Suwussa Bamrungsap

Nanotechnology in therapeutics: a focus on nanoparticles as a drug delivery system

Continuing improvement in the pharmacological and therapeutic properties of drugs is driving the revolution in novel drug delivery systems. In fact, a wide spectrum of therapeutic nanocarriers has been extensively investigated to address this emerging need. Accordingly, this article will review recent developments in the use of nanoparticles as drug delivery systems to treat a wide variety of diseases. Finally, we will introduce challenges and future nanotechnology strategies to overcome limitations in this field.

Selective photothermal therapy for mixed cancer cells using aptamer-conjugated nanorods.

Safe and effective photothermal therapy depends on efficient delivery of heat for killing cells and molecular specificity for targeting cells. To address these requirements, we have designed an aptamer-based nanostructure which combines the high absorption efficiency of Au-Ag nanorods with the target specificity of molecular aptamers, a combination resulting in the development of an efficient and selective therapeutic agent for targeted cancer cell photothermal destruction. Most nanomaterials, such as gold nanoshells or nanorods (NRs), require a relatively high power of laser irradiation (1 x 10 (5)-1 x 10 (10) W/m (2)). In contrast, the high absorption characteristic of our Au-Ag NRs requires only 8.5 x 10 (4) W/m (2) laser exposure to induce 93 (+/-11)% cell death of NR-aptamer-labeled cells. Aptamers, the second component of the nanostructure, are generated from a cell-SELEX (systematic evolution of ligands by exponential enrichment) process and can be easily selected for specific recognition of individual tumor cell types without prior knowledge of the biomarkers for the cell. When tested with both cell suspensions and artificial solid tumor samples, these aptamer conjugates were shown to have excellent hyperthermia efficiency and selectivity. Under a specific laser intensity and duration of laser exposure, about 50 (+/-1)% of target (CEM) cells were severely damaged, while more than 87 (+/-1)% of control (NB-4) cells remained intact in a suspension cell mixture. These results indicate that the Au-Ag nanorod combination offers selective and efficient photothermal killing of targeted tumor cells, thus satisfying the two key challenges noted above. Consequently, for future in vivo application, it is fully anticipated that the tumor tissue will be selectively destroyed at laser energies which will not harm the surrounding normal tissue.

Aptamer-Conjugated Nanoparticles for Cancer Cell Detection

Aptamer-conjugated nanoparticles (ACNPs) have been used for a variety of applications, particularly dual nanoparticles for magnetic extraction and fluorescent labeling. In this type of assay, silica-coated magnetic and fluorophore-doped silica nanoparticles are conjugated to highly selective aptamers to detect and extract targeted cells in a variety of matrixes. However, considerable improvements are required in order to increase the selectivity and sensitivity of this two-particle assay to be useful in a clinical setting. To accomplish this, several parameters were investigated, including nanoparticle size, conjugation chemistry, use of multiple aptamer sequences on the nanoparticles, and use of multiple nanoparticles with different aptamer sequences. After identifying the best-performing elements, the improvements made to this assays conditional parameters were combined to illustrate the overall enhanced sensitivity and selectivity of the two-particle assay using an innovative multiple aptamer approach, signifying a critical feature in the advancement of this technique.

Engineering a Unimolecular DNA-Catalytic Probe for Single Lead Ion Monitoring

The binding of proteins and small molecules by DNA is well established, but more recently, DNA molecules have been selected to catalyze biochemical reactions. These catalytic DNAs, or DNAzymes, can be activated by metal ions. In this paper, we take advantage of DNA molecular engineering to improve the properties of DNAzymes by designing a unimolecular probe for lead ion (Pb(2+))-catalyzed reaction, achieving in turn, the ability to monitor a single Pb(2+) in solution by fluorescence microscopy. Specifically, by applying a unimolecular design, a leaving substrate DNA strand labeled with a fluorophore is linked to a hairpin 8-17 DNAzyme sequence labeled with a quencher. The hairpin structure and the substrate are connected using poly T, which brings the quencher into close proximity with the fluorophore in the inactive state. The intramolecular linkage of the two strands assures efficient quenching of the fluorescence, generating almost zero background. In the presence of Pb(2+), however, the leaving substrate fragment is cleaved at the RNA site by the enzyme, releasing a fluorescent fragment for detection with repetitive cycling for signal amplification. The resulting high sensitivity with a quantifiable detection range from 2 nM to 20 microM was achieved with a high selectivity in excess of 80-fold for Pb(2+) over other metal ions. The limit of detection is about 167 times better than the previously reported similar probes (Liu, J; Lu, Y. Anal. Chem. 2003, 75, 6666-6672) and 1600 times better compared to the Pb(2+) detection limit obtained from atomic spectroscopy. Thus, this probe could provide a simple, yet rapid and sensitive measurement for Pb(2+). Furthermore, we used this probe to monitor single Pb(2+) reaction kinetics. Given this degree of sensitivity and selectivity, our new probe design may prove useful in the development of other nucleic acid-based probes for intracellular, toxicological, and environmental monitoring.

