The application of bio-nanotechnology in tumor diagnosis and treatment: a view

4.1 Nanotechnology and cancer diagnosis

Compared with traditional methods, the novel biosensor based on nanotechnology can improve the sensitivity of clinical diagnosis, in which some pathological tissues or organs can be detected earlier and more accurately. Following the development of genetic engineering, DNA biosensor is a new type of biosensor [15], [16]. The sensitivity and reliability of the DNA sensor can be evidently improved after nanomaterials were introduced into the DNA sensor design with higher DNA loading and amplified electrochemical detection signals. This DNA nano-sensor can be used to determine target DNA sequence, DNA mutation, and so on [17]. The oligonucleotide probes loaded with QDs (such as CdSe/ZnS or CdTe/CdS) can be used to study chromosomal abnormality and mutation, with good stability and biosafety. This kind of probes cannot impair the activity of antibodies, proteins, or DNA [18], [19], [20]. Using magnetic nanoparticles (NPs) as a DNA carrier or a template for DNA replication and amplification, the detection of DNA molecule and a single mismatched base can be realized by chemiluminescence technique [21], [22].

The cell biosensor based on nano-biotechnology can detect and distinguish different types of tumor cells with high sensitivity and speed and also evaluate the drug resistance of tumor cells. Then a reliable basis for cancer diagnosis and treatment can be provided [23]. As a high-throughput analytical technique based on nanotechnology, microfluidic chip has many characteristics such as lower energy consumption, shorter detection time, higher detection efficiency, lower detection cost, and so on. In clinical diagnosis, some important index of cancer such as carcinoembryonic antigen in human blood can be highly detected by the immunoassay microfluidic chip. Compared with the conventional enzyme-linked immunosorbent test (ELISA), the detection sensitivity of this immunoassay microfluidic chip is increased by 30 times. The correlation coefficient of these two methods was as high as 0.917, while the total analysis time was shortened from 45 h to 35 min [24], [25]. A liver tumor below 3 mm diameter can be sensitively found by superparamagnetic iron oxide microparticles, which is greatly significant for early diagnosis [26]. A cell sorting strategy based on anti-CD45 antibody-modified magnetic nanospheres was studied; leukocyte depletion efficiency was up to 99.9% within 30 min in simulated clinical samples, and the purity of the spiked hepatocellular carcinoma (HCC) cells was improved 265–317-fold [27]. Also, as potential biomedical nanomaterials, nano-cytotoxicity should be further studied in the future.

4.2 Fluorescent quantum dots and tumor markers or medical imaging

As a kind of NPs composed of II–VI and III–V elements (such as CdSe, CdTe, ZnSe, InP, InAs, and so on), QDs have excellent luminescence properties of fluorescence. The fluorescence intensity of QDs is higher than that of commonly used organic fluorescent dyes (such as Rhodamine B and 6G, etc.), and the stability is high. QDs have good anti-photobleaching ability, good biocompatibility, and long fluorescence lifetime [28]. It can be used as an ideal fluorescent probe and nano-carrier material and has been applied in the study of cell localization, signal transduction, and the movement and migration of intracellular molecules, with important application value in the diagnosis and treatment of diseases as shown in Figure 2 [29], [30]. Our previous study also indicated that, as drug carriers, surface functionalized CdTe QDs can improve the anti-tumor effect for tumor cells. Importantly, it can realize real-time labeling and monitoring of tumor cells and provide a new way to realize the “integration” process of monitoring and treatment during cancer therapy [31]. In particular, near infrared wavelength (700–900 nm) has a strong ability to penetrate tissue and resist bleaching, and it can avoid the interference of tissue scattering, absorption and background auto-fluorescence. Deep tissue imaging can be performed based on the near infrared QDs, with little or no side effects [32]. It has important research value in the fields of molecular imaging, non-invasive online monitoring, early diagnosis, treatment, and prognosis.

Figure 2:

A multifunctional probe consisting of a recognition unit of goat anti-mouse IgG to label the anti-EpCAM antibody, a fluorescent dye (Cy3) moiety for fluorescence (FL) imaging and upconversion nanoparticles (UCNPs) tag for both inductively coupled plasma-mass spectrometry (ICP-MS) quantification and upconversion luminescence (UCL) imaging of cancer cells. Cancer cells were counted and dual-modal imaged by using ICP-MS detection and down-conversion FL/UCL. A limit of detection of 1×10² HepG2 cells and a relative standard deviation of 7.1% for seven replicate determinations of 1×10³ HepG2 cells were obtained under the optimized conditions [29]. Reprinted from Yang et al. [29]. Copyright (2017), with permission from Elsevier.

