Activatable NIR-Ⅱ Probe for Tumor Imaging

Zhen Wang; Xi Li; Yuanyuan Li; Qi Wang; Xiaomei Lu; Quli Fan

doi:10.7536/PC201237

Progress in Chemistry ›› 2022, Vol. 34 ›› Issue (1) : 198-206 DOI: 10.7536/PC201237

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Activatable NIR-Ⅱ Probe for Tumor Imaging

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Abstract

The near-infrared window with a wavelength between 1000 and 1700 nm is usually called the second near-infrared (NIR-Ⅱ) window. In terms of biological imaging (fluorescence imaging, photoacoustic imaging, etc.), this window shows a strong attraction. Compared with traditional imaging in the visible light (400~700 nm) region and the first near-infrared (NIR-Ⅰ 700~900 nm) window, NIR-Ⅱ bioimaging provides the advantages of high resolution and deep penetration depth. However, most current "always-on" probes cannot achieve a higher signal-to-noise ratio. The imaging of tumor microenvironment-responsive smart drugs is only triggered in tumors, which can overcome this limitation. Therefore, the tumor microenvironment and the NIR-Ⅱ intelligent response probe should be fully combined, and the advantages of both will be fully utilized to improve the accurate diagnosis of tumors. This article reviews the latest research progress of activatable NIR-Ⅱ fluorescent probes in bioimaging from different pathological parameters, and presents views on the opportunities and challenges faced by this emerging field.

Contents

1 Introduction

2 Activatable NIR-Ⅱfluorescent probe

2.1 pH

2.2 Redox

2.3 Enzyme

3 Activatable NIR-Ⅱphotoacoustic imaging

3.1 pH

3.2 Redox

3.3 Exogenous substances

4 Conclusion and outlook

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Keywords

NIR-Ⅱ / fluorescence imaging / photoacoustic imaging / activatable

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Zhen Wang, Xi Li, Yuanyuan Li, Qi Wang, Xiaomei Lu, Quli Fan. Activatable NIR-Ⅱ Probe for Tumor Imaging. Progress in Chemistry, 2022, 34(1): 198-206 DOI:10.7536/PC201237

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1 Introduction

Light-based diagnostic techniques mainly include fluorescence (FL) imaging and photoacoustic (PA) imaging, which have made great contributions to biomedical research and clinical practice^[1,2]. In vivo FL and PA imaging has the advantages over traditional imaging techniques (magnetic resonance imaging, computed tomography, ultrasound scanning, positron emission tomography, etc.) In terms of wide imaging range, low cost, high sensitivity, and high spatial and temporal resolution^[3,4]. However, the interaction between light and biological tissue in the visible region (400 ~ 700 nm) and the first near-infrared window (700 ~ 900 nm, NIR-I) poses a major challenge to the imaging of deep tissues.

In order to increase the depth of optical diagnosis, the second near-infrared (NIR-II, 1000 ~ 1700 nm) window has attracted more and more attention. NIR-II fluorescence has little interaction with biological tissues, greatly reducing light scattering and autofluorescence in tissues (fig. 1), and PA imaging can ignore attenuated sound waves, thus increasing tissue penetration^{[5⇓⇓⇓~9]}^[10,11]. Therefore, NIR-Ⅱ optical imaging is more suitable for deep tissue imaging in vivo than visible light and NIR-Ⅰ optical imaging^[12⇓~14].

In the past few years, NIR-II diagnostic probes have been developed continuously, such as single-walled carbon nanotubes (SWCNs), rare-earth doped down-conversion nanoparticles (DCNPs), quantum dots (QDs), organic small molecules and semiconducting polymer nanoparticles (SPNs)^[15,16]^[17,18]^[19,20]^{[21⇓⇓~24]}^[25,26]. However, most of them are "always-on" probes and do not achieve higher signal-to-noise ratios (SNRs)^[27,28]. Therefore, tumor microenvironment and NIR-II smart response probe should be fully combined to give full play to their advantages in order to obtain high signal-to-noise ratio and improve the accurate diagnosis of tumors.

