Fluorescence Guided Sentinel Lymph Node Mapping: From Current

Subscriber access provided by University of Rhode Island | University Libraries

Review

Fluorescence guided sentinel lymph node mapping: from current molecular probes to future multimodal nanoprobes Zhifei Dai, Sadaf Hameed, Hong Chen, Muhammad Irfan, Sadia Z. Bajwa, Waheed Samraiz Khan, and Shahid Mahmood Baig Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00812 • Publication Date (Web): 03 Dec 2018 Downloaded from http://pubs.acs.org on December 10, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 18 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Bioconjugate Chemistry

Fluorescence Guided Sentinel Lymph Node Mapping: From Current Molecular Probes to Future Multimodal Nanoprobes Sadaf Hameed‡#, Hong Chen‡#, Muhammad Irfan§, Sadia Zafar Bajwa†, Waheed S Khan†, Shahid Mahmood Baig† and Zhifei Dai‡* ‡

Department of Biomedical Engineering, College of Engineering, Peking University, Beijing 100871, China.

§Department of Medicines, Gujranwala Medical College, Gujranwala 52250, Pakistan.

National Institute of Biotechnology and Genetic Engineering, Faisalabad 38000, Pakistan. KEYWORDS: Fluorescent probes, sentinel lymph node mapping, molecular probes, nanoprobes †

ABSTRACT: For SLN lymph node biopsy (SLNB), the SLN mapping has become a standard of care procedure that can accurately locate the micrometastases disseminated from primary tumor sites to the regional lymph nodes. The broad array of SLN mapping has prompted the development of a wide range of SLN tracers, rationally designed for noninvasive, and highresolution imaging of SLNs. At present, conventional SLN imaging probes (blue dyes, radiocolloids and few other smallmolecular dyes) although serves the clinical needs, these are often associated with major issues such as insufficient accumulation in SLN, short retention time, staining of the surgical field and other adverse side effects. In a recent advancement, newly designed fluorescent nanoprobes are equipped with novel features that could be of high interest in SLN mapping such as (i) a unique niche that is not met by any other conventional SLN probes (ii) their adoptable synthesis method and (ii) excellent sensitivity facilitating high resolution SLN mapping. Most importantly, lots of efforts have been devoted for translating the fluorescent nanoprobes into a clinical set-up and also imparting the multimodal imaging abilities of nanoprobes for the excellent diagnosis of life-threatening diseases such as cancer. In this review, we will provide a detailed roadmap in the progress of a wide variety of current fluorescent molecular probes and emphasize on the future of nanomaterial-based single/multimodal imaging probes that have true potential translation abilities for SLN mapping.

Introduction Cancer metastasis has become the leading cause of cancer morbidity and mortality globally and is responsible for an estimated 90% cancer deaths. Regardless of cancer types, only one out of five metastatic cancer patients are reported to survive for more than 5 years highlighting the complexity of the disease control.(1, 2) For many carcinomas, such as breast cancer, prostate cancer, lung cancer, the majority of cancer mortalities are caused by the dissemination of tumor cells from the primary site to the progressively colonized distant tissues.(3-5) Tumor metastasis usually occurs through the lymphatic or hematogenous pathway.(6) A plethora of investigations have addressed the mechanism of metastatic spread via the bloodstream to the distant tissues; however, most of the epithelial cancers develop the metastatic dissemination by spreading through lymphatic vessels to their first draining lymph node (known as sentinel lymph node, SLN).(7) Therefore, the identification of metastasis within the SLNs is a key criterion for prognostic assessment, minimally invasive tumor staging and treatment planning.(8-10) Accurate preoperative identification of lymph node metastasis is particularly important for scheduling the treatment for patients.(11) Many already reported literature have manifested that lymph node metastasis is not only considerable for distant metastasis, but also have a strong impact on local recurrence.(12) The patient’s treatment schemes are directly determined by whether their lymph nodes have metastasized, that is to identify whether they should put first on chemotherapy or radiotherapy and then surgery, or first go for surgery followed by other therapeutic methods.(13) Nevertheless, an accurate and complete clinical estimation of lymph node metastasis is vital.