In vitro studies also showed that QDs can be used to directly and quantitatively characterize tumor cells. These NPs can be used to collect the biomarkers or make the markers not degraded to improve the detection sensitivity. The time-resolved fluorescence (TRF) imaging technique was applied to immunohistochemistry in which the number of related lanthanide chelate nanoparticles is linear with the number of prostate specific antibodies [33]. Combined with QDs and multiphoton imaging, the transgenic mice expressing green fluorescent protein was studied to observe the cells and tissues around the tumor vessels. The results indicated that real-time imaging could be achieved and the tumor vessel can be distinguished from the perivascular cells and tissues by labeling with fluorescent nanocrystals. It opens up a new way for the study of tumor pathophysiology [34]. There are some diagnostic techniques (such as radiation diagnosis, X-ray tomography, and magnetic resonance imaging) that are combined with nanomaterials [35]. By controlling the dosage, the number of injection times, and the time interval, the toxicity of some NPs could be controlled to some extent.

Based on molecular recognition such as ligand and receptor, antigen and antibody reaction, the targeted imaging of lesions in vivo can be achieved by specific labeling with QDs. Folic acid can be covalently linked to PEGylated cyano propionic acid ester to form nano clusters. The affinity of these nano-covalent polymers with the folate binding protein was 10 times that of the folic acid alone [36], [37]. If the monoclonal antibody is attached to the semiconductor NPs modified by polyethylene glycol, QDs can accumulate in the tumor site and attain accurate positioning of the tumor tissue after the intravenous injection. The anti-growth factor antibody was linked with QDs, and the epidermal growth factor receptor (EGFR) could be accurately conjugated and labelled to detect biomarkers of early cervical cancer. Combined with optical imaging technology, the changes of cervical cancer can be detected at the molecular level. It is helpful for the patients before tumor metastasis [38], [39].

4.3 Nanotechnology and tumor therapy

Nanotechnology has made great progress in the research on disease treatment. Using magnetocaloric (magnetothermal) effect and photothermal effect of some nanomaterials, a series of new treatments have been developed. In particular, the use of superparamagnetic magnetic materials can achieve high sensitivity for tumor lesions imaging and diagnosis. Also, under the action of the external magnetic field, the temperature can be increased to 40–45°C to kill the tumor cells. Through the development of nanomedical devices, some complex tasks can be completed such as intracellular drug delivery, power generation, and even cleaning up the harmful deposits in blood vessels. Then, medical cell operation and biological treatment can be carried out [40], [41], [42], [43].

Semiconductor nanomaterials generally have good photocatalytic activity, which can be used as a photosensitizer applied to tumor photodynamic therapy (PDT) [44]. Combined with photosensitizer, PDT can selectively destroy the local abnormal tissue (including tumor) by selective photodynamic reaction. The basic mechanism is that the positioned photosensitizer in the targeted cells or tissues is stimulated to release the reactive oxygen species (ROS), including singlet oxygen and oxygen-free radical. Many kinds of biological molecules in tumor tissues and cells can react with ROS, in which cytotoxicity will emerge and eventually lead to the death of tumor cells and the disappearance of tumor tissues. PDT plays an increasingly important role in cancer therapy [45]. A nano photosensitizer was developed by British researchers to help treat colon cancer HT29 cells, in which poly lysine was placed into phthalocyanine to form polylysine bound tetrasulfonato-aluminum phthalocyanine entrapped nanoparticles (PCNP) NPs (45±10 nm), then porphyrin was loaded on the surface of the PCNP to form PCNP-P NPs (95±10 nm). Here, each NP can carry hundreds of photosensitive molecules, with the ability to deliver a large number of light-sensitive molecules to the tumor site. In PDT, it can carry a large number of photosensitive molecules into the tumor to kill cancer cells. The effect is better than the conventional method. The effect of PDT was enhanced by carrying two different photosensitive molecules [46].

