12/06/17 Phase 25 University of
1A Michigan
NCT03148119 Colon Cancer Prevention 05/10/17 04/24/18 Phase 1B 5
KSP-910638G heptapeptide HER2 IRDye800CW NCT03161418 Healthy Adults 06/21/17 09/20/17 Phase 26
1A
Multiplexed heptapeptides/KSP- heptapeptide dimer HER2 and IRDye800CW NCT03643068 Healthy Adults 08/22/18 10/27/18 Phase 26
QRH-E3-IRDye800/Peptide (KSP/QRH Dimer) EGFR 1A
919288G NCT03589443 Barrett Esophagus 05/10/18 05/10/19 Phase 1 50
NCT03852576 Barrett Esophagus 05/01/19 05/01/21 Phase 1B 50
QRH-882260 Heptapeptide Heptapeptide(Gln- EGFR Cy5 NCT03438435 Cholangiocarcinoma 01/20/19 11/01/20 Phase 1B 12 University of
Arg-His-Lys-Pro-Arg- Washington
Glu)
ABY-029/ABY-029 Affibody EGFR IRDye800CW NCT02901925 Glioma 12/01/16 03/01/19 Early 12 Dartmouth-
trifluoroacetate salt Phase 1 Hitchcock Medical
NCT03154411 Primary Soft-tissue Sarcoma 08/30/17 05/01/19 Early 18 Center
Phase 1
NCT03282461 Head and Neck Cancer 01/25/18 12/31/19 Early 12
Phase 1
European Journal of Pharmaceutics and Biopharmaceutics 152 (2020) 123–143
J. Jiao, et al. European Journal of Pharmaceutics and Biopharmaceutics 152 (2020) 123–143

agents. IRDye800CW-SCE, including an asymmetric urea compound

Indiana University School of

On Target Laboratories, LLC
named SCE, proved its efficiency in PSMA-positive prostate cancer cells

University of Pennsylvania
[108] (Table 6). The research team from Johns Hopkins Medical Hos-
pital synthesized a series of compounds based on urea scaffold. They
conjugated compound 3 to IRDye800CW to get YC-27 3 and test its in
vivo distribution [109] (Table 6). Two years later, they conjugated the
three compounds with five commercially available dyes such as IR-
Locations

Medicine
Dye800CW, Cy5.5, Cy7, or a derivative of indocyanine green (ICG) to
evaluate their biological performance and found compound 2,3 con-
jugates such as Cy7-3 achieved higher PSMA-specific uptake in vivo
[110] (Table 6). Bioconjugation of KUE (a low-molecular-weight, urea-
containing PSMA ligand) and ZW800-1 (a NIR fluorophore) was also
Actual Enrollment

tested in xenograft tumor mice [111] (Table 6). Another PSMA-targeted

(participants)

small molecule conjugate, OTL78, exhibited an excellent SBR of 5:1

with a half-time of 17 min which was much shorter than antibody
conjugates [112] (Table 6). 68Ga/177Lu-PSMA-I&T PET has achieved
110

147

300
48

50

50

50

50
20

great success in clinical PET imaging and radionuclide treatment of

prostate cancer. Based on parent PSMA- I&T, PSMA-I&F was created
2
2
3
1
1
1

Phase 1

Phase 1
Phase 1
Phase

Phase
Phase
Phase
Phase
Phase
Phase

and combined with Cy5 to b PSMA-I&F-Cy5 for FGS of prostate cancer

[113] (Table 6). In addition, the first enzyme-activatable NIRF dye
Complete date

which targeted at the carboxypeptidase of PSMA, 5GluAF-2MeTG, was

also successfully designed and tested [114] (Table 8).
10/31/18
11/01/15
08/01/19
09/01/20
12/01/20
12/01/18

09/01/20

12/01/20
03/20/18

3.4.2. Epidermal growth factor receptor (EGFR)

Human epidermal growth factor receptor (EGFR/ErbB-1/HER1), a
05/04/17
12/01/14
01/26/18
10/01/15
05/01/15
06/01/15

09/01/15

07/01/16
12/29/15
Start date

transmembrane protein belonging to the ErbB family of receptors, is

highly expressed in many types of cancers, such as glioblastoma, non-
small cell lung cancer, pancreatic ductal adenocarcinoma, breast
cancer, and head and neck squamous carcinoma (HNSCC) [115–117].
Patients With Suspected Malignancies of
a Diagnosis of Any Resectable Lung or

Cetuximab is an FDA-approved, humanized chimeric monoclonal anti-

body for the treatment of EGFR + tumors, such as colorectal cancer,
Bladder Cancer/Gastric Cancer

NSCLC, and HNSCC [26,118,119]. Cetuximab-800CW (Cetux-800CW)

was tested in 9 HNSCC patients, indicating its specific binding to the
EGFR [120] (Table 2, NCT01987375). Furthermore, Cetux-800CW was
Renal Cell Carcinoma
Condition or disease

