1. Introduction

To date, there is a high demand for sensitive and accurate diagnostic and therapeutic approaches to alleviate numerous medical problems. There is a significant developments observed in imaging techniques in preclinical and clinical translational research in recent times [1]. In particular, fluorescence optical imaging is widely used in in-vitro investigations and gained clinical application in optical imaging-guided surgeries improving precise removal of tissue resection without affecting normal sections [2].       Cancer is a leading devastating cause of death accounting for an estimated 9.6 million deaths in 2018 worldwide. The most common cancers are cervical, lung, breast, colorectal, prostate, skin and stomach [3-7]. Diagnosing cancer depends on either examining the tumour by histopathalogical or non-invasive imaging methods or both in combination. In routine clinical investigations, disease diagnosis includes computed tomography (CT), magnetic resonance imaging (MRI), positron emission tomography (PET), single photon emission CT (SPECT) ultrasonography, and optical imaging. These tomographic techniques help to determine morphological changes only when the tumour reaches a significant size and are detectable with good resolution and cancerous tissue contrast with respect to healthy tissue background [8-10]. However, these imaging techniques demands high cost and requires relatively long imaging time [11]. Real-time imaging is important to completely excise the tumour without affecting normal tissue, which otherwise may lead to loss of function. Furthermore, it helps in the collection of biopsy specimens for staging cancer and serves clinician in surgical and therapeutic interventions. Fluorescent small molecules and radiolabeled albumins (e.g., technetium-99m) [12], are commonly used agents identify lymph nodes as they are highly sensitive, however, it lacks specificity for tumour and low signal-to-noise ratio and associated with harmful radiation effects. Fluorescence imaging is highly sensitive and its resolution is in the order of micro-meter at the tissue surface. However, fluorescence imaging in the clinical settings are limited in use due to requirement of more depth of penetration, good signal intensity, and stability [13]. Therefore, designing the probe that overcomes all these limitations is essential for extensive clinical application specifically during surgical procedure. Fluorescent molecular probes have been widely used to target biomolecules like expressed proteins, cells, and tissues fragments [14]. Fluorescent dye such as indocynanine green is used invivo in human for visualizing vertebral arteries prior surgical procedure [15-17]. Despite such wide applications, these probes are poor in their photostability and sensitivity. Prognosis at an early stage is a major challenge and is poor for solid tumours before it metastasis [18]. Therefore, early detection at molecular stage, which could be easier to treat is very important for asymptomatic and some aggressive cancers [19]. Quantum dots application has been growing exponentially not only in industry but also in medical domain [20]. QDs have great potential for various biomedical applications such as sensors, imaging and therapy agents. A single probe that integrates multiple imaging contrasting agents is ideal for multimodality imaging applications. It also should possess reduced toxicity for successful clinical translational research in later stages [21]. The present review summarizes the advantages and limitations of current diagnostic techniques and latest developments in this field using functionalized nano formulation for various medical applications. 

2. Nanoprobes for Theranostic Application

Fluorescent dyes currently approved by the FDA for cancer imaging lack penetration depth more than 1cm [13].  QDs are versatile elements for optical cancer imaging as their optical properties are tunable and unique compared to aforementioned small molecule dyes. Quite a number of QDs formulations for theranostic application are currently available [22-26]. Due to their broad excitation and narrow emission spectrum, QDs can be used as multimodal contrast imaging agents which could be employed in positron emission tomography(PET), CT, infrared, fluorescence (optical imaging), MRI single photon emission computed tomography (SPECT) and ultrasonography (US) applications [27-29] (Figure – 1).   Hence, the size, surface chemistry, spectral properties (fluorescence ranging from the UV-blue of the mid-IR), and stability (long photoluminescence lifetime) of QDs can be easily tuned to optimize in-vivo imaging. A large Stokes shift, long fluorescence property, narrow emission band and near-infra-red emission is the fundamental properties for an ideal QD which could make it suitable for deeper imaging with high resolution [30].