Figure 1. Schematic illusion of desirable characteristics of fluorescent probes for SLN mapping The pathological status of SLN relies heavily on their accurate biopsy, which in turn depends greatly on the surgical guidance provided during the procedure.(14, 15) In a clinical setup, the SLN mapping is most frequently performed by the combination of preoperative lymphoscintigraphy and intraoperative localization of radiocolloids and blue dyes.(16, 17) This technique offers the opportunity for early detection of SLN metastasis and spares the SLN-negative patients from the unnecessary regional lymphadenectomy. SLN mapping has emerged as a valuable tool for tumor staging, treatment and diagnosis of metastasis.(18) Recent studies showed that the SLN mapping with lymphadenectomy has relatively higher sensitivity for detecting low-volume metastasis than lymphadenectomy alone, as measured with a low falsenegative (FN) rate.(19, 20)

ACS Paragon Plus Environment

Bioconjugate Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

However, currently practiced radiocolloids (99mTcnanocolloid) and dye-guided methods for SLN biopsy have their own limitations.(21) Cumulative radiation exposure for healthcare workers, issues with surgical waste disposal, high medical facility costs and restrictions on access to radioisotopes secondary to mandatory licensing, are some of the limitations of radiocolloids–guided method.(22, 23) Likewise, the associated risk of systemic allergic reaction to the dyes, a high false negative rate, low visibility in dense fatty tissues, insufficient accumulations and retention in SLN, is the limitation associated with the dye-guided methods.(24-27) Therefore, there is an urgent need to design non-invasive probes to eliminate these limitations and accelerate the achievement of accurate SLN mapping in clinical settings.(28) An ideal SLN tracer should exhibit following properties: rapid clearance from the injection site, sufficient accumulation and long retention time in SLN, excellent biocompatibility and nontoxicity in an organism, high photostability for repeated sections of imaging, good tissue penetration and high spatial/temporal resolution to precisely visualize SLNs.(29, 30) Compared with the radiocolloid and blue dyes, fluorescent probes offer the promise of radio-free, non-invasive, and real time imaging of SLNs.(31, 32) Advancements in the small moleculesbased dyes such as indocyanine green (ICG), Cy5.5, IRDye800CW, have emerged as a potential candidate for clinical SLN mapping. (33, 34) Besides molecular probes, nanomaterials owing to their unique physiochemical properties, offer new opportunities to design and develop novel nanostructured and nanoscale probes for the detection of SLNs.(35-37) A large variety of nanoprobes, including both inorganic and organic ones, have been extensively exploited in the past few decades to visualize and characterize SLNs. Moreover, novel fluorescent dyes containing nanoparticles (NPs), quantum dots and nanoprobes with different functionalities can be welldesigned for multimodality SLN mapping.(38, 39) Despite the rapid advancement and progress in the design, synthesis and implementation of fluorescent probes for SLN mapping, only a very few critical reviews are available till date. Herein, we review and summarize the key issues associated with conventional fluorescence probes and point out current research status of nano-fluorescent probes for the pre- and intraoperative detection of SLN. Besides, we also provide in-depth information on the design and characteristic of several newly designed or already wellestablished fluorescent nanoprobes which should be of immense importance in taking SLN mapping to a new height. At last, we conclude this review by providing a brief outlook on the clinical translation of potential nanoprobes, associated challenges and possible remedies.

Desirable chemical and biological characteristics of fluorescent probes For fluorescence imaging, the fluorescent dye molecules need to fulfil several criteria with respect to their chemical and biological characteristics, including wavelength, quantum yield, solubility photostability, clearance and toxicity (Figure 1).