Pituitary Neoplasms

tested for surgical navigation in 12 patients with HNSCC with an

the Pituitary Gland

average TBR of 5.2 (Table 2, NCT01987375) [121]. Cetux-800CW was

Ovarian Cancer
Ovarian Cancer

Pleural Nodule
Lung Cancer

Lung Cancer

also under stage I/II clinical trials of pancreatic adenocarcinoma, ma-

lignant glioma and HNSCC (Table 2, NCT02736578, NCT02855086,
NCT03134846). Replaced by Alexa-647 in the conjugate, Cetuximab-
Alexa-647 was tested in pancreatic ductal adenocarcinoma (PDAC)
models [122] (Table 1). In addition to cyanine dyes, cetuximab was also
ClinicalTrials.gov identifier

coupled to NIRF Ag2Se quantum dots (QDs, λex/em = 720/930 nm), to

achieve targeted imaging and therapy of orthotopic tongue cancer
[123] (Table 1).
Tumor-targeted NIRF dyes in clinical trials (small molecule conjugates).

NCT02872701
NCT02317705
NCT03180307
NCT02629549
NCT02602119
NCT02769156

NCT02769533

NCT02852252
NCT02645409

Panitumumab is another fully humanized recombinant anti-EGFR

monoclonal antibody. Five phase II and two phrase I clinical trials have
already been conducted to test best dose of panitumumab-IRDye800CW
in HNSCC (Fig. 4A,B) [124], lung cancer, pancreatic adenocarcinoma,
and malignant glioma (Table 2, NCT02415881, NCT03405142,
Fluorophore

NCT03582124, NCT03384238, NCT03510208, NCT03733210,

SO456

NCT04085887). The safety of panitumumab-IRDye800CW was vali-

dated in 15 HNSCC patients’ surgical navigation of FGS(Table 2,
NCT02415881) [125] and was used to clearly identify positive margins
Folate receptor

of the tumors in FGS of 12 patients with HNSCC (Table 2,

NCT02415881)[126]. Panitumumab-IRDye800CW was also used for
Target

surgical resection in 29 patients with oral squamous cell carcinoma and

significantly reduced margin distance from 8.0 mm to 3.2 mm
α

(NCT02415881)[127]. In addition, to handle high-grade dysplasia be-

fore HNSCC, panitumumab-IRDye800CW was used to distinguish high-
Ligand

analog
Folate

grade dysplasia from normal epithelium [128].

Smaller anti-EGFR nanobody, 7D12, was linked to IDRye800CW for
detecting orthotopic tongue cancer and cervical lymph node metastasis
Contrast agent

[129] (Table 1). The conjugate (CNDC) of S0456 and Centyrin

(83v2Cys) which is a 10KD protein scaffold binding to EGFR at the C
Table 5

OTL38

terminal human Fc fusion, was used for FGS in murine models of NSCLC
[130] (Table 1). ABY-029, a smaller probe including a synthetic

132
J. Jiao, et al. European Journal of Pharmaceutics and Biopharmaceutics 152 (2020) 123–143

Table 6
Tumor-targeted NIRF dyes in preclinical stage (small molecule conjugates).
Contrast agent Ligand Target Fluorophore λex/em(nm) Cancer type Refs

IntegriSense680 integrin αvβ3 antagonist Integrin αvβ3 VivoTag-S680 675/693 Colorectal cancer [54]
CRL-LS288 CRL(Z-360) CCK2R LS-288 747/756 Human embryonic kidney [92]
IntegriSense 680 integrin αvβ3 antagonist Integrin αvβ3 VivoTag-S680 675/693 Ovarian cancer [55]
P800SO3 SO3- Cartilage P800 (self invented) NR Prostate/breast cancer [98]
KUE-ZW800-1 Lysine-urea-glutamate (KUE) PSMA ZW800-1 770/778 Prostate cancer [111]
800CW-SCE SCE (an asymmetric urea compound) PSMA IRDye800CW 775/780 Prostate cancer [108]
800CW − E2 E2 analogue ethinyl estradiol amine ERα IRDye800CW 778/832 Breast cancer [91]
OTL78 coined DUPA PSMA SO456 776/793 Prostate cancer [112]
IntegriSense750 integrin αvβ3 antagonist Integrin αvβ3 NR 745/800 HNSCC [56]
ProSense750EX integrin αvβ3 antagonist cathepsin NR 745/800 HNSCC [56]
ProSense750FAST integrin αvβ3 antagonist cathepsin NR 750/770 HNSCC [56]
Dasatinib-MHI-148 Dasatinib Src and Lyn kinases MHI-148 760/808 Breast cancer [94]
YC-27 3 compound 3 PSMA IRDye800CW 775/780 Prostate cancer [109]
Cy7-3 compound 3 PSMA Cy7 759/788 Prostate cancer [110]
IR-34 —— mitochondria IR-34 770/830 NSCLC [97]
SAHA-800CW SAHA Histone deacetylases (HDACs) IRDye800CW 775/780 HCC [93]
Cy5-AFTN Afatinib(AFTN) HER1/HER2 Cy5 650/670 Breast cancer [135]
PSMA-I&F. PSMA-I&T PSMA Cy5 640/656 Prostate cancer [113]