3. Surface Modification and Shell Capping of QDs

QDs are generally hydrophobic and therefore require suitable surface modification prior to biological applications. Also, most QDs include heavy metal ions which are toxic (e.g. Cd and Te). When these QDs encounters UV excitation source, these heavy metal ions escape from the crystal and may cause cytotoxicity. Hence several studies have been done on developing suitable QDs surface modification which improves stability, solubility in aqueous medium, biocompatibility, fluorescence intensity with reduced cytotoxicity [28, 31-35]. Hence, some fundamental properties are required to target solid cancerous tumour; viz., fluorescent core material that have broad excitable and emission range from ultra-violet to near-infra-red region (NIR) (e.g. Ag2Se); Semiconducting shell with improved quantum efficiency and enhanced photostability; Hydrophilic ligands for attaching biomolecules such as follate, anti-body etc., with the QDs (e.g. polyethylene glycol), and biomolecule conjugation for active targeting (e.g. folate, antibody etc.). For enhancing fluorescent property of QDs, a wide band gap semiconductor shell coating is practiced since 1990 [36, 37]. Coating with suitable material shell also enhances photostability and photoluminescence quantum yields [38]. To functionalize QDs with suitable biomolecules to enable it to target specific proteins expressed on the surface of the cancer cells, they can be surface functionalized depends on various aspects of tumour microenvironment such as heterogeneous upregulation of surface proteins [39], activity of enzymes [40] and pH [41]. Hence, for wide theranostic biomedical applications, QDs are defined as nanoparticles with fluorescent core, semiconductor shell, surface modified for dispersion in water and functionalized with suitable biomolecule to target expressed proteins by cancer cells.  QDs are also said to leak especially through permeable tumour vessels and are retained due to reduced lymphatic drainage, which is known as the enhanced permeability and retention (EPR) effect [42]. However, tumour microenvironment factors and EPR effect alone may not be sufficient to target tumours. Hence recent studies have intended for incorporating active targeting moieties to improve tumour site accumulation and cancer cell-specific interactions with cancerous tissue compared to healthy tissue. QDs coated with polydentate phosphine aided in visual guidance throughout complete resection in the sentinel lymph node real-time mapping procedure in large animals [43].

4. Multimodal Nano Probes

Currently there are several medical imaging diagnostic tools in use for routine clinical utility. Among them, fluorescence imaging (FLI), magnetic resonance (MR), and computed tomography (CT) imaging are the most commonly applied scanning techniques. However, each modality has its own limitation and cannot provide complete information [44]. For example FL imaging can provide high sensitive but poor resolution images [45, 46].  CT imaging can provide a high spatial resolution and anatomical information for the tissues but with poor soft tissue contrast and lack sensitivity [47]. MR on the other hand can provide soft tissue contrast but its sensitivity is poor [48]. The main challenge in developing multi-modality probes is that they must integrate multi imaging contrast agents. For instance, CT requires much larger amount contrast agents for efficient signal readout than what optical imaging require. This large amount of contrast agent may further increase the toxicity level and solubility issues. Hence, biocompatibility is critical to warrant the safety in their further in vivo bioimaging applications. Hence, development of multi modal imaging probes that combines the advantages of every imaging modality would definitely improve the accuracy and sensitivity in disease diagnosis.  Studies have shown the utility of these multifunctional nanoprobes combining fluorescence, magnetic resonance and computed tomography that are promising and versatile agents for diagnosing tumours [49].  The use of these nano-probes in multimodal imaging is illustrated in figure – 2.