In this review, we summarize the research progress and challenges of activatable NIR-Ⅱ diagnostic probes in biosensing and biomedicine. The design and biological application of various intelligent responsive NIR-II imaging probes are described in detail according to different pathological parameters, and the challenges and future prospects of this emerging field are also discussed.

2 NIR-Ⅱ fluorescent probe capable of being activated

FL imaging has the characteristics of high signal-to-noise ratio and high sensitivity, which plays an indispensable role in the field of biological imaging. So far, a variety of FL contrast agents have been developed, including organic molecules and rare earth nanoparticles. However, the limited penetration depth of these contrast agents has prompted researchers to further develop NIR-II based contrast agents. Therefore, more and more NIR-II contrast agents have been developed. However, these "always-on" probes will always produce background signal interference that can not be ignored in the imaging process. In order to solve these interferences, intelligent response contrast agents combined with tumor microenvironment (abnormal pH, redox or enzymes) have attracted more and more attention^[29,30]^[31]. Changes in physiological parameters are manifested at the cellular, tissue and organ levels and are associated with pathological processes. More importantly, these parameters are important biomarkers for different kinds of diseases, such as cancer, inflammation and cardiovascular diseases.

2.1 pH

pH is an important physiological parameter in vivo, and it is also an important biomarker to explore the tumor microenvironment^[32,33]. Compared with normal cells, tumor cells have higher metabolic capacity and tend to be more acidic^[32,34]. The pH value of extracellular matrix in tumor tissues (6.5 ~ 7.0) was lower than that in normal tissues (7.2 ~ 7.4). In addition, lysosomes of tumor cells (pH 3.8-4.7) were more acidic than those of normal cells (pH 4.5-6.0).

Based on this difference, Zhang et al. developed a series of pentamethyl cyanine fluorophores (Fig. 2), which significantly overcome the severe solvatochromism, thus achieving stable absorption and emission above 1000 nm, increasing the fluorescence brightness by about 44 times, and having superior photostability in aqueous solution^[35]. The probe enables lymphatic imaging with high penetration depth (8 mm), high contrast, and high stability. Among them, BTC1070 showed acid response ability, and the intensity of NIR-Ⅱ fluorescence increased with the increase of acidity. By fitting the integrated intensity ratios of different pH values, the optimal sensing pH range of 1 to 4 was obtained, and noninvasive ratiometric fluorescence imaging and in vivo quantification of intragastric pH were achieved with reliable accuracy compared with standard pH meters.

2.2 Oxidation-reduction

The redox state in organisms is of great significance to maintain the homeostasis of the internal environment and life activities. Biological redox species mainly include reactive oxygen species (ROS), reactive sulfur species (RSS) and reactive nitrogen species (RNS)^[37,38]^[30,39]^[40]. Among them, ROS and RNS are the main oxidizing substances, and RSS is the main reducing substance. ROS is a general term describing the chemical substances formed after incomplete reduction of oxygen, including superoxide anion (

O 2 -

), hydrogen peroxide (H₂O₂) and hydroxyl radical (· OH).RNS mainly includes nitrogen dioxide (NO₂), peroxynitrite (ONOO^-), nitric oxide (NO) and nitroso (HNO), and RSS includes thiols (GSH, CYS, HCY), disulfides (RSSR) and persulfides (H₂S₂) and H₂S. These substances play key roles in various physiological and pathological processes. Therefore, the development of smart probes with high specificity and sensitivity for monitoring the abnormal expression of redox species in living systems is of great significance for the early diagnosis of drug toxicity.

In 2019, Liu Et al. reported the first · OH-responsive NIR-II fluorescent probe (Hydro-1080), which was first obtained by reducing the fluorescent molecule Et-1080 (Fig. 3A). Due to the reduction of conjugation and coplanarity, the probe had no obvious fluorescence in the NIR-II region^[36]. The fluorescence of the conjugated system was recovered after the formation of Et-1080 by the oxidation of · OH. It can adjust the fluorescence intensity independent of the emission wavelength. Hydro-1080 has excellent sensitivity (LOD = 0.5 nmol/L) (fig. 3B) and selectivity (fig. 3C) to · OH, is able to track subtle changes in liver (· OH) induced by external stimuli, and provides clear images with high contrast. This probe has been successfully used to monitor · OH production induced by lipopolysaccharide (LPS) and acetaminophen (APAP) excess. Compared with 808 nm laser excitation, the probe exhibited better signal to noise ratio under 1064 nm laser excitation and higher fluorescence intensity with increasing APAP dose. Finally, the feasibility of Hydro-1080 imaging was further verified by in vivo mouse experiments (Fig. 3D).

