Multifunctional Nanoprobes for Cancer Cell Targeting, Imaging and Anticancer Drug Delivery

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Multifunctional Nanoprobes for Cancer Cell Targeting, Imaging and Anticancer Drug Delivery

Pavel Linkov, Marie Laronze-Cochard, Janos Sapi, Lev Sidorov, Igor Nabiev

To cite this version:

Pavel Linkov, Marie Laronze-Cochard, Janos Sapi, Lev Sidorov, Igor Nabiev. Multifunctional

Nanoprobes for Cancer Cell Targeting, Imaging and Anticancer Drug Delivery. Physics Procedia,

Elsevier, 2015, 73, pp.216 - 220. �10.1016/j.phpro.2015.09.160�. �hal-03112420�

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Physics Procedia 73 ( 2015 ) 216 – 220

Peer-review under responsibility of the National Research Nuclear University MEPhI (Moscow Engineering Physics Institute) doi: 10.1016/j.phpro.2015.09.160

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Pavel Linkov et al. / Physics Procedia 73 ( 2015 ) 216 – 220 217

Nomenclature

DNA deoxyribonucleic acid

GM Goeppert-Mayer unit is equal to cm⁴ · s · photon^í¹ ML monolayer

PL photoluminescent

SILAR successive ionic layer adsorption and reaction TPE two-photon-excitation

QD quantum dot

QY quantum yield

The uncontrolled growth of cancer cells, started due to certain DNA stimuli. Anticancer drugs either kill cancer cells or modify their growth. Inhibitors of enzymes, responsible for DNA replication have become central parts of a cancer treatment (Arcidiacono et al. (2012)). Heterocyclic moieties fused with aromatic ring system with various heteroatoms like N, S, and O have possessed a wide spectrum of pharmacological activity. Synthesis of nitrogen containing heterocyclic compounds has been a subject of great interest due to important roles in cell biology during DNA replication, DNA transcription and segregation of chromosomes during mitosis (Neidle et al. (2005)). Inhibition of enzyme expression by stabilizing of both single- and double-strand DNA can trigger cellular signal transduction pathways leading to cancer cell death.

Intracellular transport of different biologically active molecules is one of the key problems in drug delivery in a cancer treatment. However, protection properties of the cellar membranes restrict the direct intracellular delivery of therapeutic compounds. Intracellular delivery using membrane-permeable vectors is a perspective method that is successfully employed to transport various therapeutic functional agents into cancer cells. Delivery methods can be categorized as involving viral (Luo et al. (2000)) or non-viral (Kaneda (2010)) carrier systems. Non-viral delivery methods such as peptide- and lipid-based systems have received more attention over the past decades than viral methods due to safety reasons. Advantages of non-viral systems are ease and flexibility of assembly, low toxicity. In case of properly selected way of cell penetration, an active transport vector can take drug molecule or even small particles into cells through the membrane (Farkhani et al. (2014)). Furthermore, vector can provide the affinity to DNA and can ensure nuclear targeting of the drug. Linking an imaging (fluorescence) agent to the pharmacological-agent-conjugated recognized vectors ensures real-time tracking of the delivery process of the active substance into the nucleus.

Conventional fluorophores, such as fluorescent dyes and fluorescent or bioluminescent proteins were originally used for this purpose. However, these fluorophores have some disadvantages imposing substantial limitations on the use of these tags. Quantum dots (QDs), semiconductor photoluminescent (PL) nanocrystals, are more promising candidates due to their unique fluorescent characteristics, such as size-tunable light emission, high signal brightness, and resistance to photobleaching. It is especially worthy to note that QDs have orders of magnitude higher values of two-photon absorption cross section (Hafian et al. (2014)) than organic molecules and, hence, can be used as effective biomedical fluorescent labels operating in the two-photon excitation mode. Two-photon excitation of biomedical markers allows fluorescence in the visible spectral range to be obtained upon irradiation of the probe in the near-infrared region of the spectrum, where the biological tissue transparency window is located (Smith et al. (2009)). The nonlinear dependence of the fluorescence intensity on the two-photon absorbance can significantly increase the contrast of tissue imaging for medical diagnosis by increasing the ratio of the label fluorescence signal to that of the background fluorescence of biological tissues. The resistance of CdSe-based QDs to photobleaching and their long-term stability in aqueous media make it possible to perform in vivo molecular tracking for extended periods of time (Jaiswal et al. (2004); Gao et al.

(2004)). Advanced core/shell QDs exhibit good biocompatibility and water solubility; standard bioconjugation strategies can be used for attaching biomolecules to them. It has been shown that conjugates of water-soluble CdSe/ZnS QDs and single-domain antibodies used as two-photon-excitation (TPE) probes enable clear discrimination of tumor areas from normal tissues (Hafian et al. (2014)).