QDs can be synthesized for bimodal or multimodal applications suitable for infrared fluorescence, positron emission tomography (PET), SPECT, CT, MRI and ultrasonography imaging. Studies have proven QDs utilization in real-time imaging of cancer in-vitro and in-vivo [50-52].  Appropriate biomolecules are attached with QDs for targeting specific cancer markers which are found in the tumour microenvironment. For example, prostate-specific antigen [53-55], HER2 [56, 57], folic acid [58, 59] and CD44 [60]. The biomolecules that are attached to QDs are monoclonal antibody, or immunoglobulin , or peptide [61] [61, 62] depending on the target cell expressions.  QDs encapsulated with paramagnetic liposomes are used to monitor tumour angiogenesis using MRI [63, 64]. Stroh M and colleagues showed permeability of the tumour microvasculature in its microenvironment using different sized QDs [62]. Functionalized QDs with paramagnetic dendritic wedges and (Asn-Gly-Arg) peptides were used to demonstrate angiogenesis in tumour microenvironment and in myocardial infarction using MR molecular imaging [65, 66]. Apart from MR applications, QDs also labelled for positron emission tomography (PET) imaging for monitoring tumour angiogenesis [67], vascular endothelial growth factor [68], by functionalizing QDs with suitable biomolecules (Figure – 3). The advantage of using functional QDs also extended to therapeutics [69] and enhanced specificity for targeting [70].

5. Functionalization for Cell Targeting, Imaging, Biocompatibility

QDs can be conjugated with peptides, proteins, antibodies, aptamers (oligonucleotides), polyethylene glycol (PEG) and small molecules [31]. These surface modified QDs are widely used for different modality imaging, targeting and tracking, both in-vitro and in-vivo [72]. It is important to select appropriate strategy for achieving solubility and stability of QDs in aqueous solutions under physiological condition for various biological applications. Hydrophobic interaction (with amphipilic molecules) and ligand exchange strategies are the two main surface modification procedures of QDs, based on the requirement the method of their synthesis vary. The surface modification protocol is chosen without impairing the photo-physical properties of QD. These conjugated molecules have binding sites to achieve cell targeting, increasing uptake of QDs in cells [73]. The amine and carboxyl groups present in these biomolecules couple with surface modified QDs by forming simple amide bonds [74].  For instance, DNA has several coupling sites such as amines, hydroxyl and phosphate in which it can bind non-specifically with QDs and other micro-environmental molecules present [75]. The complimentary binding of the DNA fragment in the DNA functionalized QDs are detectable by calorimetric detection assays [76]. Another important conjugating biomolecule is the poly ethylene glycol (PEG) which proved to enhance the cellular uptake and increase the retention time of these QDs in the body [77]. These PEG conjugated QDs are shown to have enhanced endothelial permeability retention (EPR) rate and are biocompatible [78]. However, these PEGylated QD needs amine, thiol or carboxyl functional groups to achieve covalent ligation. Apart from these, carbohydrates such as dextran have been used widely to provide solubility and biocompatibility to the QDs [79].  These dextrans when coated on QDs said to tolerate a wide range of pH without affecting their fluorescence and fluorescence resonance energy transfer (FRET) properties [80]. Hence, depending on the application of QDs, their surface is modified which ensures the functional aspect to the QDs. Table -1 summarises the various nano formulation suitable for different imaging modalities that can image in single mode or in combination.  Multimodal molecular imaging pools two or more kinds of imaging techniques to for new fusion mode of imaging, which can pay way for obtaining further information in diagnosis and prognosis. Recently, multimodal molecular imaging has been extensively used to improve medical investigation and clinical practice. Multimodal molecular imaging has been successfully applied to diagnose various medical problems such as cardiovascular diseases [124], psychiatric abnormalities [125, 126], surgical resection of the tumour [127] etc. In summary, multimodal molecular imaging has a future potential development and will definitely bring a major breakthrough in the field of medical imaging and molecular science.