affibody targeted EGFR Z03115-Cys was also tested in EGFR positive Afatinib (AFTN), as a dual HER1 and HER2 tyrosine kinase inhibitor
orthotopic glioma [131] and soft tissue sarcomas [132]. Smaller size approved by FDA in 2013, was designed to bind Cy5 to be a dual-tar-
contributed to quicker imaging and clearance than Cetux-800CW. The geted small molecular NIRF dye and validated in vivo xenograft tumor
tumors were clearly visualized at 1-h post-injection with SBR of 8–16. [135] (Table 6). Besides, NIRF emitted MnCuInS/ZnS QDs were also
After successful preclinical studies in rats, ABY-029 was then evaluated conjugated with a HER2 antibody to image HER2-positive breast cancer
in clinical trials of glioma, primary soft-tissue sarcoma, and HNSCC cells [136] (Table 1).
(Table 4, NCT02901925, NCT03154411, NCT03282461). Now the re-
sults were not revealed.
3.4.4. Three examples above: Peptide conjugates and small molecule
conjugates are more suitable for intraoperatively molecular imaging
3.4.3. Human epidermal growth factor receptor 2 (HER2) Based on studies above, we found that it usually takes antibody
Human epidermal growth factor receptor 2 (HER2/ErbB2), which is conjugates a few days while just a few hours for peptide conjugates and
highly expressed in nearly 30% of breast cancer patients, has been a small molecule conjugates to get the highest SBR in molecular imaging
biomarker and therapeutic target [133]. Trastuzumab is a humanized (Fig. 4). The main reason for this is that full antibodies with large Fc
monoclonal antibody of HER2. Transtuzumab-IDRye800CW, with a fragments are built for therapeutic purpose. The huge size of antibodies
TBR of 2.92 ± 0.29, was used for intraoperative NIRF imaging at the may limit their potential in intraoperative molecular imaging. The ad-
submillimeter level in SK-BR-3 (Her2-overexpressing) breast cancer vantage of antibody conjugates is that many therapeutic antibodies
cell- and KATO-III (Her2-overexpressing) gastric cancer cell-bearing have been approved by FDA for therapeutic purpose. Once linked to
nude mice [30]. More peptide conjugates were introduced in HER2 mature dyes such as IRDye800CW, antibodies can easily be introduced
targeted NIRF dyes. A real-time targeted optical dye using an IR- into clinical trials for molecule imaging.
Dye800CW-labeled HER2 binding peptide was synthesized to generate To overcome bulky size of antibody conjugates, the topical use of
tumor-specific contrast and validate its performance characteristics in antibody conjugates was studied. Under fluorescence molecular endo-
preclinical HER2-positive xenograft breast cancer of tumor-bearing scopy (FME), Wouter B Nagengast and colleagues employed bev-
mice [134] (Table 3). KSP-910638G, a heptapeptide that binds to Her2, acizumab-IRDye800CW to recognize esophageal adenocarcinoma
was linked to IRDye800CW to detect breast cancer (Table4, (EAC) and compared its performance in systemic tracer administration
NCT03161418). KSP-QRH-E3-IRDye800CW, a multiplexed heptapep- and topical tracer spraying(Table 2, NCT02129933)[137]. As a result,
tide that targets EGFR and Her2, was used to observe healthy people the incubation time between tracer administration and molecular
and patients with Barrett Esophagus (Table4, NCT03643068, imaging was reduced from 2 days to 5 min [137]. The topical tracer
NCT03589443, NCT03852576). QRH-882260, a heptapeptide that approach was able to elevate early lesion detection rate by 33% and
binds to EGFR, was linked to Cy5 to detect colon cancer (Table4, provides an excellent method for antibody based NIR fluorescent probes
NCT02574858, NCT03148119), Barrett esophagus (Table4, to decrease their long incubation time for imaging [137].
NCT03589443) and cholangiocarcinoma (Table4, NCT03438435). In our opinion, peptide conjugates and small-molecule conjugates

Table 7
Tumor-targeted NIRF dyes in preclinical stage (dual-modality dyes).
Contrast agent Other Imaging ligand Target Fluorophore λex/em(nm) Cancer type Refs

111In-DTPA-trastuzumab-800CW SPECT/CT trastuzumab HER2 IRDye800CW 778/832 Breast cancer [148]