Many nanomaterials, especially semiconductor nanomaterials such as nano titanium dioxide (TiO₂) and zinc oxide (ZnO), have good photocatalytic activity [47], [48]. Compared with the ordinary photosensitizer, nanomaterials have good stability, high catalytic performance, and low cost and cytotoxicity. More importantly, it can selectively destroy the tumor but not endanger the normal organization. PDT has played an important role in the treatment of skin cancer, prostate cancer, lung cancer, head and neck cancer, breast cancer, and other tumors [49]. PDT enriches the application scope of the photosensitizer and plays an important role in the combined therapy of tumors. In our study, a new type of TiO₂ nano-whisker material was prepared by modification and doping of the crystal structure, which can improve the photocatalytic effect of TiO₂ nanomaterials and effectively kill the tumor cells [50].

4.4 Nano-drug delivery system and tumor therapy

Because of its high specific surface area and surface and interface effect, nanomaterials have a natural advantage as drug carriers. Some surface-modified NPs can evade the identification of macrophages and can better target the tumor tissues except the mononuclear phagocytic system. Polyvinylpyrrolidone and PEG (poly(lactic-co-glycolic acid)-b-poly(ethylene glycol)) NPs can be loaded with different drug molecules. In vitro and in vivo melanoma experiments showed that these drug nanocompounds had obvious anti-tumor effect as shown in Figure 3 [51], [52]. Dextran adriamycin loaded into chitosan NPs (about 100 nm diameter) can significantly increase the drug absorption by Hela cells and liver cancer cells [53]. Aclarubicin loaded into the activated carbon NPs was given to mice with lymph node metastasis by local injection. The results showed that this drug delivery system can be selectively distributed on the local lymphatic system and form a high drug concentration in this region, while aclacinomycin concentration is very low in other parts of the mouse body [54]. The common chemotherapeutic agents such as daunorubicin and doxorubicin (DOX) were loaded into different types of nanomaterials (such as Fe₃O₄ NPs [55], ZnO NPs [56], carbon nanotubes [57], and so on). In vivo and in vitro studies showed that nano-drug complexes can significantly increase the effective concentration of chemotherapeutic agents in tumor cells and improve the cytotoxicity effect of drugs for tumor cells. Moreover, the nano-drug complex has no obvious side effects on the normal visceral tissues of the experimental rats, and the safety of the chemotherapeutic drugs is improved [58], [59], [60].

Figure 3:

Preparation of aptamer-functionalized, DOX-loaded NPs via nanoprecipitation of amphiphilic block copolymers, followed by carbodiimide-mediated aptamer coupling. As a targeted drug delivery system, these nanoparticles were roughly 100–125 nm in diameter and have a high encapsulated efficiency for DOX. Reprinted by permission from Weigum et al. [51].

The current report indicated that drug resistance can also be largely overcome by the nanomaterial itself or by the formation of nano-drug complex. For example, it is believed that P-gp reverse by NPs may be attributed to the high drug concentrations in the vicinity of the cell membrane following the drug’s transport into the cell. This locally high concentration of drugs is thought to saturate the P-gp glycoprotein [61]. Some nanomaterials were combined with chemotherapy drugs and MDR reversal agents to enhance the effect of MDR, in which poly(D,L-lactide-co-glycolide) (PLGA) can encapsulate adriamycin and cyclosporin A to form a nanocomposite drug. The in vitro and in vivo results indicated that these nanocomposite drugs can increase the local drug concentration in tumor cells and play the role of P-gp antagonists. The reversal of MDR was enhanced because of the synergistic effect of these two agents [62]. In other reports, water-in-oil-in-water (w/o/w) double-emulsion method was used to coload antisense-miRNA-21 and gemcitabine (GEM; 2′-deoxy-2′,2′-difluorocytidine) in PEGylated-PLGA-NPs, and PEGylated-PLGA NPs co-encapsulated with antisense-miRNA-21 and gemcitabine (GEM) was synthesized. The therapeutic efficacy in human HCC (Hep3B and HepG2) cells in vitro was tested. The cellular uptake of NPs displayed time-dependent increase of NP concentration inside the cells. These co-encapsulated NPs revealed increased treatment efficacy in HCC cells as shown in Figure 4 [63]. In addition, the nanocomposite drug between magnetic Fe₃O₄ NPs and DOX can reverse MDR of leukemia K562/A02 cell line. Through the signal pathway of transferrin receptor, the mRNA level of MDR gene (MDR1) was reduced, and the MDR was reversed [64], [65].

Figure 4:

Water-in-oil-in-water (w/o/w) double emulsion method was used to coload antisense-miRNA-21 and GEM in PEGylated-PLGA-NPs. These co-encapsulated nanoparticles revealed increased treatment efficacy in HCC cells, compared to cells treated with either antisense-miRNA-21- or GEM-loaded NPs at equal concentration, indicating that down-regulation of endogenous miRNA-21 function can reduce HCC cell viability and proliferation in response to GEM treatment. Reprinted with permission from Devulapally [63]. Copyright (2016) American Chemical Society.