In this paper, we present the structure of a new type of multipurpose nanoprobe for targeted delivery of therapeutic compounds into live cancer cells, which can be tracked using one- and two-photon confocal microscopies.

2. Development of multifunctional nanoprobes

We are developing a new structure of multifunctional nanoprobes allowing optical imaging. The multifunctional nanoprobe includes (1) a therapeutic functional agent (derivatives nitrogen heterocycle) capable of interacting with DNA in live cells; (2) a vector molecule responsible for selective targeting and internalization of anticancer agents into cancer cells; (3) innovative PL agents (QDs). These three functional components have to be carefully conjugated in such a way that the recognized vector system ensures penetration of the multifunctional probe into a cell. The drug agent targets the probe to the intercellular location, and the bright PL of QDs makes it possible to track the intracellular

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penetration of the nanoprobe and visualization of its interaction with the target by means of single- or multiphoton microscopy (Montenegro et al. (2013)).

2.1. Synthesis of CdSe/ZnS/CdS/ZnS core/multishell QDs

The construction of bright nanoprobes requires compact QDs with a high PL quantum yield (QY) and long-term stability in the aqueous medium. This was accomplished in the following steps: (1) synthesis of homogeneous defect- free CdSe QD cores; (2) coating of the cores with ZnS/CdS/ZnS multishells; (3) analyses of single-photon and two- photon emission properties of QDs of different sizes.

We have synthesized defect-free QDs with different core sizes (1.8, 2.3, and 2.7 nm in diameter) and different emission wavelengths in the visible spectral region. The QD cores were obtained by the hot-injection method using cadmium n-hexadecylphosphonate and selenium tri-n-octylphosphine complex as precursors, tri-n-octylamine as a stabilizing additive, and 1-octadecene as a solvent (Stsiapura et al. (2006)). Then, purified QD cores were coated with a novel ZnS/CdS/ZnS multishell having overall thickness of three monolayers (MLs) and providing an extraordinary high potential barriers (due to the first and last ZnS MLs), enhanced by a strong quantum confinement effect; the 3-ML thickness was sufficient for reliable protection of excited carriers (electrons and holes) from the environment. The shell coating procedure was based on the SILAR (Successive Ionic Layer Adsorption and Reaction) approach for controllable deposition of inorganic shells over QD cores. As a result, we obtained series of highly luminescent CdSe/ZnS/CdS/ZnS QDs with emission wavelength maxima at 540, 575, and 610 nm and PL QYs of 53, 97, and 80%, respectively (Samokhvalov et al. (2014)). The absorption and photoluminescence spectra of the obtained core/multishell samples are shown in Fig. 2.

400 450 500 550 600 650 700 750 800

Abs QD-540 Abs QD-570 Abs QD-610 PL QD-540 PL QD-570 PL QD-610

Onm

Absorbance / Photoluminescence intensity

Fig. 1. Absorption and PL spectra of CdSe/ZnS/CdS/ZnS samples.

Next, we studied the two-photon properties of three series of the synthesized QDs in chloroform (Linkov et al.

(2013)). The two-photon excitation action cross-section, the most important property in terms of multiphoton imaging, was measured to be 11000, 16000, and 13000 GM (1 GM = 10⁵⁰ cm³ s/photon) (Larson et al. (2003)) for the QD-540, QD-570 and QD-610, respectively, which is comparable with the best samples of CdSe/ZnS and is substantially higher than these values for organic fluorophores, which are typically no more than 100 GM (Smith et al. (2009)).

This finding allows us to conclude that the synthesized QDs have large two-photon excitation action cross sections, which makes the synthesized nanocrystals an excellent photoluminescent material for engineering of bright nanoprobes for multiphoton microscopy. Thus, the core/multishell QDs with excellent PL in the one- and two-photon modes can serve as materials for fabricating small-sized fluorescence labels for the aforementioned triple system.

2.2. Synthesis of a potential anticancer drugs and their assembly into a multifunctional nanoprobe

At present, we are starting the next stage, the synthesis of nitrogen containing heterocycle, which are potential anticancer agents. They will be obtained in a sequence of reactions of addition and substitution of functional groups allowing anchorage of vectors specifically recognized by cancer cell and QDs.

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Pavel Linkov et al. / Physics Procedia 73 ( 2015 ) 216 – 220 219 These der

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multiphotoni imaging pro biocompatibi The emergin new prospect Acknowledg This study 4.624.2014/K References

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