111In-farletuzumab-IRDye800CW SPECT/CT farletuzumab FRα IRDye800CW 778/795 Ovarian cancer [149]
111In- RDC018 SPECT/CT bispecific Ab(bsAb)TF12 TROP-2/ HSG IRDye800CW 778/795 Prostate cancer [150]
pH sensitive-RGD-ICG NIRF (pH activated) RGD peptides Integrin αvβ3 ICG 788/813 Gastric cancer [53]
ABIR-targeting NIR pH-activatable probe NIRF (pH activated)) RGD peptides Integrin αvβ3 Indole intermediate2 778/791 Breast cancer [57]
GGKGPLGLPG-Fe3O4-Cy5.5 NIRF (pH activated)) GGKGPLGLPG (peptide) MMP9 Cy5.5 678/695 Colorectal cancer [157]
89Zr-bevacizumab-IRDye800CW SPECT/CT PET/CT bevacizumab VEGF IRDye800CW 780/795 HNSCC [151]
89Zr-cetuximab-IRDye800CW SPECT/CT PET/CT cetuximab EGFR IRDye800CW 780/795 HNSCC [151]
68 Ga-PSMA11-IRDye800CW PET/CT PSMA11 PSMA IRDye800CW 780/795 Prostate Cancer [155]
FA-NBs-IR780 Ultrasound Folic acid FR IR780 640/780 Breast cancer [156]

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Table 8
Tumor-targeted NIRF dyes in preclinical stage (activatable dyes).
Contrast agent Ligand Target Fluorophore λex/em(nm) Cancer type Refs

Quenched DDAO Arachidonate Arachidonic Acid cPLA2 DDAO 600/660 Triple negative breast cancer [167]
LBH589 − Cy5.5 LBH589 Histone deacetylases (HDACs) Cy5.5 678/695 breast cancer [168]
GANP γ-glutamate (γ-Glu) γ-Glutamyl transpeptidase (GGT) mCy-Cl 695/720 Ovarian/Colorectal/glioblastoma [158]
MMP-750 NR MMP NR 749/775 Malignant gliomas [163]
IR825-FNP-I FNP-I low pH IR825 808/— breast cancer [172]
NIR-GA GA low pH Changsha dye 709/733 HCC [173]
5GluAF-2MeTG 2MeTG PSMA 5GluAF 465/515 prostate cancer [114]
EGF-ATTO655 EGF EGFR ATTO655 600/684 epidermoid carcinoma [164]
cRGD-MP cRGD Integrin αvβ3 ATTO655 600/684 glioma [165]
CE7Q/CQ/S erlotinib EGFR Cy7 759/788 NSCLC [174]