In the same year, Wang et al. reported a ONOO^- responsive NIR-II inorganic quantum dot fluorescence probe (V&A@Ag₂S) (fig. 4A)^[41]. Initially, the fluorescence of V&A@Ag₂S appears to be off due to the energy transfer from Ag₂S to the A1094 fluorophore. After intravenous injection, V&A@Ag₂S rapidly accumulated in the inflamed vascular endothelium of brain injury (TBI) based on vascular adhesion factor (VCAM 1) -mediated endocytosis, after which the nanoprobe achieved rapid recovery of NIR-II fluorescence of Ag₂S QDs through bleaching of A1094 by peroxynitrite (ONOO^-), a TBI precursor biomarker (Fig. 4 B). In cell experiments, the probe produced intense NIR-II fluorescence within 30 min (fig. 4C), showing a time-dependent enhancement of the NIR-II fluorescence signal. In further experiments in mice, the V&A@Ag₂S showed the advantages of high speed, high sensitivity and high signal-to-noise ratio compared with the "always-on" probe in brain trauma imaging.

As one of the reductive thiols, H₂S plays an important role in stabilizing redox in vivo. Overexpression of endogenous H₂S is one of the key features of colon cancer^[42,43]. However, studies using optical probes activated by endogenous H₂S for specific diagnosis of colon cancer are rare. In 2018, Fan et al. reported a boron dipyrromethene probe (ZX-NIR) sensitive to H₂S (Fig. 5A)^[42]. In the presence of H₂S, the NIR-II fluorescence of ZX-NIR at 900 ~ 1300 nm was activated (Fig. 5B), thus realizing the precise visualization of HCT116 tumor in vivo, and the NIR-II fluorescence intensity ratio between tumor and normal tissue was as high as 5.7 (Fig. 5C), which provided a new idea for the treatment of colon cancer.

2.3 Enzyme

Enzymes play an important role in a variety of biological processes and are associated with a variety of pathological conditions^{[45⇓⇓⇓~49]}. For example, β-galactosidase (β-gal) is widely present in human lysosomes. Abnormally active β-gal is often associated with ovarian cancer, so β-gal is an important biomarker for the detection of ovarian cancer. The development of highly sensitive β-gal probes can play a positive role in the early prediction and monitoring of ovarian cancer. Therefore, Gu et al. reported an NIR-II fluorescent probe BOD-M-β-gal (fig. 6a) for monitoring the behavior of β-Gal in vivo^[44]. BOD-M-β-Gal incorporates β-galactose residues into boron-dipyrromethene (BODIPY) -based NIR fluorophores via a self-eliminating linker. β-Gal can trigger the hydrolysis of β-galactose residues in the probe, followed by a 1,6-elimination reaction to release the BODIPY-based fluorophore, resulting in the formation of BOD-M-SH with strong NIR-II fluorescence (fig. 6B, C). In further in vivo experiments, the signal of BOD-M-β-Gal was collected at 1 min after injection into the tail vein of mice, and the signal reached the maximum at 10 min, and the SNR was as high as 6.43 at the depth of 2 mm (Fig. 6d), which proved the high sensitivity and specificity of BOD-M-β-Gal in ovarian cancer imaging and provided ideas for early research of ovarian cancer.

