Quantum dots (QDs) are practically nanoparticles, which are obtained by reducing sizes until they reach the order of nanometers. Their unique optical properties inspire scientists especially in medical sciences in applications such as tumor detection, cardiovascular imaging, and cancer targeting. Here, we first discuss scanning acoustic microscopy (SAM) results to evaluate the potential of SAM in the detection of lead-sulfide (PbS), graphene, and cadmium-telluride/cadmium sulfide (CdTe/CdS) quantum dot aggregates. The success of imaging quantum dots by SAM indicated the potential of SAM in monitoring the microenvironment of the disease and also the therapeutic effect of the drug-loaded QDs. Therefore, secondly we present confocal laser scanning microscopy results of graphene QDs engulfed in macrophages, which are in high numbers in the microenvironment of tumors, to evaluate the potential of graphene QDs in tumor targeting.
- graphene quantum dots
- drug delivery
- molecular markers
- biological imaging
QDs attract great attention as contrast and therapeutic agents since they have unique properties including good light stability, low toxicity, strong fluorescence intensity, and changing emission wavelength with QD size, ranging from 10 to 100 Å in radius [1, 2]. Therefore, they have great potential in the fields of biological imaging, molecular markers [3, 4, 5], and drug delivery [6, 7, 8]. They can be used in tumor detection , cardiovascular imaging , and cancer targeting . Noble metal nanoparticles have been widely used as biomarkers due to their high optical absorption and biocompatibility [12, 13]. Alternatively, graphene quantum dots (GQDs), exhibiting lower cytotoxicity than cadmium (Cd)-, selenium (Se)-, and lead (Pb)-based quantum dots do , are extensively used in biomedical applications [15, 16]. Using these quantum dots as contrast agents in imaging techniques such as magnetic resonance imaging (MRI) , optical coherence tomography (OCT) , positron emission tomography (PET), single-photon emission computed tomography (SPECT), optical imaging (e.g., with fluorescence and Raman detection) and photoacoustic imaging (PAI) [18, 19, 20] proved their potential as diagnostic agents. QD aggregate formation is beneficial for drug delivery with drugs loaded on the surface of QDs [6, 7]. The QD aggregates can be used as multifunctional nanosystems for biomedical imaging and drug delivery for simultaneous detection, monitoring, and treatment of diseases.
Scanning acoustic microscopy (SAM) is a noninvasive imaging modality, which gives information about the morphology and the mechanical properties of the specimen simultaneously with a micrometer resolution. Focused high-frequency ultrasound signals are sent onto the specimen and there is no requirement of special staining for the target material. An image of an area of around 5 mm × 5 mm is captured in a couple of minutes. The two-dimensional maps of speed of sound (SOS) [21, 22, 23, 24, 25, 26, 27, 28, 29, 30] or acoustic impedance [31, 32] can be obtained. For resolving cells and organelles, higher frequencies of 100 to 1200 MHz [33, 34, 35, 36, 37, 38, 39, 40] should be used.
Here, we first discuss the ability of SAM in the detection of lead-sulfide (PbS), graphene, and cadmium-telluride/cadmium sulfide (CdTe/CdS) quantum dots. SAM monitors QD aggregates with a micrometer resolution and also provides mechanical information by calculating acoustic impedance values. This achievement brings into mind that when these QDs are targeted to a diseased area with an attached drug, the detection of the microenvironment of a disease and also the therapeutic effect of the drug-loaded QDs are both feasible by SAM. When tumors are considered, there are macrophages in their microenvironment, which are cells of the immune system responsible in the detection, phagocytosis, and destruction of harmful organisms, and these can be targeted with QDs. Therefore, secondly we present confocal laser scanning microscopy results of macrophages filled with GQDs. Confocal laser scanning microscopy shows GQDs can be engulfed in macrophages and have potential in tumor targeting.
2. Why and how do optical properties change with quantum limitation?
Imagine the structure of a crystal, a regular structure with a certain distance between the atoms, and shrink this well-shaped structure many times, over and over again, and compress it to the smallest volume as much as possible. When we do it in real life, we encounter an effect called quantum limitation. The crystal you have imagined becomes so small that it obeys quantum rules of the micro-world rather than rules of the macro-world. Practically, we are not talking about a three-dimensional object in these dimensions anymore but a one-dimensional point. Probably, you do not mind if we call our shrinked crystal a quantum dot. This is how quantum dots emerge by reducing crystals to nanoscale (10−9m). Many features of these points, especially their optical properties, open up new medical applications. From medical imaging to therapy, quantum dots demonstrate the potential that nanotechnological advances can achieve, in medical sciences.