are more suitable for intraoperatively optical imaging. Even though cysteine) targeting dye, p8-SWNT, for in vivo NIR II fluorescence
many clinical trials of antibody conjugates were conducted, most of imaging and increased the post-operative tumor-related survival [144]
them are in phase 1/2 for safety or dose-finding studies. The develop- (Table 3). FD-1080(λex/λem = 1064 nm/1080 nm) with both ex-
ment of PSMA targeted, EGFR targeted and HER-2 targeted probes citation and emission in the NIR II spectrum has been applied in vivo
demonstrated more researchers focused on peptide conjugates and molecular imaging [145]. Compared with NIR dyes with excitation
small molecule conjugates for their rapid imaging. In clinics, it is time- wavelength under 1000 nm, FD-1080 gave deeper penetration depth
consuming for us to have to inject targeted agents a few days before and superior imaging resolution [145]. Moreover, ER3+-doped DCNP
surgeries when some patients with malignances may need receive FGS (λem = 1550 nm), a NIR II b (λem = 1500 nm-1700 nm) nanoprobe
as soon as possible. Due to individual difference in pharmacokinetics, it for in vivo fluorescence imaging was invented for FGS with a pene-
is also difficult for us to judge the proper time when antibody con- tration depth of 3.5 cm [146]. NIR II dyes with longer wavelength can
jugates achieve highest SBR for FGS in the several days after injection. greatly increase penetration depth which is essential for recognizing
In hence, peptide conjugates and small molecule conjugates may be the PSM and positive lymph nodes in FGS.
developing trend in the nearest future. More preclinical studies and Even though NIR II can show deeper organs and lymph drainage,
clinical trials need to focus on them. most NIR II dyes were still in material invention stage and researchers
are focusing on decreasing in-vivo toxicity of NIR II dyes. The NIR-II
fluorescent agents, which were excreted slowly and were greatly re-
4. Deeper: Two methods to increase penetration depth of cancer
tained within the reticuloendothelial system such as the spleen and
targeted NIRF dyes
liver, still need to be ameliorated in future research. To the best of our
knowledge, there has been no clinical trials of NIR II dyes. However,
NIR II dyes and dual-modality dyes can significantly increase pe-
with the application of novel materials, both the accuracy and quality
netration depth of tumor-targeted NIRF dyes, leading to deeper ima-
of novel NIR or NIR-II dyes will be greatly improved in the near future
ging. The penetration depth can progressively be ameliorated by the
[147]. Once a mature NIR II dye were introduced in clinical trials,
application of NIR II dyes which target the second window NIRF range
tumor-targeted NIR II fluorescent dyes would make another essential
of 1000‐1700 nm [106]. Longer wavelength means deeper penetration,
breakthrough for FGS.
less photon scattering, weaker tissue autofluorescence background and
higher SBR (Fig. 5A–D). Dual-modality dyes were also introduced and
most of them used another label such as radiotracers in the tumor- 4.2. Dual-modality methods
targeted dyes. The radiotracers can undoubtedly increase detection
depth by SPECT/PET prior to surgery or radio-guided surgery with FGS Dual-modality probes comprise a ligand with a NIRF label and an-
(Fig. 5E–F). other type of label (e.g., radiotracers, pH activatable dye). The NIRF
label can delineate the margins of tumors intraoperatively through NIR
optical imaging, while the other tracer can be examined by SPECT/CT/
4.1. Near infrared II dyes
MRI/PET (Fig. 5 E-F).
Compared with traditional NIR I dyes, NIR-II dyes possess less tissue
scatter and autofluorescence at these wavelengths, which could im- 4.2.1. Dual-modality conjugates including NIRF dyes and radiotracers
111
prove the detection of small, deep tumors in animal models [84]. NIR-I In is a radioactive isotope of indium (In). 111In-DTPA-trastu-
dyes are NIR dyes with only a 0.2-mm maximum depth of penetration zumab-IRDye800CW, a dual-labeled trastuzumab-based imaging agent,
that is not deep enough for many of the oncological surgeries, while was synthesized and its binding affinity to HER2-overexpressing cells as
NIR-II light can emit at a maximum of 4 mm, and even deeper. The well as its characterization in preclinical HER2 positive xenograft breast
fluorophores in the NIR-II window will allow surgeons to observe cancer of tumor-bearing mice were validated [148] (Table 7). Hekman
deeper anatomical structures with better clarity and novel imaging et al performed 111In-farletuzumab-IRDye800CW, a FRα-targeted dual-
capabilities that are unavailable with NIR-I fluorescent dyes [106]. NIR- modality imaging agent, to detect ovarian cancer and guided resection
II contrast agents that were reported include CH1055-PEG (Fig. 5 A-D, in an intraperitoneal ovarian cancer model [149] (Table 7). Moreover,
λex/λem = 750 nm/1055 nm), IR-FGP (λex/λem = 745 nm/ pretargeting strategies were introduced for high SBR. 111In-RDC018-
1050 nm), H2a-4 T (λex/λem = 763 nm/1033 nm), downconversion IRDye800CW, which can conjugate to TF12, an anti-TROP-2x anti-HSG
nanoparticles (DCNPs; λex/λem = 808 nm/1060 nm), lanthanide na- bispecific antibody, was injected in vivo after 16 h of injection of TF12
noparticles (LNPs; λex/λem = 980 nm/1162 nm), Q4NPs (λex/ to delineate the margin of a trop-2-expressing metastatic growing
λem = 808 nm /1100 nm), SCH1100 (λex/λem = 808 nm/1100 nm), tumor model [150] (Table 7). In addition, 111Indium-DOTA-Labetu-
NaCeF4, uPAR-CH1055, CH4T (λex/λem = 808 nm/1055 nm) zumab-IRDye800CWwhich targets CEA, was used in FGS of colorectal
[106,138–143]. Lorenzo Ceppi and colleagues applied Single-walled cancer patients (Table 2, NCT03699332). Beyond 111In conjugates,
carbon nanotubes (SWNTs, λex/λem = 880/970 nm) to form a SPARC 89
Zr-bevacizumab-IRDye800CW and 89Zr-cetuximab-IRDye800CW
(specific peptide which binds the secreted protein, acidic and rich in were synthesized to target VEGF and EGFR in HNSCC [151] (Table 7).

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J. Jiao, et al. European Journal of Pharmaceutics and Biopharmaceutics 152 (2020) 123–143

Fig. 5. Representative figures from the imaging

process with a NIR-II fluorescent dye and a dual-
modality dye in preclinical stage. (A, B)
Lymphatic NIR-II imaging with CH1055-PEG.
(A) A digital photograph of a nude mouse with a
xenograft U87MG tumor located on the
shoulder. (B) NIRF imaging with CH1055-PEG.
The green asterisk denotes the location of the
U87MG tumor. (C, D) Superior NIRF imaging
with CH1055-PEG in through-skull non-invasive
imaging of brain tumor at 4 mm depth. (C) Color
photograph of a nude mouse preceding high-
magnification NIR-II imaging, with an outline
over suture lines. (D) Brain vasculature imaging
through the scalp and skull with shaved heads
using CH1055-PEG [106]. The figures are used
under the permission of Copyright © 2015,
Springer Nature. (E, F) Proof-of-concept of a
68
dual-modality dye, Ga-PSMA-11-IR-
Dye800CW, in FGS of healthy pigs. This research
was originally published in JNM. Ann-Christin
Baranski et al. PSMA-11–Derived Dual-Labeled
PSMA Inhibitors for Preoperative PET Imaging
and Precise Fluorescence-Guided Surgery of
Prostate Cancer [155]. J Nucl Med. 2019;
60:758–763. © SNMMI. Both NIR-II dyes and
dual-modality dyes can provide deeper imaging
in FGS. (For interpretation of the references to
color in this figure legend, the reader is referred
to the web version of this article.)