In 2018, Cai et al. reported a nitroreductase (NTR) -activated NIR-II fluorescent probe IR1048-MZ, which conjugates the nitroimidazole group with IR-1048 dye, and the electron-withdrawing group MZ induces electron transfer, resulting in fluorescence quenching and reducing the absorbance of IR-1048^[50]. When MZ is reduced to an amine group by nitroreductase, the NIR-II fluorescence signal of IR1048-MZ is restored (fig. 7A). In the actual test, IR1048-MZ has a weak absorption in the range of 800 ~ 1050 nm, and no fluorescence emission at 1046 nm. However, the probe showed an absorption peak near 980 nm after the addition of NTR in an anoxic environment, and showed a 106.9-fold fluorescence enhancement at 1046 nm after irradiation with 980 nm laser (fig. 7 B, C). The probe provided more accurate and deeper imaging of tumor location and boundary tissue in vivo experiments in mice (Fig. 7d), and laid the foundation for subsequent photothermal therapy (PTT) of tumors.

3 Activatable NIR-Ⅱ photoacoustic imaging

PA imaging is a hybrid biological imaging method combining optical excitation and ultrasonic detection, which is one of the most promising alternatives to conventional optical imaging^[51,52]. PA imaging solves the problems of optical diffusion threshold and the limited penetration depth of conventional optical imaging by detecting the acoustic wave generated by the imaging target after absorbing the excitation light. In biological tissues, the scattering of ultrasound is much smaller than that of light (about 1000 times). Semiconductor polymers, metal nanostructures and inorganic semiconductors have been widely used in tumor imaging. For example, Pu et al. Have developed many semiconducting polymers with excellent PA properties for tumor imaging^[53,54]. However, most of the developed PA imaging probes are NIR-I photoresponsive materials. Compared with the NIR-I photoresponsive material, the NIR-II photoresponsive agent is not interfered by endogenous signals due to less absorption of NIR-II light by biological tissues in vivo. At the same time, the penetration depth of NIR-II light is larger. Therefore, more and more NIR-ⅡPA imaging probes have been developed in recent years.

Compared with the "always-on" probe, the PA imaging probe activated by the tumor microenvironment has no obvious PA signal in normal tissues, so the PA imaging probe activated by the tumor microenvironment shows a higher signal-to-noise ratio.

3.1 pH

The pH of tumor is lower than that of normal tissue, which is often used as a trigger factor for PA contrast agent^[56,57]. Xing et al. reported a self-assembled charge-transfer nanocomposite (CTN) based on 3,3,5,5-tetramethylbenzidine (TMB), which exhibited NIR-IIPA signal at the tumor site through pH activation^[58]. Compared with neutral or weak alkaline conditions (pH ≥ 7), the prepared CTN showed a strong absorption change at 750 ~ 1200 nm at pH = 5. The signal of mice at the tumor site increased with the time of CTN injection and reached the peak at 4 H after injection, while there was almost no PA signal in normal mice without tumor at the same site after 4 H of injection, which improved the signal to noise ratio between tumor and normal tissues. At the same time, CTN is degradable under physiological conditions (e.g., pH = 7.4), alleviating the biosafety issue of nanoparticle accumulation in vivo.

In 2019, Dong et al. reported a Mo₂C derived polyoxometalate as a pH-activated NIR-ⅡPA imaging agent (POM) for tumor imaging (Figure 8 a)^[55]. Under mild acidic conditions in the tumor, the polyoxometalate derived from Mo₂C shows strong absorption at 1064 nm (Fig. 8B), which can be used as NIR-ⅡPA imaging. As shown in Figure 8C, the PA signal in the tumor increased with time after injection of the Mo₂C derived polyoxometalate, reaching a maximum at 8 H, and the probe had good chemodynamic efficacy.

3.2 Oxidation-reduction

In the tumor microenvironment, the high expression of redox is another factor that triggers the intelligent second near-infrared photoacoustic imaging probe^[60]. In 2020, Zheng et al. designed and constructed an activatable diagnostic and therapeutic nanosystem (AB-DS@BSA-N₃NYs) on the basis of one-pot method and surface functional modification to improve the second near-infrared photoacoustic imaging (Figure 9 a)^[59].