How come a crystal, fitting into a very tiny volume, has different optical properties? We have already mentioned that the shrinking crystal follows the quantum laws. There are orbits (remembering the Bohr model of the atom) at different energy levels surrounding the nucleus of the atom, and these orbits contain electrons (Figure 1). When electrons fall from a high energy level to a low energy level, they expose the energy difference between levels in the form of photons. We name and perceive this situation as the luminescence of an object.
During shrinking, the atoms of our crystal begin to get stuck in a gradually shrinking volume. As a result, the energy levels get closer, merge, and form the energy bands. With these energy bands formed, the energy gaps of the atom, , are changed (Figure 2). The energy gaps of the atom are the basis for the photons emitted, that is, the optical properties of the atom. In summary, the change in the distribution of energy levels of the atoms of our infinitely shrinking crystal causes optical properties to change.
So what does this optical change mean to us? Primarily, two important energy bands are formed under the quantum limitation, which can be summarized as the easily changed energy gap by the type of material we choose and the size of the particles when producing quantum dots (i.e., our crystal). By changing the energy gap of the material, photon energy is changed as well as its color. Consequently, we can change the color of the light emitted by the quantum dots according to our needs. This is a very important development for areas such as medicine, where imaging tools are highly needed. Figure 3 shows inverted fluorescent microscope images of QD aggregates of the same material with different QD sizes. Figure 3a shows the fluorescence image of CdTe/CdS QD (5 nm) aggregates, obtained with excitation source of 546 nm, while Figure 3b shows the fluorescence image of CdTe/CdS QD (2 nm) aggregates, obtained with excitation source of 430 nm. Change in QD size induces different energy gaps, therefore requiring different excitation wavelength. Similarly, change in material induces different energy gaps and, therefore, requires different excitation wavelength. Figure 4 shows the fluorescence image of water-based colloidal suspension of PbS QDs (5 nm) obtained with excitation source of 630 nm.
3. Theranostic approaches: imaging, targeted drug delivery, and therapy
Would it be sufficient to be limited to the field of imaging with quantum dots that have a lot of potential? No. Let’s take, for example, a tumor tissue. Our goal is to use quantum dots first, in imaging and diagnosis of tumors and, then, in therapy. It would be great if quantum dots targeted to the tumor can both designate the tumor boundary and be helpful for shrinking the tumor. We call the medical approaches that combine imaging, diagnosis, and therapy as theranostic approaches and quantum dots have great potential in this area .
The dimensions of QDs in the order of nanometer, which we use as high-quality imaging tools due to their optical properties, also help us in therapy. In today’s standard practice, the therapy process of tumor tissue involves heavy chemotherapeutic drugs, thus bringing a corrosive treatment process. In contrast, there is the possibility of giving targeted drugs with quantum dots. The targeted drug delivery technique, using structures such as quantum nanotubes, is based on fixing the drug to the surface of the quantum dots and sending the drug only to tumor cells.
What kind of quantum dots do we have for medical applications? The carbon element that forms the organic basis of life stands out among the quantum dots we have managed to produce until now. Graphite, the single-layer polymer of carbon, has proven its usefulness over time. Now, graphene quantum dots try to prove themselves. Thanks to in vivo studies in mice, graphene quantum dots, which we have been aware of their potential for a certain period of time, will be the pioneers of theranostic approaches in medicine [42, 43].
Graphene quantum dots, which have many roles in tumor treatment, will play a strategic role in diagnosis and treatment, from imaging of tumor tissue to transporting chemotherapeutic drugs to tumor cells with active targeting method. However, targeted drug delivery method has already been accomplished with nanoscale tools such as nanotubes. What makes GQDs special in cancer treatment is photothermal treatment. Photothermal treatment means heating the tumor area with the laser beam (photons) sent to tumor, whose boundary can be designated by luminous QDs (Figure 5). The miracle of GQDs is that they offer all of these possibilities together and enable cancer treatment with minimal surgical intervention. In particular, low toxicity values allow us to predict that GQDs will be widely used in living organisms in the future. GQDs already proved their applicability in tumor tissue treatment in mice. Breast cancer in humans is among the diseases that are predicted to be treated using GQDs. On the other hand, GQDs can also be used as contrast and drug delivery agent in indocyanine green angiography (ICG), instead of indocyanine green due to its enhanced healing rate (Figure 6). While answering our initial question, we would not be wrong if we say that our earnings are brand new medical practices that give excitement and hope.
