Targosomes: Anti-HER2 PLGA nanocarriers for bioimaging, chemotherapy and local photothermal treatment of tumors and remote metastases.

Developing combined cancer therapy strategies is of utmost importance as it can enhance treatment efficacy, overcome drug resistance, and ultimately improve patient outcomes by targeting multiple pathways and mechanisms involved in cancer growth and progression. Specifically, the potential of developing a combination chemo&photothermal therapy using targeted polymer nanoparticles as nanocarriers offers a promising approach for synergistic cancer treatment by combining the benefits of both therapies, such as targeted drug delivery and localized hyperthermia. Here, we report the first targeted anti-HER2 PLGA nanocarriers, called targosomes, that simultaneously possess photothermal, chemotherapeutic and diagnostic properties using only molecular payloads. Biocompatible poly(lactic-co-glycolic acid), PLGA, nanoparticles were loaded with photosensitizer phthalocyanine, diagnostic dye Nile Blue, and chemotherapeutic drug irinotecan, which was chosen as a result of screening a panel of theragnostic nanoparticles. The targeted delivery to cell surface oncomarker HER2 was ensured by nanoparticle modification with the anti-HER2 monoclonal antibody, trastuzumab, using the one-pot synthesis method without chemical conjugation. The irradiation tests revealed prominent photothermal properties of nanoparticles, namely heating by 35 °C in 10 min. Nanoparticles exhibited a 7-fold increase in binding and nearly an 18-fold increase in cytotoxicity for HER2-overexpressing cells compared to cells lacking HER2 expression. This enhancement of cytotoxicity was further amplified by >20-fold under NIR light irradiation. In vivo studies proved the efficacy of nanoparticles for bioimaging of primary tumor and metastasis sites and demonstrated 93% tumor growth inhibition, making these nanoparticles excellent candidates for translation into theragnostic applications.

Phage-mimicking nanoagents for rapid depolymerase specificity screening against multidrug resistant bacteria.

With the rise of drug resistance, bacteriophages and bacteriophage-derived proteins may become an efficient successor to traditional antibiotics. While the enormous natural diversity of the phages allows matching virtually any bacteria, identification of the potentially life-saving phage is currently a tedious and time-consuming challenge that often cannot be performed within a reasonable time. Here we show a rapid 1-min bacteriophage screening assay based on specially constructed phage-mimicking nanoagents and surface plasmon resonance effect. Within the assay, a panel of phage-mimicking gold nanoparticles, possessing the specificity and enzymatic activity of a particular phage, is mixed with a suspension of the bacteria of interest. The spectral behaviour of the assay mix allows measurement of two critical parameters of the nanoagents and the corresponding bacteriophages: 1) direct assessment of their specificity due to convergence of the particles on the cell walls, and more importantly, 2) real-time evaluation of their enzymatic activity for the destruction of the cell capsule via detection of nanoagent detachment from the surface of bacteria. The proposed assay overcomes the current time limitations of the phage-bacteria matching procedures and thereby can facilitate faster development and adoption of phage-based therapies as a much-needed alternative to traditional antibiotics.

Dynamic light scattering biosensing based on analyte-induced inhibition of nanoparticle aggregation.

A new approach to direct quantitative detection of small molecules (haptens) by dynamic light scattering biosensing is presented. The proposed technique implements a homogeneous competitive immunoassay and is based on optical detection of specific inhibition of nanoparticle aggregation induced by the analyte in a sample. The technique performance was tested both in buffer and milk for detection of chloramphenicol - antibiotic relevant to food safety diagnostics. Good specificity, sensitivity (LOD in milk is 2.4 ng/ml), precision (4.0 ± 1.2%), ruggedness (8.3%), and 96% recovery in conjunction with a record wide dynamic range (3 orders of magnitude) of the nanosensing technique were demonstrated. Such characteristics complemented by the assay simplicity (no washing step) and a short assay time make the approach attractive for application as an analytical platform for point-of-care and field-oriented diagnostics. Graphical abstract.

Nanoparticle-based drug delivery via RBC-hitchhiking for the inhibition of lung metastases growth.