Based on tumor accumulation mediated by high permeability and retention effect (EPR) of tumor tissue, tumor overexpression of GSH can accelerate the generation of H₂S in nanoparticles by reacting with coated diallyl trisulfide (DATS) to obtain stronger absorption and NIR-II emission (fig. 9b, C). Meanwhile, the in-situ released H₂S can not only be used for gas therapy, but also reduce —N₃(-) to —NH₂(+), thereby enhancing the tumor-specific aggregation of NYs. Therefore, accurate NIR-II FL/PA dual-mode imaging can be achieved. In the in vivo experiment, both NIR-II and PA signals increased in a concentration-dependent manner with the gradual increase in the concentration of NYs, reaching a maximum at 6 H after injection (Fig. 9d). Interestingly, the charge reversal occurring during the reaction not only favors tumor uptake of positively charged AB-DS@BSA-N₃NYs, but also significantly improves tumor aggregation and retention time. The feasibility and efficiency of the probe for in vivo photoacoustic imaging are further demonstrated.

Xing et al. developed a smart H₂O₂ activator and acid enhancer (SHT) (fig. 10a)^[61]. Horseradish peroxidase and TMB were co-loaded in the mesoporous silica nanocarrier. TMB was oxidized by H₂O₂ in the presence of horseradish peroxidase, resulting in a strong absorption at 1064 nm (fig. 10 B). At 4-6 H after injection, the PA signal at 1064 nm reached a peak, and the significant difference in PA intensity could be observed between tumor and normal tissues (Fig. 10c). The results show that the intelligent H₂O₂ activation acid-increasing agent has high sensitivity.

3.3 Xenobiotics

Drug-resistant Staphylococcus aureus (MRSA) is a serious threat to human health, so it is important to develop effective methods to eliminate the infection and monitor the treatment process^[62]. Recently, Sheng et al. reported an activatable NIR-II photochromic strategy of micro gold/silver nanorods (Au/Ag NRs) for photochemical synergistic therapy of MRSA infection and in situ monitoring of the therapeutic process^[63]. Au/Ag NRs were efficiently activated by exogenous ferricyanide solution and continuously released free Ag⁺ to eliminate MRSA, triggering NIR-II photothermal and PA performance enhancement. The activated NIR-II photothermal effect, in turn, accelerates the release of free Ag⁺ from Au/Ag NRs for synergistic clearance of Gram-positive S. aureus. After co-processing, a 20-fold increase in NIR-IIPA signal was observed between the implant site and normal tissue, with a maximum signal-to-noise ratio of 9.5, allowing sensitive monitoring of the Ag⁺ release process. The prepared gold/silver nanocomposite material has good stability and biocompatibility, and has good application prospect.

4 Conclusion and prospect

The tumor microenvironment activated NIR-II probe not only has better tissue penetration depth, but also has better tumor and background SBR in optical imaging. Smart probes that produce a signal only when a specific biomarker or event occurs show the advantage of a specific signal and are closely related to the concentration of the biomarker, which is not possible with traditional probes. It is worth noting that the development of smart probes is still in its infancy, and challenges and opportunities coexist. The following problems need to be solved.

(1) Develop more probes that can be used. Due to the difficulty of probe design, the number of probes used for NIR-II fluorescence or photoacoustic imaging is very limited, which is the main obstacle to explore its further application.

(2) Obtain better biocompatibility. Although many of the existing NIR-II probes show better imaging performance, there is still little valuable information on the pharmacokinetics of excretion and long-term toxicology of these NIR-II probes. Especially, the toxicity of inorganic NIR-II fluorescence in vivo has not been observed, which greatly limits the application of inorganic nanomaterials. Therefore, it will be an important research field to clarify the toxicity and pharmacokinetics of excretion, and then to design and develop highly biocompatible, safe and biodegradable NIR-II contrast agents.

(3) Higher quantum efficiency is obtained. High fluorescence quantum efficiency can greatly improve the resolution of TBR and in vivo imaging, avoid the loss of dynamic biological information, and reduce the dose of nanoprobes in vivo. At present, the quantum yields of most developed NIR-II fluorescent probes are below 5%, especially for the commonly used SWCNs, there is still much room for improvement. Therefore, the development of probes with higher quantum yield is still the key to future research.

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