Additionally, A11 cMb, anti-PSCA minibody, was conjugated with (Table 2, NCT02497599). Bombesin-like peptide (BBN) was first con-
Cy5.5 and radiolabeled with 124I or 89Zr and 124I-A11 cMb-Cy5.5 was jugated with Cy5.5 to targeting gastrin-releasing peptide receptor
successfully performed for immunoPET/fluorescence imaging of pros- (GRPR) in prostate cancer bearing mice [50]. Then, 68Ga-BBN-IR-
tate cancer xenografts expressing high level of PSCA [152]. Another Dye800CW which targets GRPR, was also used to illuminate the sur-
dual-modality probe, 124I-A2cDb-800, based on A2 cys-diabody geries of patients of glioblastoma (Table 4, NCT02910804).
(A2cDb) targeting PSCA was screened for PSCA expression in pan-
creatic ductal adenocarcinoma xenograft models by immuno-PET
[153]. However, flow cytometry results indicate that elevated dye-to- 4.2.2. Dual-modality conjugates based on 68Ga-PSMA
68
protein ratios can also lead to reduced tumor uptake as a result of de- Ga-PSMA positron emission tomography (PET) has been widely
clined immunoreactivity and stability [154]. In clinical trials, 111In- applied in the diagnosis and radiotherapy management in prostate
DOTA-Girentuximab-IRDye800CW, which targets carbonic anhydrase cancer patents. Based on N,N9-bis [2-hydroxy-5-(carboxyethyl)benzyl]
IX, was used to illuminate the surgeries of patients of renal carcinoma ethylenediamine-N, N9-diacetic acid (HBED-CC)–based PET tracer
68
Ga-Glu-urea-Lys (Ahx)-HBED-CC (68Ga-PSMA-11), dual-labeled

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J. Jiao, et al. European Journal of Pharmaceutics and Biopharmaceutics 152 (2020) 123–143

Fig. 6. Representative figures from the imaging

process with an enzyme activatable and a pH ac-
tivatable NIRF dye. (A, B) Situ NIRF imaging with
an enzyme activatable NIRF dye, LUM015, in FGS
for breast cancer. (A) Fluorescent image captured
from transected ex vivo resected invasive ductal
breast cancer mass from an example patient. (B)
Pathology slide taken from same resected mass;
the oval hole within the mass is a processing ar-
tefact [169]. The figures are used under the per-
mission of Copyright © 2018, Springer Nature. (C,
D) Ex vivo imaging of bladder specimens with an
antibody conjugate, ICG-pHLIP, in FGS for high-
grade urothelial carcinomas [171]. (C) Brightfield
and (D) NIRF images. The figures are used under
the permission of Copyright © 2016, Proc Natl
Acad Sci U S A. Both “activatable” methods can
provide stronger SBR in FGS.

PSMA-11 was designed and validated in LNCap and PC3 cell mouse However, dual-modality dyes with radiotracers may not be welcomed
tumor models [155] (Fig. 5 E-F, Table 7). 68Ga-PSMA-11-IRDye800CW in clinics because the bulky size of SPECT/PET equipment may limit its
achieved highest PSMA-specific tumor enrichment at 2 h after injection. potential use. Meanwhile, surgeons are also not willing to be exposed to
Due to the tiny size of PSMA-11, urine excretion facilitated quick radioactive nuclide for a long time and we prefer to use radiation-free
clearance of the free dyes in blood [155]. However, the tumor uptake tumor-specific NIRF dyes. If a single modality dye without radiotracers,
decreased between 2 h and 6 h after injection, which left less than 5 h such as PSMA-11-IRDye800CW, can also be designed and used in the
for patient to receive PSMA PET and FGS. The limited time can be second injection while 68Ga-PSMA-11-IRDye800CW was used in the
elaborated by second injection of dual-modality tracers or simplify the first injection, not only the necessary time for surgeries can be con-
surgical process [155]. 68Ga-PSMA-11-IRDye800CW has the potential firmed but also the unnecessary radiation can be avoided for surgical
to be the first dual-modality NIRF dyes applied in FGS of prostate teams in surgeries. We believe that the combination of dual-modality
cancer patients because both 68Ga-PSMA-11 and IRDye800CW had dyes and single-modality dyes targeted on the same biomarker in FGS
been widely validated in clinical trials. can be one of the developing trends in the future.