Delivery of particle-based theranostic agents via their transportation on the surfaces of red blood cells, commonly referred to as RBC-hitchhiking, has historically been developed as a promising strategy for increasing the extremely poor blood circulation lifetime, primarily, of the large-sized sub-micron agents. Here, we show for the first time that RBC-hitchhiking can be extremely efficient for nanoparticle delivery and tumor treatment even in those cases when no circulation prolongation is observed. Specifically, we demonstrate that RBC-hitchhiking of certain small 100 nm particles, unlike that of the conventional sub-micron ones, can boost the delivery of non-targeted particles to lungs up to a record high value of 120-fold (and up to 40% of the injected dose). To achieve this remarkable result, we screened sub-200 nm nanoparticles of different sizes, polymer coatings and ζ-potentials and identified particles with the optimal RBC adsorption/desorption behavior. Furthermore, we demonstrated that such RBC-mediated rerouting of particles to lungs can be used to fight pulmonary metastases of aggressive melanoma B16-F1. Our findings could change the general paradigm of drug delivery for cancer treatment with RBC-hitchhiking. It is not the blood circulation lifetime that is the key factor for nanoparticle efficiency, but rather the complexation of nanoparticles with the RBC. The demonstrated technology could become a valuable tool for development of new strategies based on small nanoparticles for the treatment of aggressive and small-cell types of cancer as well as other lung diseases.

Magnetometry based method for investigation of nanoparticle clearance from circulation in a liver perfusion model.

Nanoparticles (NPs) are among the most promising agents for advanced theranostics. However, their functioning in vivo is severely inhibited by the mononuclear phagocyte system (MPS), which rapidly removes all foreign entities from blood circulation. Little is known about the sequestration mechanisms and the ways to counteract them. New methods are highly demanded for investigation with high scrutiny of each aspect of NP clearance from blood. For example, while liver macrophages capture the majority of the administered particles, reliable investigation of this process in absence of other MPS components is hard to implement in vivo. Here, we demonstrate a novel method for real-time investigation hepatic uptake of NPs in an isolated perfused liver based on an extremely accurate magnetometric registration technique. The signal is obtained solely from the magnetic NPs without any 'background' from blood or tissues, which is a significant advantage over other techniques, e.g. optical ones. We illustrate the method capacity by investigation of behavior of different particles and show good correlation with in vivo studies. We also demonstrate notable suitability of the method for studying the NP clearance from the flow in the user-defined mediums, e.g. those containing specific serum components. Finally, the method was applied to reveal an interesting effect of short-term decrease of liver macrophage activity after the first interaction with small amounts of NPs. The developed perfusion model based on the high-performance magnetometry can be used for finding new mechanisms of NP sequestration and for development of novel 'stealth' nanoagents.

Data on characterization and validation of assays for ultrasensitive quantitative detection of small molecules: Determination of free thyroxine with magnetic and interferometric methods.

The presented data refer to optimization and quantitative characterization of a rapid lateral flow assay based on high-affinity bifunctional ligand and magnetic nanolabels, which was developed for detection of small molecules of thyroid hormones. The results were obtained by several techniques, including the magnetic particle quantification method, spectral-correlation interferometry and spectral-phase interferometry, dynamic light scattering, enzyme linked immunosorbent assay. The long-term stability of "antibody - magnetic nanoparticle" conjugates is shown. The assay specificity is confirmed, and verification of successful combination of magnetic particles and antibodies is demonstrated. The kinetic and equilibrium dissociation constants are determined for interactions between thyroxine and monoclonal antibodies. The obtained data could be used for design of other platforms for detection of small molecules.

Synthesis of Magnetic Nanoparticles Stabilized by Magnetite-Binding Protein for Targeted Delivery to Cancer Cells.

A new method for obtaining biomodified magnetite nanoparticles for targeted delivery to cells was developed. The method is based on the use of the C-terminal fragment of the Mms6 protein, which is involved in the magnetite biomineralization during the synthesis of magnetosomes in magnetotactic bacteria Magnetospirillum magneticum AMB-1, and the barnase*barstar high-affinity protein pair. The Mms6 protein fragment is required for stabilizing magnetite, and the barnase*barstar pair mediates the interaction between nanoparticles and the component for modification. The efficiency of this method was confirmed in the synthesis of magnetite nanoparticles recognizing the HER2/neu tumor marker and in the selective labeling of HER2/neu with these nanoparticles on the surface of cancer cells.

Ultrasensitive detection enabled by nonlinear magnetization of nanomagnetic labels.

Geometrically confined magnetic particles due to their unique response to external magnetic fields find a variety of applications, including magnetic guidance, heat and drug delivery, magneto-mechanical actuation, and contrast enhancement. Highly sensitive detection and imaging techniques based on the nonlinear properties of nanomagnets were recently proposed as innovative strong-translational potential methods applicable in complex, often opaque, biological systems. Here we report on the significant enhancement of the detection capability using optical-lithography-defined, ferromagnetic iron-nickel alloy disk-shaped particles. We show that an irreversible transition between strongly non-collinear (vortex) and single domain states, driven by an alternating magnetic field, translates into a nonlinear magnetic response that enables ultrasensitive detection of these particles. The record sensitivity of ∼3.5 × 10-9 emu, which is equivalent to ∼39 pg of magnetic material is demonstrated at room temperature for arrays of patterned disks. We also show that unbound disks suspended in the aqueous buffer can be successfully detected and quantified in real-time when administered into a live animal allowing for tracing of their biodistribution. The use of nanoscale ferromagnetic particles with engineered nonlinear properties opens prospects for further enhancing the sensitivity, scalability, and tunability of noise-free magnetic tag detection in high-background environments for various applications spanning from biosensing and medical imaging to anti-counterfeiting technologies.