Page 2 of 18

Wavelength

The fluorescence light needs to penetrate a maximum distance through the tissues. Absorption and scattering are the limiting factors that hinder the propagation of the light through the tissues.(40) However, with increases the wavelength of the penetrating light, which is in the red or infrared region of the spectrum, the absorption and scattering of the light could be minimized.(41) Therefore, fluorescent probes are usually designed with a wavelength between 700-900nm for imaging of SLNs.(42)

Quantum Yield

The fluorescence quantum yield (QY) is another criterion to determine the efficiency of fluorescent probes.(43) The maximum fluorescence QY is considered as 1.0 (100%), consequently any agent with QY ranges from 0.1 to 1 should retain fluorescence. As stated by the Rose model,(44) high QY can significantly improve the signal-to-noise ratio (SNR) and resolution of bioimaging, minimizing the dosage of fluorescent probe administrated in vivo, as well as avoid the possible loss of bioinformation.(45) Till now, the majority of the reported fluorescent probes possess a QY of 10-20% in serum, with higher QY being quite rare,(46) leaving a plenty of room for the development of new fluorescent probes with improved QYs and photostability.

Solubility

It is evident that fluorescent probes should be soluble for administration into the lymphatic systems. Solubility is a substantial issue for both inorganic and organic fluorescent probes.(47) Most of the cyanine dyes have poor solubility in water-based buffering system.(48) Therefore, additional chemical groups, typically sulphonates, can be substituted to the base chemical structure of the probes to dramatically increase the solubility.(49) Of note, even though the substitution of the solubilizing group to the fluorescent probes can enhance biocompatibility, it can also greatly alter the biodistribution and pharmacokinetics, so these properties should be well-characterized in the formulation or buffer utilized for in vivo administration.(50)

Photostability

Many small molecular fluorescent probes undergo irreversible photochemical destruction or photobleaching, consequently complicate the visualization of fluorescent probes as they eventually destroyed by the exposure of light requisite to excite them into fluorescing.(51) Therefore, a fluorescent probe with lower photochemical destruction and higher stability would be beneficial for their practical use.(52)

Clearance and Toxicity

The clearance of the fluorescent probes and their potential toxicity in the living system is the first and foremost factor that hinders their clinical implementation.(53) Albeit, a wide range of existing fluorescence dyes evince remarkable imaging performance, however, the toxicity concerns including their chemical components and stability as well as morphological parameters should be carefully considered for further preclinical investigations and clinical utility.(54) In general, researchers should strive for nanoprobes without any heavy-metal elements, have high explicit pharmacokinetics and hold high chemical stability.(38)

2 ACS Paragon Plus Environment

Page 3 of 18 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Molecular fluorescent probes for SLN mapping

with high sensitivity and specificity rate. Nearly more than 50 clinical trials of ICG as NIR fluorescent probe in

Figure 2. A Sentinel lymph node (arrow) mapping in the axilla of a woman with breast cancer after peri-tumoral injection of ICG: HSA as described in 15. NIR fluorescence = 800 nm channel; 67 msec exposure time. The NIR fluorescence image was pseudo-color. (83) Molecular fluorescent probes have also been studied for fluorescence signal generation and contrast modulation to identify SLNs. Recently, near-infrared (NIR) fluorescence imaging has been employed to manifest exquisite sensitivity enabling non-invasive estimation of SLN architecture and function.(55, 56) NIR light, with the wavelength range of 700-900nm, provides numerous significant advantages over the other available imaging modalities, including high signal-to-background ratio (SBR), low optical scattering, and high photon penetration. In many clinical studies, NIR molecular probes, at a very low concentration, are systematically administrated to a specific tissue or organ and invisible NIR fluorescence can be used to “see” any desired structures without staining of the surgical field.(57) Compared with other imaging modality, fluorescence imaging has a wide range of benefits derived from its combination of high spatial and temporal resolution.(58) Till date, ICG and methylene blue have been used clinically as an effective lymphatic tracer for SLN mapping in breast cancer, colon cancer, gastric cancer, head and neck, cervical cancer, vulvar cancer, and lungs cancer.(59) However, it is still very challenging to develop probes that can truly selectively and sensitively discriminate the dysfunctionality in the SLNs. During the past few years, significant research efforts have been devoted to developing molecular fluorescent probes. In the following section, we briefly address the rationale and prior use of ICG, IRDye800CW, and other cyanine probes for intraoperative visualization and non-invasive imaging of SLNs in various carcinomas.