4. Scanning acoustic microscopy results
SAM experiments are performed by a scanning acoustic microscope (AMS-50SI), developed by Honda Electronics (Toyohashi, Japan). SAM has two measurement modes; sound speed mode and acoustic impedance mode. In Figure 7, SAM system in acoustic impedance (AI) mode is demonstrated. It is composed of an 80 MHz transducer with quartz lens, a pulser/receiver, an oscilloscope, a computer, and a display monitor. An 80 MHz transducer, which has a spot size of 17 μm and a focal length of 1.5 mm, generates and also collects the reflected acoustic waves, therefore acting as a pulser/receiver. Distilled water is chosen as the coupling medium between the quartz lens and the substrate. X-Y stage controlled by a computer is responsible of the two-dimensional scanning of the transducer. The reflected signals from the reference material and the target are analyzed by the oscilloscope. Finally, acoustic intensity and impedance maps with 300 × 300 sampling points are visualized with a lateral resolution of approximately 20 μm.
SAM images of the QD aggregates, as can be seen in Figure 8, are received using acoustic impedance mode of SAM. The size of graphene and CdTe/CdS (green) QDs is 2 nm, the size of PbS QDs is 3 nm, and the size of CdTe/CdS (orange) QDs is 5 nm with quantum efficiencies higher than 50%. The images are constructed using the acoustic reflections from both surfaces of the reference (water) and QD aggregates on the polystyrene substrate. The acoustic impedance distributions indicate different acoustic properties due to the variation of elasticity of QD aggregates of different materials and sizes. Biocompatible graphene-based QDs can be used in clinical applications, while PbS, CdTe/CdS (green), and CdTe/CdS (orange) QDs are characterized here to certify the success of SAM. The success of SAM in monitoring the QD aggregates is promising for the early diagnosis of patients with potentially vulnerable plaques or tumors. The development of atherosclerotic plaques can be traced with the distribution and activity of macrophages by staining them with gold nanoparticles . Similarly, macrophage infiltration, which is a hallmark of cancer progression , can be detected staining macrophages with QDs [47, 48]. SAM probe, used in surgeries to determine the margins of the diseased regions, which are stained by QDs, will provide physicians a better precision in their judgments.
SAM measures the acoustic impedance of the target by comparing the reflected signal from the target with the signal reflected from the reference material, in acoustic impedance mode. The reflected signal from the reference material is
where is the signal transducer generates, is the acoustic impedance of water (1.50 MRayl), and is the substrate’s acoustic impedance (2.37 MRayl). The signal reflected by the target is
Consequently, the target’s acoustic impedance can be calculated as
5. Confocal laser scanning microscopy results
THP-1-derived macrophages, treated with different concentrations of graphene quantum dots (100, 250, 500, 750, 1000 μg/mL) for 24 hours, are visualized using confocal laser scanning microscope. According to Figure 9, QGDs are successfully internalized by THP-1-derived macrophages. Different layers of the z-stack (focus stacking) image show that dots are engulfed and stored in vesicles. Both fluorescent and bright-field images from the z-stack indicate that GQDs are distributed beneath cell membrane three-dimensionally.
For the determination of concentration range required for the visualization, GQDs are administered to macrophages in different concentrations such as 0 (ctrl), 250, 500, 750, and 1000 μg/mL. Figure 10 shows the signals coming from GQDs, which are engulfed by THP-1-derived macrophages at different concentrations. 500, 750, and 1000 μg/mL treatments can be seen visually, where the signal intensity of a 250 μg/mL treatment is not clearly observable.
The potential of QDs as contrast and therapeutic agent make these nanometer-sized particles very attractive for especially medical areas. Specifically, the low toxicity of graphene is favorable for in vivo studies. QDs have a tendency to aggregate, and the increase in concentration enhances aggregate formation, which becomes more likely to be observed. Imaging of these targeted QD aggregates provides detection and treatment of diseases. With a high-frequency transducer, SAM can detect smaller aggregates or even single QDs. The possibility of treating patients noninvasively by tracing and monitoring QDs, as in photothermal therapy, is a very important advantage. Consequently, multitasking QDs are excellent agents and worth being developed for biomedical applications.
All experiments were performed in the Department of Molecular Biology and Genetics and Department of Physics in Bogazici University.
Conflict of interest
The authors declare no conflict of interest.
I want to thank M. D. Kamran Aghayev (Biruni University, Istanbul, Turkey) for the invitation to the brain aneurysm surgery. I want to thank Melita Parlak (Bogazici University, Istanbul, Turkey) for performing all experiments.