4.2.3. Other types of dual-modality conjugates 5. Stronger: “Turn on/off (Activatable) method” shows stronger
Tumor-targeted NIRF dyes can also be combined with ultrasound TBR
contrast to create dual-modality dyes. FA-NBs-IR780 was developed to
be dual-mode agent which includes IR780 and micro-bubbles (MBs) for Activatable NIRF dyes can contribute to stronger imaging with
both molecular imaging and ultrasound imaging [156]. Nanoprobes higher SBR because the previously quenched dyes with less background
were also introduced to construct dual ratiometric fluorescent probes. fluorescence can only be activated by the specific enzymes or low pH in
Fe3O4 nanoparticles were used to connect Cy5.5, Fe3O4, folate, peptide the tumors. Activatable NIRF probes can be divided into enzyme-acti-
substrates and the N-carboxyhexyl derivative of 3-amino-1,2,4-triazole- vatable dyes and pH-activatable dyes (Fig. 6). Enzyme-activatable dyes
fused 1,8-naphthalimide (ANNA)[157] (Table 7). The fluorescence of and pH-activatable dyes can separately be activated by specific en-
ANNA, which was quenched while attached to the surface of an Fe3O4 zymes in cancer cells and low pH in the microenvironment around
nanoparticle, was activated when the peptide linker was cleaved by cancer cells. Activatable NIRF probes have been widely developed for
MMP-9[157]. Meanwhile, Cy5.5 was also conjugated with the activated the in vivo detection of malignant tumors in preclinical experiments.
pH dye to visualize protease activities in vivo [157] (Table 7). How- Some of them such as LUM015 have already been in clinical trials
ever, the in vivo toxicity of nanomaterials is still under consideration (Fig. 6A,B, Table 9).
and need to be ameliorated in the future.
5.1. Enzyme-activatable dye
4.2.4. The potential developing trends of dual-modality dyes
Dual-modality NIRF dyes may have potential use for its two probes. Enzyme-activatable dyes, specifically “turned on” by tumor cells,

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J. Jiao, et al. European Journal of Pharmaceutics and Biopharmaceutics 152 (2020) 123–143

were developed to decrease background fluorescence and increase TBR.

Gamma-glutamyl transpeptidase (GGT) is an enzyme that is recognized

Massachusetts General

Massachusetts General
as a potential biomarker for many cancers on cell membranes. A GGT-
Duke University activatable NIRF agent was invented by conjugation of a GGT-re-
cognitive substrate gamma-glutamate (γ-Glu) and an NIR cyanine
fluorophore (mCy-Cl) with a self-immolative linker p-aminobenzyl al-
Locations

Hospital

Hospital
cohol (PABA) [158] (Table 8). A dual-channel, tumor-targeted GGT-
activatable fluorescent agent (Glu-DFB-biotin) was designed to be
lighted up by hepatic cancer cells in vivo [159]. A silicon, rhodamine-
based NIRF probe, gGlu-HMJSiR, was also designed for GGT, enabling
the discrimination of tumors with different enzymatic profiles [160].
Matrix metalloproteinases (MMPs), which are overexpressed in various
Actual Enrollment

tumor types, play a critical role in facilitating cancer invasion and

(participants)

metastasis. Fangfang Xia et al designed and developed pH-responsive

gold nanoclusters-based nanoprobes, CDGM NPs, including MMP2
polypeptide (CPLGVRGRGDS), for tumor targeting, pH-sensitive drug
15
60

21

release, photodynamic therapy and fluorescence imaging [161]

(Table 3). Additionally, Kuo-Hwa Wang et al designed a novel MMP3-
Phase 1/ 2
Phase 1
Phase 1

sensitive NIRF probe that includes a synthesized peptide and Cy5.5 to

Phase

detect stromal cell activation in early-stage epithelial ovarian cancer

(EOC) [162] (Table 3). Moreover, the precise resection of malignant
Complete date

glioma was conducted in FGS using MMPSense 750 FAST (MMP-750),

08/01/15
10/01/17

11/01/18

an MMP targeting the NIRF agent [163] (Table 8). MMP-750 could
precisely delineate the tumor margins in surgery, resulting in complete
removal of the tumors [163]. Based on photo-induced electron transfer
mechanism, Hyunjin Kim and colleagues synthesized a zwitterionic
06/01/12
09/01/17

05/01/16
Start date

NIRF conjugated probe, ATTO655-EGF (λex/λem = 600/684 nm), and

enable it for tumor-targeted imaging squamous cell carcinoma with a
TBR of 6.37[164] (Table 3). ATTO655-EGF was synthesized by NHS
Sarcoma/Soft Tissue Sarcoma/Breast Cancer

reaction and activated when amino acid quenchers (Trp and Tyr) was
cut off by proteinase K after receptor-mediated endocytosis [164]
Colorectal Cancer /Pancreatic Cancer/

(Table 8). Another targeted probe including ATTO655, cRGD-MP, was

also invented and validated for specific fluorescence imaging of integrin
αvβ3-overexpressing cancer cells [165] (Table 8). Cytosolic phospholi-
pase A2 (cPLA2), which plays an important role in several malignant
tumors, is an active signaling phospholipase in charge of the release of
Condition or disease

Esophageal Cancer

arachidonic acid from peri-nuclear membranes [166]. Arachidonic acid-

DDAO, in which arachidonic acid is linked to DDAO (a NIR fluor-
Breast Cancer

ophore), was synthesized and shown to be selectively activated by

triple-negative breast cancer cells in vitro, indicating its potential for in
vivo imaging [167] (Table 8). For histone deacetylases (HDACs),
LBH589 − Cy5.5 was used for in vivo NIRF imaging in triple-negative
ClinicalTrials.gov identifier

breast cancer [168] (Table 8).In clinical trials, LUM015, a GGRK that is
conjugated to Cy5 and activated by cathepsin, was the only enzyme-
activatable dye in the clinical trials [169] (Table 9, Fig. 6 A-B,
NCT01626066
NCT02438358

NCT02584244

NCT01626066, NCT02438358, NCT02584244).