Indocyanine Green in SLN mapping Indocyanine Green (ICG) is a near-infrared tricarbocyanine dye approved by the Food and Drug Administration (FDA) for clinical applications in 1954.(60) In the past few years, ICG exhibiting strong absorption in the NIR spectra has gained profound attention for many diagnostic and therapeutic applications, including angiography in ophthalmology, evaluation of cardiac output and hepatic function, perfusion-related analysis of tissues and organs, photodynamic and photothermal therapy.(61-65) Additionally, ICG is also highly desirable for SLN mapping

diagnostic and surgical medicines with promising progress in the mapping lymphatic dysfunctionality have enlightened its endless clinical advantages (Table 1).(33, 66-75) A significant number of studies have been conducted to highlight the use of ICG as NIR fluorescence with an improved detection rate in various carcinomas such as breast cancer, melanoma, gastric cancer and lung cancer. (76, 77) ICG in SLN mapping for Breast Cancer Breast cancer so far is the most common malignancy in women worldwide. In Asia, Africa, and South America, the incidence rates of breast cancer are increasing, nevertheless, most likely due to changes in the lifestyle and delayed breast cancer screening programs.(78) Likewise, mortality rates of breast cancer are also continuously increasing in these areas, partly because of the limited or no access to the most advanced diagnostic and surgical medicines and equipment.(79) The presence or absence of axillary lymph node involvement remains the most critical prognostic factor that can determine the recurrence and survival in patients with breast cancer. Over the past decades, SLN mapping has comparable diagnostic accuracy for acquiring nodal status in breast cancer. The major advantage of ICG as a lymphatic tracer for breast cancer is the real time identification of lymph flows from the breast to the axilla. Therefore, SLNs can be visualized and resected more precisely, particularly in cases with multiple lymph drainage pathways, where ICG can identify multiple SLNs with high detection rate.(80) For instance, a low concentration of ICG ( 300 nm ) hardly leave the injection site. (118) (Figure 6)

Ongoing research efforts in nanotechnology and material chemistry facilitate the development of novel nanomaterials-based fluorescence probes, which significantly accelerates the implementation of fluorescence imaging in clinical SLN mapping.(113) Nanomaterials hold unique physicochemical properties such as nanoscale size, high stability and significant surfaceto-volume ratio, which make them appealing for SLN


Page 7 of 18 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Bioconjugate Chemistry Figure 6. Schematic illustration of SLN mapping using imaging nanoprobes. Imaging nanoprobes with hydrodynamic diameters of 5–10 nm size scale can flow into adjacent nodes in the chain through the SLN, with diameter > 300 nm rarely leave the injection site, with hydrodynamic diameters of 10–50 nm exhibit rapid uptake into the SLN and do not leave (118).

Table 2: Summary of SLN mapping by using fluorescent nanoprobes. Study

Fluorescence

Carrier

Tumor type

Targeting agents

Target

Size (nm)

Imaging system

Animal model

Ref

Tsuchimoc hi et al. 2013

ICG

silica

NA

PAMAM

NA

30~50

PDE

mouse

(119)

Dai et al. 2014 Liu et al. 2015 Kong et al. 2015 Shou et al. 2018 Shi et al. 2018

5-AF

dextran

NA

VEGF-C

~180

NA

mouse

(120)

Cy754

mesoporous silica cholesterol

breast cancer gastric cancer melanoma

NA

VEGF-C R NA

35

mouse

(121)

NA

NA

~30

Endra Nexus 128 NIR

dog, pig

(122)

thiophene

DPP-2F

68

NA

mouse

(123)

RGD

αvβ3

10~21

IVIS Lumina XR III

mouse

(124)