Smart Multifunctional Nanostructure for Targeted Cancer Chemotherapy and Magnetic Resonance Imaging

Targeted chemotherapy and magnetic resonance imaging of cancer cells in vitro has been achieved using a smart multifunctional nanostructure (SMN) constructed from a porous hollow magnetite nanoparticle (PHMNP), a heterobifunctional PEG ligand, and an aptamer. The PHMNPs were prepared through a three-step reaction and loaded with the anticancer drug doxorubicin while being functionalized with PEG ligands. Targeting aptamers were then introduced by reaction with the PEG ligands. The pores of the PHMNPs are stable at physiological pH, but they are subject to acid etching. Specific binding and uptake of the SMN to the target cancer cells induced by aptamers was observed. In addition, multiple aptamers on the surface of one single SMN led to enhanced binding and uptake to target cancer cells due to the multivalent effect. Upon reaching the lysosomes of target cancer cells through receptor-mediated endocytosis, the relatively low lysosomal pH level resulted in corrosion of the PHMNP pores, facilitating the release of doxorubicin to kill the target cancer cells. In addition, the potential of using SMN for magnetic resonance imaging was also investigated.

Competition-mediated pyrene-switching aptasensor: probing lysozyme in human serum with a monomer-excimer fluorescence switch.

Lysozyme (Lys) plays crucial roles in the innate immune system, and the detection of Lys in urine and serum has considerable clinical importance. Traditionally, the presence of Lys has been detected by immunoassays; however, these assays are limited by the availability of commercial antibodies and tedious protein modification and prior sample purification. To address these limitations, we report here the design, synthesis, and application of a competition-mediated pyrene-switching aptasensor for selective detection of Lys in buffer and human serum. The detection strategy is based on the attachment of pyrene molecules to both ends of a hairpin DNA strand, which becomes the partially complementary competitor to an anti-Lys aptamer. In the presence of target Lys, the aptamer hybridizes with part of the competitor, which opens the hairpin such that both pyrene molecules are spatially separated. In the presence of target Lys, however, the competitor is displaced from the aptamer by the target, subsequently forming an initial hairpin structure. This brings the two pyrene moieties into close proximity to generate an excimer, which, in turn, results in a shift of fluorescence emission from ca. 400 nm (pyrene monomer) to 495 nm (pyrene excimer). The proposed method for Lys detection showed sensitivity as low as 200 pM and high selectivity in buffer. When measured by a steady-state fluorescence spectrum, the detection of Lys in human serum showed a strong fluorescent background, which obscured detection of the excimer signal. However, time-resolved emission measurement (TREM) supported the potential of the method in complex environments with background fluorescence by demonstrating the temporal separation of probe fluorescence emission decay from the intense background signal. We have also demonstrated that the same strategy can be applied to the detection of small biomolecules such as adenosine triphosphate (ATP), showing the generality of our approach. Therefore, the competition-mediated pyrene-switching aptasensor is promising to have potential for clinical and forensic applications.

Detection of Lysozyme Magnetic Relaxation Switches Based on Aptamer-Functionalized Superparamagnetic Nanoparticles

Magnetic relaxation switch (MRSw) detection is based on aggregate formation or dissociation when magnetic nanoparticles (MNPs) bind to target molecules. In the aggregated state, the dephasing rate of nearby proton spins is higher than in the dispersed state, resulting in a decrease in the spin-spin relaxation time, T(2). In this work, an MRSw-based nanosensor for lysozyme (Lys) protein detection was achieved using iron oxide nanoparticles conjugated with either Lys aptamer or linker DNA, which can hybridize with the extended part of the aptamer to form clusters. Upon the addition of Lys, the aptamers bind with their targets, leading to disassembly of clusters and an increase in T(2). A detection limit in the nanomolar range was achieved for Lys detection in both buffer and human serum. The determination of Lys level in different types of cancer cell lysates was also performed to demonstrate detection in real clinical samples.

Nanoparticle-aptamer conjugates for cancer cell targeting and detection.

Aptamers are DNA or RNA oligonucleotide sequences that selectively bind to their target with high affinity and specificity. They are obtained using an iterative selection protocol called SELEX. Several small molecules and proteins have been used as targets. Recently, a variant of this methodology, known as cell-SELEX, has been developed for a new generation of aptamers, which are capable of recognizing whole living cells. We have used this methodology for the selection of aptamers, which show high affinity and specificity for several cancer cells. In this chapter, we describe (1) the process followed for the generation of aptamers capable of recognizing acute leukemia cells (CCRF-CEM cells) and (2) the method of enhancing the selectivity and sensitivity of these aptamers by conjugation with a dual-nanoparticle system, which combines magnetic nanoparticles (MNP) and fluorescent silica nanoparticles (FNP). Specifically, the selected aptamers, which showed dissociation constants in the nanomolar range, have been coupled to MNPs in order to selectively collect and enrich cells from complex matrices, including blood samples. The additional coupling of the aptamer to FNPs offers an excellent and highly sensitive method for detecting cancer cells. In order to prove the potential of this rapid and low-cost method for diagnostic purposes, confocal microscopy was used to confirm the specific collection and detection of target cells in concentrations as low as 250 cells. The final fluorescence of the cells labeled with the nanoparticles was quantified using a fluorescence microplate reader.