Synthesis and Characterization of Hybrid Core-Shell Fe3O4/SiO2 Nanoparticles for Biomedical Applications.

The creation of markers that provide both visual and quantitative information is of considerable importance for the mapping of tissue macrophages and other cells. We synthesized magnetic and magneto-fluorescent nanomarkers for the labeling of cells which can be detected with high sensitivity by the magnetic particle quantification (MPQ) technique. For stabilization under physiological conditions, the markers were coated with a dense silica shell. In this case, the size and zeta-potential of nanoparticles were controlled by a modified Stober reaction. Also, we developed a novel facile two-step synthesis of carboxylic acid-functionalized magnetic SiO2 nanoparticles, with a carboxyl polymer shell forming on the nanoparticles before the initiation of the Stober reaction. We extensively characterized the nanomarkers by transmission electron microscopy, electron microdiffraction, and dynamic and electrophoretic light scattering. We also studied the nanoparticle cellular uptake by various eukaryotic cell lines.

Synthesis of magnetic silica nanomarkers with controlled physicochemical properties.

Magnetic markers which can be detected with an extremely high sensitivity with the method of magnetic particle quantification (MPQ) were synthesized. Using a controlled Stober reaction, a set of magnetic silica markers of different sizes and zeta potentials was obtained. The use of a carboxymethyl dextran polymer to stabilize the magnetite particles during the synthesis made it possible to substantially reduce the detection limit of the obtained construct, which opens up new opportunities for creating effective diagnostic nanoagents.

MPQ-cytometry: a magnetism-based method for quantification of nanoparticle-cell interactions.

Precise quantification of interactions between nanoparticles and living cells is among the imperative tasks for research in nanobiotechnology, nanotoxicology and biomedicine. To meet the challenge, a rapid method called MPQ-cytometry is developed, which measures the integral non-linear response produced by magnetically labeled nanoparticles in a cell sample with an original magnetic particle quantification (MPQ) technique. MPQ-cytometry provides a sensitivity limit 0.33 ng of nanoparticles and is devoid of a background signal present in many label-based assays. Each measurement takes only a few seconds, and no complicated sample preparation or data processing is required. The capabilities of the method have been demonstrated by quantification of interactions of iron oxide nanoparticles with eukaryotic cells. The total amount of targeted nanoparticles that specifically recognized the HER2/neu oncomarker on the human cancer cell surface was successfully measured, the specificity of interaction permitting the detection of HER2/neu positive cells in a cell mixture. Moreover, it has been shown that MPQ-cytometry analysis of a HER2/neu-specific iron oxide nanoparticle interaction with six cell lines of different tissue origins quantitatively reflects the HER2/neu status of the cells. High correlation of MPQ-cytometry data with those obtained by three other commonly used in molecular and cell biology methods supports consideration of this method as a prospective alternative for both quantifying cell-bound nanoparticles and estimating the expression level of cell surface antigens. The proposed method does not require expensive sophisticated equipment or highly skilled personnel and it can be easily applied for rapid diagnostics, especially under field conditions.

A comprehensive study of interactions between lectins and glycoproteins for the development of effective theranostic nanoagents.

A comprehensive study of the interactions between lectins and glycoproteins possessing different glycosylation profiles in the composition of nanoparticles was carried out in order to find specifically interacting protein pairs for the creation of novel classes of multifunctional nanoagets that based on protein-assisted selfassembly. We obtained information about specific interactions of certain lectins with selected glycoproteins as well as about the ability of certain monosaccharides to competitively inhibit binding of glycoproteins with lectins. These protein-mediated interactions may be involved in the formulation of self-assembled nanoparticles for therapy and diagnostics of various diseases.

[Detection of pyrethroids by spectral correlation interferometry].

A label-free method based on spectral correlation interferometry has been developed for highly sensitive detection of pyrethroids by competitive immunoassay on the surface of sensor chips made of widely available microscopy glass cover slips. It is shown that the method allows independent optimization of each step of the sensor surface modification. This fact may be used to increase the efficiency of development of protocols for a wide spectrum of immunoassays that employ glass surface as a solid phase. Detection of 3-phenoxybenzoic acid, which is one of the most stable metabolites of a large number of pyrethroids, on the surface of the optimized sensor chips has been demonstrated on the level of 15 pg/ml. That is 50 times better than the sensitivity of the enzyme-linked immunosorbent assay (ELISA).