IRDye900 PDFT Cy5.5

CuS

gastric cancer

Moreover, the recent advancement in targeted nanoprobes justifies the significance of surface functionalization for more specific and selective detection of SLNs.(125) In fact, the surface properties of nanoprobes are closely associated with their stability, toxicity and biodistribution.(10) Recent studies have used target ligands, i.e. peptides, antibodies and small receptor ligands to modify the surface of nanoprobes to differentiate metastatic lymph nodes by specifically targeting metastatic tumor cells.(126, 127) A wide range of nanomaterials based on organic, inorganic as well as on multifunctional nanoprobes have been prepared, reported and applied for real-time identification of SLNs.(128, 129) Among them, quantum dots (QDs), silica nanoparticles (SiO2 NPs), metal nanoparticles (MNPs) and lipid as well as polymer-based nanoparticles with a number of desired features are the latest addition to the list of fluorescence nanoprobes.(37, 116, 127) An overview of recent investigations of fluorescent nanoprobes in SLN mapping is displayed in Table 2.(119-124) In the subsequent section of this review, we will focus on various nanoprobes employed in SLN mapping.

Organic Nanoprobes Although the development of organic nanoprobes has attracted immense attention, only a few of them are

promptly available to the scientific community owing to poor hydrophilicity and photostability.(130) Besides, most conventional organic molecules manifest notorious drawbacks at relatively high concentrations or in the aggregated states, in such a way that their fluorescence is greatly self-quenched, this phenomenon is frequently referred as aggregation-caused quenching (ACQ) effect.(131, 132) In order to overcome this shortcoming, novel strategies have been adopted to prepared organic nanoprobes with better photostability and biocompatibility. One of the strategies for achieving better fluorescence signals involves the use of carrier materials for these organic dyes. Therefore, the selection of carrier materials is one of the crucial factors that can regulate the mapping performance of these fluorescence nanoprobes. Several carrier materials, such as liposomes, polymers and proteins, have been used in the design of nanoprobes with improved SLN mapping ability. Below, we will briefly discuss these material groups and present their best matches regarding their potential application for SLN mapping. Conjugated polymers, with - conjugation along the backbone of the polymer chain, are compelling fluorescent probes with prospective application in biological fluorescence imaging. Owing to small size, bright fluorescence emission, and biocompatibility, conjugated polymers are considered as one of the most broadly



explored nanocarriers for delivering fluorescent probes to the SLNs. Kim et al, reported the application of cyanovinylene-backbone polymeric NPs (60nm) for real time SLN mapping in NIR wavelength range. These polymeric NPs were administrated intradermally in the forepaw of the mice. Owing to high fluorescence intensity at 365nm, these polymeric NPs can be trailed even by naked eye, enabling accurate detection of SLNs in an observational time frame.(133) Liposomes also hold enormous potential for theranostics owing to their strong capability to encapsulate nearly any small molecules. These biocompatible particles may substantially vary in size from few nanometers to several micrometers. Their physical and chemical properties have been thoroughly investigated and reported for drug delivery and theranostics.(134) Fluorescence agents can either covalently or non-covalently integrated into the different compartments (either in the shell or in the core) of liposomes. Moreover, their surface can easily be tailored and functionalized with biological recognition molecules, such as antibodies, glycans and peptides for specific targeting. Recently, lymph node mapping by using chlorophyll-incorporated liposomes as performed by Chu et al.(135) They found that the liposomes with an average size of 22nm are capable of emitting strong fluorescence at 680nm approximately for SLN mapping in a mouse model. Additionally, they observed the low cytotoxicity in macrophage and normal liver cell lines, thereby suggesting an immense potential for clinical SLN mapping.