Tumor-targeted NIRF dyes in clinical trials (activatable dyes).

5.2. pH-activatable dye

Low pH activatable dyes are another group of “turn on/off” probes.

Fluorophore

Due to the Warburg effect, cancer cells can produce high levels of lactic
acid through glycolysis in tumors, leading to a low pH in the tumor
Cy5

surroundings. pH low insertion peptides (pHLIPs) are a group of

membrane-conjugating peptides that specifically target acidic cells ei-
Cathepsin

ther in vitro or in vivo by inserting across cellular membranes when the

Target

pH of the extracellular environment is low [170]. Jovana Golijanin et al

developed ICG-pHLIP contrast which can be activated by low pH and
marked high-grade urothelial carcinomas in human cystectomy speci-
mens of 22 patients to improve the early diagnosis of bladder cancer
peptide
Ligand

GGRK

(Fig. 6C,D) [171]. The sensitivity and specificity of ICG-pHLIP were

found to be 97% and 80%, respectively [171]. IR825-FNP-I, a nano-
particle conjugate, was developed as a pH sensitive NIRF dye for FGS of
Contrast agent

breast cancer [172] (Table 8). Meanwhile, NIR-GA, a small molecular

LUM015

conjugate was also designed for FGS of hepatocellular carcinoma (HCC)

Table 9

[173]. NIR-GA mainly localized in lysosome where low pH can activate

fluorescent dye by H + -triggered spirolactam ring [173] (Table 8).

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J. Jiao, et al. European Journal of Pharmaceutics and Biopharmaceutics 152 (2020) 123–143

Fig. 7. Distribution schematic diagram of tumor-targeted NIRF dyes in the human body.

Tumor specific ligands can also be combined with pH sensitive agents. Funding
A novel NIRF/pH dual-responsive nanocomplex, CE7Q/CQ/S, enabled
targeted NIRF imaging and photothermal therapy (PTT) at the same This work was supported by the National Natural Science
time [174]. Foundation of China (grant nos. 81772734).

5.3. The potential developing trends of activatable/ “turn on/off” dyes 8. Role of the Funder/Sponsor

Activatable/ “turn on/off” dyes, namely smart probes, can largely The funding organizations had no role in the design and conduct of
decease unwanted background fluorescence. For this special trait, the study; collection, management, analysis, and interpretation of the
tumor-targeted activatable NIRF dyes with higher SBR may be another data; preparation, review, or approval of the manuscript; and decision
essential developing trend in tumor-specific NIRF agents. Some recent to submit the manuscript for publication.
studies also showed the potential to combine dual-modality dye or re-
cognition motif with activatable dye to invent an dual-modality acti- Ethical approval
vatable probe [175] or tumor-specific excretion retarded (TER) probe
[176]. This combination may better delineate margins of tumors and This article does not contain any studies with human participants or
lymph node metastases. animals performed by any of the authors.

Author contributions
6. Summary and outlook

The manuscript was written through contributions of all authors. All

In this article, we summarized all tumor-targeted NIRF dyes in
authors have given approval to the final version of the manuscript.
clinical trials and preclinical stage in a time-related framework to show
the developing trend of the dyes (Fig. 1) and the distribution of tumor-
targeted NIRF dyes in the human body (Fig. 7). The imaging with Declaration of Competing Interest
tumor-targeted NIRF dyes are becoming quicker, deeper and stronger.
Due to smaller ligands, peptide and small molecule conjugates would be The authors declare that they have no known competing financial
more appropriate for quicker molecular imaging. NIR II dyes and the interests or personal relationships that could have appeared to influ-
dual-modality method can provide deeper imaging and the activatable ence the work reported in this paper.
method can provide stronger imaging with higher SBR. To realize the
clinical application of tumor-targeted NIR dyes intraoperatively in on- Acknowledgment
cological surgeries, more multi-center, phrase II/III clinical trials are
urgent in the nearest future. Although some obstacles exist, clinical The authors are grateful to Prof. Changhong Shi (Division of Cancer
translation of tumor-targeted NIRF dyes in FGS can undoubtedly pro- Biology, Laboratory Animal Center, Fourth Military Medical University)
long thousands of patients’ lives. and Dr. Rui Li (Department of Chemistry, University of Cambridge) for

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their critical reading of the manuscript. 1072–1084, https://doi.org/10.7150/thno.9899.

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