HCC is caused by many factors, such as viral and chemical carcinogens, which lead to uncontrolled growth of HCC. HCC has a high malignancy degree, and surgical resection often loses the best time for treatment following the high recurrence rate and poor prognosis. Early diagnosis and treatment is an effective way to improve the survival rate of HCC patients [66], [67]. Nanotechnology gradually played an important role in the diagnosis and treatment of liver cancer. Vascular endothelial growth factor (VEGF) can serve as a biomarker in the diagnosis and treatment of HCC. The immunomagnetic reduction method constructed by the magnetic NPs and the high temperature superconducting quantum interference device can quantitatively detect the low VEGF concentration of HCC rat model. The detection limit was between 2 pg/ml and 8000 pg/ml, and the detection sensitivity was higher than that of the traditional ELISA method [68]. Based on proteomics, nano liquid chromatography-mass spectrometry technology can be used to analyze 59 known or unknown proteins expressed in HCC cell membrane. Then, the occurrence and development of HCC can be detected for the early diagnosis of cancer. Also, the high throughput screening of new drug evaluation can be used by this technology [69]. As a kind of specific protein, GPC3 (Glypican-3) is overexpressed on the surface of liver cancer cells and plays a great significance for the early diagnosis of HCC. αGPC3 (the monoclonal antibody of GPC3) labeled with biotin was attached to the surface of superparamagnetic NPs, and HepG2 cells highly expressing GPC3 can be targeted by these nanocomposites. These targeted nanocomposites can bring out dual imaging function, in which tumors can be early diagnosed. Also, the tumor site can be really time monitored during the operation. It plays an important clinical value in HCC diagnosis, treatment, and prognosis [70].

On the surface of liver cells, there is a galactose receptor that can bind to the glycoprotein ligand containing galactose residues at nonreduced ends. This conjugation could actively transfer into the liver cells. Galactose amine (GAL)-albumin (AN) NPs were synthesized by conjugation with GAL and AN, then loaded with DOX. This GAL-DOX-AN nano-drug system can be used for liver cancer’s targeted therapy and achieve the active transport of anticancer drugs [71]. The PLGA NPs loaded with paclitaxel and galactose can increase the encapsulation efficiency of paclitaxel and control drug release by reducing the hydrophobicity of particles surface and increasing the potential. Thus, there is a significant targeting and cytotoxicity for HepG2 cells [72]. There are binding sites for glycyrrhetinic acid (GA) and glycyrrhizic acid on the liver cell membrane. Thus, GA and glycyrrhizic acid can be used as the guiding groups in the liver-targeted drug delivery system. DOX-loaded GA-modified alginate (ALG) NPs (DOX/GA-ALG NPs) have a good targeting effect on HepG2 cells and can significantly inhibit the proliferation of tumor cells. Importantly, the heart cells and the liver cells surrounding the tumor were not affected by administration of DOX/GA-ALG NPs, whereas myocardial necrosis and apparent liver cell swelling were observed after DOX⋅HCl administration as shown in Figure 5 [73]. Epidermal growth factor (EGF) can stimulate the growth of epithelial cells and promote the occurrence of tumor. EGFR can be specifically combined with EGF, transforming growth factor and other ligands. There is expression of EGFR in normal tissues, but the expression in tumor tissues is much higher than that in normal tissues [74]. In vivo experiments showed that paclitaxel NPs targeted to EGFR were firstly clustered in the liver, then in kidney and tumor tissues. The peak value was reached in the tumor tissue after administration of 3 h [75].

Figure 5:

DOX released from DOX/GA-ALG NPs was pharmacologically active and therefore able to target and induce cell death in tumor cells. Both DOX/GA-ALG NPs and DOX⋅HCl suppress tumor growth compared with the saline group, and DOX/GA-ALG NPs exhibited the greatest antitumor effects. (A) Image of Kunming mice bearing H22 liver tumor in situ and a tissue slice of liver tumor in situ. (B) Representative photographs of excised H22 tumors in situ from mice treated with saline, GA-ALG NPs, DOX⋅HCl and DOX/GA-ALG NPs. Reprinted from Zhang et al. [73]. Copyright (2012), with permission from Elsevier.