Page 8 of 18

with IRDye800 organic dye. SLN mapping procedure with IRDye800 and NIR-PNG nanoprobes. (b, f) In vivo NIR fluorescence images (pseudocolor) at 30 min postinjection of the IRDye800 dye (0.5 μg in 50 μg of PBS) (b) and NIR-PNG nanoprobes (50 μg, conjugated with 0.5 μg of IRDye800, in 50 μg of PBS) (f). The arrows indicate the axillary SLN. (c, g) Color images of a mouse after methylene blue dye injection and skin removal after an injection of the IRDye800 (c) and NIR-PNG nanoprobes (g). (d–i) Ex vivo NIR fluorescence (d, h) and color (e, g) images of dissected LN and fat tissue of a mouse injected with the IRDye800 (d, e) and NIR-PNG nanoprobes (h, i). (j) Ex vivo NIR fluorescence images of dissected SLN on time points in the range 1–48 h postinjection.(118) Over the past decade, NIR-emitting self-assembled polymer nanogels have been widely exploited for in vivo animal imaging.(136) In a recent study, Noh et al designed and synthesized self-assembled pullulan-cholesterol nanosized (30nm in diameter) polymer nanogel (PNG) (Figure 7). (118) They also conjugated IRDye800, a NIR dye, on the surface of PNG (NIR-PNG) and demonstrated the improved photostability and higher retention time of NIR-PNGs in SLNs than IRDye800. The NIR-PNG nanoprobes revealed fast fluorescence SLN imaging in both small animal (mouse, 2min) and big animal (pig, 1min). The results also confirmed the nontoxic behavior of pullulan in mouse dendritic cells and adenocarcinoma cells. However, further animal studies on toxicity evaluation are desired. Target-activatable fluorescence nanoprobes, which can be turned on and off by enzymatic activity, redox potential, or pH level, has attracted immense attention in recent years. Several known strategies are generally adopted for designing target-activatable nanoprobes, such as photoninduced electron transfer (PET), self-quenching systems, and Forster resonance energy transfer (FRET).(137, 138) Herein, Hagimori et al. synthesized fluorophore-quencherbased activatable nanoprobe for SLN imaging. In this approach, TAMRA was selected as a fluorophore and BHQ2 or QSY7 as a quencher and synthesized fluorophorequencher conjugated γ-PGA complexes by the electrostatic self-assembly system and named as TAMRA-G4-DTPA/PEIBHQ2/γ-PGA complex, and TAMRA-G4-DTPA/PEI-QSY7/γPGA.(139) These complexes with an average particle size of 40nm were clearly identified in popliteal LN of mouse modal during the in vivo fluorescence imaging studies. Figure 8 manifested the weak background signal, suggesting the retention of γ-PGA complex within the lymph nodes without any leakage to the capillary vessels.

Figure 7. Schematic illustration and chemical structure of NIR-PNG based on pullulan-cholesterol nanogels conjugated


Page 9 of 18 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 8. (a) Scheme for TAMRA-G4-DTPA/PEI-BHQ2/γPGA and TAMRA-G4-DTPA/PEI-QSY7/γ-PGA complexes with Negative ζ-Potential (b) Fluorescent imaging after injection of TAMRA-G4-DTPA/PEI-Y7/γ-PGA complex into mouse footpads (arrows). TAMRA fluorescence signal (red) was clearly visible in mouse popliteal LN (arrow heads). Ex vivo fluorescent image acquired after the excision of lymph nodes (A: injected side, B: non-injected side (control). (139) Most of these investigations reveal the versatility of fluorescence organic nanoprobes for SLN mapping in experimental animal models. However, the clinical translation of these organic nanoprobes is still facing formidable hurdles, perceived toxicities and regulatory issues, which need to be addressed. Overall, there is a huge space to design a new generation of organic nanoprobes to obtain more enhanced and accurate SLN imaging.(140)

Inorganic Nanoprobes Organic nanoprobes have been extensively investigated and reported and are a major contributor in the clinical success

of fluorescence-based imaging of SLNs. However, the application of inorganic NPs as imaging modality is acquiring a major momentum in diverse areas of medicines and surgery in the recent few years.(141) Unlike many already reported organic nanoprobes, the unique physicochemical properties such as stability, inertness, and ease of surface functionalization make inorganic nanoprobes superior for real time fluorescence imaging of living bodies.(142) Of particular interest, quantum dots, (143) metal NPs(144), and silica NPs(145) have been developed as inexpensive, efficient, stable and tunable exogenous imaging agents for biological systems. Semiconductor Quantum dots (QDs), with a diameter less than 10nm, are exceptionally bright fluorescence nanoprobes that can overwhelm some of the spectral and depth limitations of conventional fluorophores.(146) QDs are capable of tuning their emission wavelength throughout the NIR-spectrum by simply adjusting their size and composition.(147) Consequently, the ease of modifying their spectra and high quantum yield enables multicolor visible spectrum that can be visualized with the naked eye, thereby allowing real-time multicolor mapping of SLNs.(148) Additionally, their large surface/volume ratio allows their conjugation with other small molecules (ligands), such as peptides, antibodies or nucleic acid for a fluorescence mapping application.(149) Therefore, QDs can offer real-time and simultaneous visualization of the lymphatic system, thus can shift the paradigm of precise surgical procedures. Kim et al, systematically investigated the possibility of utilizing the NIR-QDs (Em=850nm) for mapping of SLNs.(41) Intradermal injection of only 400pmol NIR-QDs permitted the real-time imaging of SLNs even 1cm below the skin (Figure 9). This study was considered as a major breakthrough, since it provides direct visual guidance to surgeons for SLN mapping with minimal incision