Imaging Modality	Contrast Probe	Reference
Computed Tomography (CT)	Conjugation of nanoparticle with X-ray-absorbing atoms and liposomes/ emulsion/ lipoproteins/ polymers	de Vries A et al [81], Elrod DB et al [82], Attia M et al. [83], Sung June Kim et al [84]
	Polymer-coated bismuth sulfate (Bi2 S3)	Rabin et al [85]
	Gold labelled 2-deoxy-d-glucose	Hyafil et al [86]
	Gold labelled 2-deoxy-d-glucose	Li J et al [87]
	Gold nanoparticles with a prostate-specific membrane antigen	Kim D et al [88]
	Gold nanoparticles with liposomal iodine	Kayyali MN et al [89]
	Iodine-contained diatrizoic acid (DTA) conjugated to glycol chitosan (GC - DTA)	Choi D et al [90]
	Nano composite of folic acid (FA), and iron platinum-dimercaptosuccinnic acid/PEGylated graphene oxide	Yue L et al [91]
Magnetic Resonance Imaging (MRI)	Diffusion weighted imaging (DWI), Apparent diffusion coefficient (ADC), Hydrogen proton magnetic resonance spectroscopy (MRS), Magnetization transfer ratio (MTR), other active nuclei such as hydrogen, phosphorous, sodium, carbon, and fluorine	[92-98]
	Superparamagnetic agents for altering proton relaxation time T1, T2, or T2*	[99]
	Non-specific, tumour-specific, antibody conjugated, specific proteinases or pH sensitive, T-cell/stem-cell labelled contrast agents	[100-103]
	Gold core with slilica coated trans-1,2-bis(4-pyridyl)-ethylene (MRI –Photoacoaustic Raman Imaging - MPR)	Moritz F Kircher et al [104]
	Nano composite of folic acid (FA), and iron platinum-dimercaptosuccinnic acid/PEGylated graphene oxide	Yue L et al [91]
Positron Emission Tomography (PET)	2-fluoro-2-deoxy-glucose ([18F]-FDG)	[71, 105-107]
Single Photon Emission Computed Tomography (SPECT)	Technetium 99 m, Copper 64, Gallium 68, Iodine 124	[108-112]
	Iodine 123, Indium (In-111), Gallium 67	[113, 114]
Ultrasonography (US)	GC-DTA Encapsulated with perfluoropentane (Bimodal probe for CT and US)	Choi D et al [90]
	Antibody attached micro bubbles	[115, 116]
	Peptide attached micro bubbles	[117, 118]
Optical Imaging Fluorescence, Bioluminescence, Fluorescent Molecular Tomography (FMT), etc.	Lanthanide-based probes, Arginyl peptides to cross-linked iron oxide amine (amino-CLIO), Fluorescent dye-doped silica (DySiO2), green fluorescent protein (GFP)	[119-123]

Table 1: Functionalized contrast probes for different Molecular Imaging Modality.

6. Summary

Molecular imaging using QDs is found to be effective for cancer imaging both in-vitro and in-vivo conditions. Though this technique is simple, low-cost, and highly sensitive in comparison to other imaging techniques it bench side to bed side transformation is still underway due to many reasons such as fluorescence particles inside the body, limitations in deep tissue penetration, etc. However, for studies involving detecting cancer biomarkers non-invasively which are expressed on cancer cell membrane at the early stage of cancer, application of QDs is promising.  NIR-emitting QDs are used widely for imaging solid tumour, tumour vasculatures and sentinel lymph nodes. Surface modification is necessary to achieve high stability, biocompatibility, clearance, specificity in tumour targeting, biodistribution, suitability of multimodal imaging, etc. In summary, the surface of the QDs needs to be engineered specific to each application and acts as a single unit that compliments the limitation of the imaging modality and suitable for unique bioimaging applications. Future research should be focused on not only multimodal but also functionalized imaging probes for effective targeting tumour for diagnosis and therapy. Non-invasive cancer imaging, drug delivery, real-time guidance for the surgery, continuous monitoring for drug therapy, imaging metastasis, detecting circulating tumour cells, imaging angiogenic vasculatures, sentinel lymph node is the basic requirement for effective cancer management. All these requirements could be satisfied in future by using suitable multimodal QDs. It is anticipated to achieve translational research activity in the near future using appropriate imaging probes for managing cancer and other dreadful disease.

7. Conclusion

 The multimodal QDs devoloped in future should satisfy all the requirements suitable for cancer theranostic applications. It is anticipated to achieve translational research activity in the near future using appropriate imaging probes for managing cancer and other dreadful disease.

Acknowledgments

The author would like to thank the Director- Research, Chettinad Academy of Research and Education for his moral support and encouragements.

 

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