Figure 9. Images of mouse injected intradermally with 10 pmol of NIR QDs in the left paw. Left, pre-injection NIR autofluorescence image; middle, 5 min post-injection white light color video image; right, 5 min post-injection NIR fluorescence image. An arrow indicates the putative axillary sentinel lymph node. Fluorescence images have identical exposure times and normalization. (b) Images of the mouse shown in a 5 min after reinjection with 1% isosulfan blue and exposure of the actual sentinel lymph node. Left, color video; right, NIR fluorescence images. Isosulfan blue and NIR QDs were localized in the same lymph node (arrows). (c) Images of the surgical field in a pig injected intradermally with 400 pmol of NIR QDs in the right groin. Four time points are shown from 9 top to bottom: before injection (autofluorescence), 30 s after injection, 4 min after injection and during image-guided resection. ACS Paragon Plus Environment For each time point, color video (left), NIR fluorescence (middle) and color-NIR merge (right) images are shown. Fluorescence images have identical exposure times and normalization. To create the merged image, the NIR fluorescence image was pseudocolored lime green and superimposed on the color video image. The position of a nipple (N) is indicated.(41)


inaccuracies.(41) Likewise, several investigations have been carried out to identify the potential application of different materials in combination with QDs for SLN mapping. Therefore, a lot of core/shell-structured QDs have been designed with an alloyed core of group II-VI or III-V elements and an intermediate/outer shell of other semiconducting elements. Consequently, Anne et al demonstrated that 655nm emitting carboxyl-coated CdSe /ZnS QDs, with an average diameter of 16nm, allow the realtime visualization of SLNs after subcutaneous injection of only 20 pmol QDs in mouse modal. However, to overcome the potential toxicity of Cd-based QDs, PEG coated and Cd-free QDs are exploited thoroughly for accurate visualization of SLNs.(150, 151) To validate this, Bezdentnaya et al. synthesized 650-800nm emitting CuInS2/ZnS core/shell QDs with an average diameter of 5.5nm. PEG coated CuInS2/ZnS-QDs demonstrated higher retention in the lymph nodes for accurate identification of SLNs in 4T1-metastaic mouse modal.(151) Moreover, as compared to CdTeSe/ZnS-QDs, CuInS2/ZnS-QDs revealed 5times lower cytotoxicity even at a dose of 100nM against MRC-5 cells.(150) However, slow elimination (3 months) of CuInS2/ZnS-QDs from the mouse preclude them from further imaging applications for a human clinical trial, thereby, require additional research efforts to achieve desired results. (152, 153) Silica (Si) itself offers eternal opportunities for easy modification and loading of various theranostic and therapeutic moieties for a number of biological applications. (154) Usually, many fluorescent dyes can reside inside the SiNPs for biological imaging.(155) These dye-doped Si-based probes can improve the imaging sensitivity and provide highly amplified signals as compare to single dyes alone.(156) Thereby, Si-based nanoprobes owing to high photostability, non-toxic nature, enhanced light conversion efficiency and reduced photobleaching of fluorescence dyes are imperative as a host material in SLN imaging.(156) Towards this goal, ultra-small SiNPs (

Fluorescence Guided Sentinel Lymph Node Mapping: From Current

Recommend Documents